WO2025231433A1

WO2025231433A1 - Organ perfusion and therapeutic delivery device

Info

Publication number: WO2025231433A1
Application number: PCT/US2025/027602
Authority: WO
Inventors: Heiko YANG; Marshall Stoller
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2024-05-03
Filing date: 2025-05-02
Publication date: 2025-11-06
Anticipated expiration: 2026-11-03

Abstract

Methods and devices are provided for perfusion of an organ. The perfusion device uses a perfusion circuit comprising a bypass loop with a flow valve or pinch valve that can be adjusted to control the flow rate of the perfusate to and from an organ while maintaining flow through an oxygenator for oxygenating the perfusate. Also provided is a sterile bag for holding the organ, which is connected to the perfusion circuit. The bag can be customized for any size organ and provides three-dimensional compression to reduce organ edema. The perfusion device can be used for normothermic perfusion as well as sub-normothermic and hypothermic perfusion. Methods of organ gene therapy and gene editing during ex vivo perfusion are also provided.

Description

ORGAN PERFUSION AND THERAPEUTIC DELIVERY DEVICE

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit under 35 U.S.C. § 119(c) of provisional application 63/642,381, filed May 3, 2024, which application is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. U2CDK133488 awarded by the National Institutes of Health. The government has certain rights in the invention.

INTRODUCTION

[0003] Donor organs are typically placed on ice until they are transplanted into a recipient. Ex vivo normothermic perfusion using a machine keeps organs warm and viable by continuously pumping blood through them. Advantages of ex vivo normothermic perfusion include that the organ has less ischemic time without blood flow, which may allow the organ to survive longer and better maintain function outside of the body. Perfusion of an organ on a machine also allows the quality and function of an organ to be evaluated before the organ is transplanted into a patient. [0004] Normothermic ex vivo organ perfusion is a growing frontier for biomedical investigation and therapeutic development. However, the high cost of commercial ex vivo perfusion equipment, the limited availability of human organs, and the steep technical learning curve present considerable barriers to the widespread adoption of this technology.

SUMMARY

[0005] Methods and devices are provided for perfusion of an organ. The perfusion device uses a perfusion circuit comprising a bypass loop with a flow valve or pinch valve that can be adjusted to control the flow rate of the perfusate to and from an organ while maintaining flow through an oxygenator for oxygenating the perfusate. Also provided is a sterile bag for holding the organ, which is connected to the perfusion circuit. The bag can be customized for any size organ and provides three-dimensional compression to reduce organ edema. The perfusion device can be used for normothermic perfusion as well as sub-normothermic and hypothermic perfusion. Methods of organ gene therapy and gene editing during ex vivo perfusion are also provided. [0006] In one aspect, a perfusion device for circulating perfusate to and from a donor organ is provided, wherein the perfusion device comprises a perfusion circuit comprising: a) a sterile container to hold the donor organ; b) a perfusate reservoir that collects the perfusate flowing out from the donor organ; c) a venous tube, wherein the venous tube can be connected to a vein of the donor organ and the perfusate reservoir, wherein the venous tube carries the perfusate from the vein to the perfusate reservoir; d) an oxygenator that oxygenates the perfusate, wherein the oxygenator comprises an inlet for receiving oxygen from an oxygen reservoir, an inlet for receiving the perfusate circulating through the perfusion circuit, and an outlet for delivering oxygenated perfusate to the perfusion circuit; e) a pump that propels the perfusate circulating through the perfusion circuit, wherein the pump is connected to the perfusate reservoir by a first tubing, and wherein the pump is connected to the inlet of the oxygenator by a second tubing; and f) arterial tube, wherein the arterial tube can be connected to an artery of the donor organ and the outlet of the oxygenator, wherein the arterial tube carries the oxygenated perfusate from the outlet of the oxygenator to the artery; g) a bypass tube wherein the first bypass tube is connected to the outlet of the oxygenator and the perfusate reservoir; and h) a pinch valve, wherein the pinch valve is positioned between a first portion of the bypass tube and a second portion of the bypass tube. [0007] In another aspect, a perfusion device for circulating perfusate to and from a donor organ is provided, wherein the perfusion device comprises a perfusion circuit comprising: a) a sterile container to hold the donor organ; b) a perfusate reservoir that collects the perfusate flowing out from the donor organ; c) a venous tube, wherein the venous tube can be connected to a vein of the donor organ and the perfusate reservoir, wherein the venous tube carries the perfusate from the vein to the perfusate reservoir; d) an oxygenator that oxygenates the perfusate, wherein the oxygenator comprises an inlet for receiving oxygen from an oxygen reservoir, an inlet for receiving the perfusate circulating through the perfusion circuit, and an outlet for delivering oxygenated perfusate to the perfusion circuit; e) a pump that propels the perfusate circulating through the perfusion circuit, wherein the pump is connected to the perfusate reservoir by a first tubing, and wherein the pump is connected to the inlet of the oxygenator by a second tubing; and f) an arterial tube, wherein the arterial tube can be connected to an artery of the donor organ and the outlet of the oxygenator, wherein the arterial tube carries the oxygenated perfusate from the outlet of the oxygenator to the artery; g) a sterile gravity bag; h) a first bypass tube wherein the first bypass tube is connected to the sterile gravity bag and the outlet of the oxygenator; and i) a second bypass tube wherein the second bypass tube is connected to the sterile gravity bag and the perfusate reservoir. [0008] In certain embodiments, the pump is a centrifugal pump or a peristaltic pump. In some embodiments, the pump maintains an artery pressure between in a range between 60 mm Hg to 75 mm Hg.

[0009] In certain embodiments, the perfusion device further comprises one or more heating elements or cooling elements. In some embodiments, the one or more heating elements comprise a water bath, a warming plate, or a combination thereof. In some embodiments, the one or more cooling elements comprise an ice bath, a thermoelectric temperature controller, or a combination thereof. In some embodiments, the perfusion device comprises: a first heating element or cooling element, wherein the first heating element or cooling element maintains the sterile container at a desired temperature; and a second heating element or cooling element, wherein the second heating element or cooling element maintains the perfusate reservoir at a desired temperature.

[0010] In certain embodiments, a heating element or cooling element is adjusted to maintain the temperature of the donor organ in a range from 4 °C to 40 °C. In some embodiments, the temperature is maintained in a range from 20 °C to 40 °C, 35 °C to 37 °C, 20 °C to 32 °C, 13 °C to 24 °C, or 4 °C to 10 °C during perfusion. In some embodiments, a heating element maintains the temperature of the donor organ at about 37 °C.

[0011] In certain embodiments, the perfusion device further comprises a connector, wherein the perfusate reservoir is connected to the sterile container by the connector.

[0012] In certain embodiments, the perfusion device further comprises a sample port, wherein the sample port is connected to the arterial tube.

[0013] In certain embodiments, the perfusion device further comprises a pressure sensor. In some embodiments, the pressure sensor is connected to the sample port.

[0014] In certain embodiments, the oxygenator is a pediatric oxygenator.

[0015] In certain embodiments, the sterile container further comprises a drainage port. In some embodiments, the drainage port is connected to an inlet of the perfusate reservoir by a drainage line tubing.

[0016] In certain embodiments, the donor organ is a kidney. In some embodiments, the artery is a renal artery and the vein is a renal vein. In some embodiments, a ureteral catheter or cannula that can be connected to a ureter of the kidney and a urine collection container that can be connected to the ureteral catheter or cannula by a urine drainage line tubing. In some embodiments, the flow rate of the perfusate through the renal artery is maintained in a range from 5 ml/minute to 1000 ml/minute. [0017] In certain embodiments, the donor organ is a kidney, a heart, a liver, a lung, a stomach, a small intestine, a large intestine, a pancreas, or a gonad, or a portion thereof.

[0018] In certain embodiments, the perfusion device further comprises an air bubble sensor that can detect air bubbles in the perfusate.

[0019] In certain embodiments, the perfusion device further comprises an infusion container, wherein the infusion container is connected to the perfusion circuit by infusion line tubing. In some embodiments, the infusion container is a syringe, wherein the syringe is connected to the infusion line tubing by a luer lock. In some embodiments, the infusion line tubing is connected to the outlet of the oxygenator and the infusion container.

[0020] In certain embodiments, the perfusion device further comprises an infusion pump or syringe pump to propel an infusion contained in the infusion container through the infusion line tubing into the perfusate. In some embodiments, the infusion container contains an infusion comprising a therapeutic agent or nutrient. In some embodiments, the infusion comprises heparin, prostacycline, glucose, insulin, a bile salt, an amino acid, a fatty acid, a steroid, a diuretic, a recombinant nucleic acid, a vector, a gene editing system, or any combination thereof. In some embodiments, the recombinant nucleic acid, vector, or gene editing system is encapsulated in a lipid nanoparticle (LNP). In some embodiments, the recombinant nucleic acid is a DNA or RNA encoding a therapeutic protein or regulatory RNA. In some embodiments, the recombinant nucleic acid comprises a viral vector or plasmid. Exemplary viral vectors include, without limitation, an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, or a retroviral vector. In some embodiments, expression of the therapeutic protein or regulatory RNA is inducible. In some embodiments, the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of a therapeutic protein. In some embodiments, the gene editing system comprises a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator- like effector nuclease (TALEN). In some embodiments, wherein the organ is a kidney, the recombinant nucleic acid or gene editing system is delivered in the perfusate to a nephron.

[0021] In certain embodiments, the perfusion device further comprises a stand, wherein the sterile container is placed on the stand.

[0022] In certain embodiments, the sterile container is a bag.

[0023] In certain embodiments, the perfusion device further comprises a pole, wherein the pole is used to hang the bag. [0024] In certain embodiments, the sterile container further comprises a plurality of magnets, wherein the plurality of magnets applies compression to reduce edema of the donor. organ.

[0025] In certain embodiments, the donor organ is obtained from a live organ donor or an organ donor after circulatory death.

[0026] In certain embodiments, a venous cannula is used to connect the venous tube to a vein of the donor organ, and an arterial cannula is used to connect the arterial tube to an artery of the donor organ.

[0027] In certain embodiments, the perfusion device further comprises a plurality of magnetic tubing connectors for connecting the tubing of the perfusion circuit, wherein a first magnetic tubing connector is attached to a first tubing and a second magnetic tubing connector is attached to a second tubing, wherein the first tubing and the second tubing are connected to each other by magnetically joining the first magnetic tubing connector to the second magnetic tubing connector. In some embodiments, the first magnetic tubing connector and the second magnetic tubing connector, when magnetically joined, can be rotated to reduce kinking of the first tubing and the second tubing. In some embodiments, each magnetic tubing connector comprises a rubber gasket, wherein tubing connections made with the plurality of magnetic tubing connector are watertight up to a pressure of at least 250 millimeter of mercury (mm Hg).

[0028] In certain embodiments, the perfusion device further comprises a sensor for measuring temperature of the perfusate, flow-rate of the perfusate, pH of the perfusate, concentration of oxygen in the perfusate, concentration of glucose in the perfusate, concentration of hemoglobin in the perfusate, concentration of sodium in the perfusate, concentration of potassium in the perfusate, concentration of calcium in the perfusate, concentration of carbon dioxide in the perfusate, percent saturation of oxygen in the perfusion, or concentration of lactate in the perfusate, or any combination thereof.

[0029] In another aspect, a method of using a perfusion device, described herein, for perfusion of an organ is provided, the method comprising: placing the organ in the sterile container; adding perfusate to the perfusate reservoir; connecting the venous tube to a vein of the organ and the perfusate reservoir; connecting the arterial tube to an artery of the organ and the outlet of the oxygenator; connecting the oxygen reservoir to the inlet of the oxygenator; and turning on the pump to circulate the perfusate through the perfusion circuit and to and from the donor organ.

[0030] In certain embodiments, the method further comprises turning on a first heating element or cooling element, wherein the first heating element or cooling element maintains the sterile container at a first desired temperature; and turning on a second heating element or cooling element, wherein the second heating element or cooling element maintains the perfusate reservoir at a second desired temperature. In some embodiments, the first heating element or cooling element and the second heating element or cooling element are adjusted to maintain the temperature of the sterile container and the perfusate reservoir in a range from 4 °C to 40 °C during perfusion. In some embodiments, the temperature is maintained in a range from 20 °C to 40 °C, 35 °C to 37 °C, 20 °C to 32 °C, 13 °C to 24 °C, or 4 °C to 10 °C during perfusion. In some embodiments, a heating element maintains the temperature of the donor organ at about 37 °C.

[0031] In certain embodiments, the sterile container further comprises a drainage port, wherein the method further comprises connecting the drainage port to the perfusate reservoir with a drainage line tubing.

[0032] In certain embodiments, the method further comprises: connecting a sample port to the arterial tube; and connecting a pressure sensor to the sample port.

[0033] In certain embodiments, the sterile container further comprises a drainage port, wherein the method further comprises connecting the drainage port to the inlet of the perfusate reservoir with a drainage line tubing.

[0034] In certain embodiments, the donor organ is a kidney. In some embodiments, connecting the arterial tube comprises connecting the arterial tube to a renal artery, and connecting the venous tube comprises connecting the venous tube to a renal vein. In some embodiments, the method further comprises connecting a ureteral catheter or cannula to a ureter of the kidney; and connecting a urine drainage line tubing to the ureteral catheter or cannula and a urine collection container, wherein urine is collected in the urine collection container. In some embodiments, the method further comprises adjusting the flow valve such that flow rate of the perfusate through the renal artery is maintained in a range from 60 ml/minute to 700 ml/minute.

[0035] In certain embodiments, the donor organ is a kidney, a heart, a liver, a lung, a stomach, a small intestine, a large intestine, a pancreas, or a gonad, or a portion thereof.

[0036] In certain embodiments, the method further comprises adjusting the pinch valve such that flow rate of the perfusate through the renal artery is maintained in a range from 5 ml/minute to 1000 ml/minute.

[0037] In certain embodiments, the method further comprises connecting infusion line tubing to an infusion container and the perfusion circuit; adding an infusion to the infusion container; and using a syringe pump or infusion pump to propel the infusion into the perfusate. In some embodiments, the infusion comprises a therapeutic agent or a nutrient. In some embodiments, the infusion comprises heparin, prostacycline, glucose, insulin, a bile salt, an amino acid, a fatty acid, a steroid, a diuretic, a recombinant nucleic acid a vector, a gene editing system, or any combination thereof. In some embodiments, the recombinant nucleic acid, vector, or gene editing system is encapsulated in a lipid nanoparticle (LNP). In some embodiments, the recombinant nucleic acid is a DNA or RNA encoding a therapeutic protein or regulatory RNA. In some embodiments, the recombinant nucleic acid comprises a viral vector or plasmid. Exemplary viral vectors include, without limitation, an adeno-associated viral vector, an adenoviral vector, a lenti viral vector, or a retroviral vector. In some embodiments, expression of the therapeutic protein or regulatory RNA is inducible. In some embodiments, the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of a therapeutic protein. In some embodiments, the gene editing system comprises a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN). In some embodiments, wherein the organ is a kidney, the recombinant nucleic acid or gene editing system is delivered in the perfusate to a nephron.

[0038] In certain embodiments, the method further comprises genetically modifying the organ during perfusion. In some embodiments, the organ is genetically modified to convert a disease-associated allele to a wild-type allele or an immunologically non-compatible allele to a compatible allele.

[0039] In certain embodiments, the method further comprises administering a therapeutic agent locally to a site on the organ during ex vivo perfusion of the organ.

[0040] In certain embodiments, the therapeutic agent is toxic when administered to a subject in vivo. In some embodiments, the therapeutic agent is a gene therapy agent, a chemotherapeutic agent, or a radiotherapeutic agent.

[0041] In certain embodiments, the method further comprises pressurizing hollow spaces within the organ. In some embodiments, wherein the organ is a kidney, pressurizing comprises pressurizing a renal pelvis or collecting system of the kidney.

[0042] In certain embodiments, the method further comprises surgically repairing the organ prior to transplantation into a recipient.

[0043] In certain embodiments, the method further comprises placing the sterile container on a stand.

[0044] In certain embodiments, the sterile container is a bag, wherein the method further comprises hanging the bag on a pole. [0045] In certain embodiments, the method further comprises adding a plurality of magnets to the sterile container, wherein the plurality of magnets applies compression to reduce edema of the donor organ during the perfusion.

[0046] In certain embodiments, the donor organ is obtained from a live organ donor or an organ donor after circulatory death.

[0047] In certain embodiments, a venous cannula is used to connect the venous tube to a vein of the donor organ, and wherein an arterial cannula is used to connect the arterial tube to an artery of the donor organ.

[0048] In certain embodiments, a plurality of magnetic tubing connectors are used for connecting the tubing of the perfusion circuit, wherein a first magnetic tubing connector is attached to a first tubing and a second magnetic tubing connector is attached to a second tubing, wherein the first tubing and the second tubing are connected to each other by magnetically joining the first magnetic tubing connector to the second magnetic tubing connector. In some embodiments, the method further comprises rotating the first magnetic tubing connector relative to the second magnetic tubing connector, when the first magnetic tubing connector and the second magnetic tubing connector are magnetically joined to reduce kinking of the first tubing and the second tubing.

[0049] In certain embodiments, the method further comprises measuring temperature of the perfusate, flow-rate of the perfusate, pH of the perfusate, concentration of oxygen in the perfusate, concentration of glucose in the perfusate, concentration of hemoglobin in the perfusate, concentration of sodium in the perfusate, concentration of potassium in the perfusate, concentration of calcium in the perfusate, concentration of carbon dioxide in the perfusate, percent saturation of oxygen in the perfusion, or concentration of lactate in the perfusate, or any combination thereof.

In certain embodiments, a higher overall flow rate of the perfusate is maintained through the perfusion circuit and a lower flow rate of the perfusate is maintained through the organ. [0050] In another aspect, a bag to hold an organ is provided, wherein the bag comprises: a plurality of magnets, wherein the plurality of magnets applies compression to reduce edema of the donor organ during perfusion of the organ; a drainage port, wherein the drainage port can be connected to a perfusion circuit such that blood from bleeding of the organ or perfusate in the bag flows through the drainage port into the perfusion circuit.

[0051] In certain embodiments, the bag has a size designed to fit the organ.

[0052] In certain embodiments, the organ is from an adult, child, or infant. [0053] In certain embodiments, the organ is a kidney, a heart, a liver, a lung, a stomach, a small intestine, a large intestine, a pancreas, or a gonad, or a portion thereof.

[0054] In certain embodiments, the bag is sterilized.

[0055] In certain embodiments, the bag is maintained at a desired temperature. In some embodiments, the temperature of the bag is maintained in a range from 4 °C to 40 °C. In some embodiments, the temperature of the bag is maintained in a range from 20 °C to 40 °C, 35 °C to 37 °C, 20 °C to 32 °C, 13 °C to 24 °C, or 4 °C to 10 °C. In some embodiments, the bag is heated to a temperature of about 37 °C.

[0056] In certain embodiments, the bag further comprises perfusate.

[0057] In certain embodiments, the bag further comprises an organ.

BRIEF DESCRIPTION OF THE DRAWINGS

[0058] FIGS. 1A-1D. Blueprints for low-cost normothermic ex vivo kidney perfusion (NEVKP) systems. FIGS. 1A-1C. Three NEVKP circuits (prototype A (FIG. 1A), prototype B (FIG. IB) and prototype C (FIG. 1C)) reflecting the chronological evolution of our circuit design. FIG. ID. Experimental workflow from harvesting tissue to initiating NEVKP.

[0059] FIGS. 2A-2E. Perfusion of non-heparinized kidneys. FIG. 2A. Mottled appearance of a flushed kidney harvested from an animal without systemic heparinization compared to a flushed kidney from a heparinized animal. FIG. 2B. Perfusion pressure during first 60 minutes of NEVKP when flow rate was maintained at a constant 100 mL/min. Representative photo of pressure-related hemorrhage in a non-heparinized kidney. FIG. 2C. Normalized perfusion flow rate during first 60 minutes of NEVKP in heparinized and non-heparinized kidneys when perfusion pressure was held constant between 70 and 80 mmHg. Representative photo of non-heparinized kidney perfused for 60 minutes without hemorrhage. FIG. 2D. Representative H&E stains of porcine renal tissue according to perfusion condition. Scale bars = 100 pm. FIG. 2E. Comparison of urine production start times during NEVKP for heparinized and non- heparinized kidneys.

[0060] FIGS. 3A-3D. Normothermic ex vivo kidney containment bag. FIG. 3A. Front view of bag design. FIG. 3B. Surface temperature of kidney during NEVKP during first 20 minutes (n=4). FIG. 3C. Side cross-sectional view of kidney within the bag with ultrasound probe. FIG. 3D. Representative grayscale ultrasound with color Doppler image of kidney during NEVKP.

[0061] FIGS. 4A-4G. Prolonged NEVKP of porcine and human kidneys. FIG. 4A. Aggregate pH, pCh, pCCh, and urine output over time from five porcine NEVKP experiments. FIG. 4B. Perfusate and urine potassium concentration over time from experiment P5. FIG. 4C. Representative H&E stain of glomerulus and renal tubules after 4 and 36 hours of NEVKP. FIG. 4D. Aggregate pH, pO2, pCCE, and urine output over time from four human donor NEVKP experiments. FIG. 4E. Gross examination of kidney from experiment Hl. A small lower pole artery was ligated during preparation for NEVKP. Yellow quadrangles indicate well perfused tissue and the unperfused area of the lower pole. FIG. 4F. H&E stains of the perfused and unperfused areas from experiment Hl after 18 hours of NEVKP. FIG. 4G. NEVKP-associated weight gain of human and porcine kidneys.

[0062] FIGS. 5A-5D. Autotransplantation of porcine kidneys after NEVKP. FIG. 5A. Experimental schematic of NEVKP and autotransplantation experiments with comparison of urinary potassium (mmol/L), creatinine (mg/dL), and specific gravity. FIG. 5B. Representative photo of an absorbent pad placed beneath the animal’s cage with a wet area (lower left) indicative of urine. This photo was taken on Day 2 after autotransplantation of a 24-hour NEVKP kidney. FIG. 5C. Representative image of Doppler waveform and resistive index of the 24-hour NEVKP autograft on Day 2 after autotransplantation. FIG. 5D. H&E stain of 24-hour autograft taken on necropsy. Scale bars = 100 pm. SCS = Static Cold Storage, RI = Resistive Index.

[0063] FIG. 6. Perfusate pCb, an indicator of oxygenation efficiency, within first 15 minutes of NEVKP for new and used oxygenators.

[0064] FIG. 7. Learning curve for NEVKP. Duration and experimental outcomes of first 17 porcine NEVKP experiments.

[0065] FIG. 8. Normothermic ex vivo kidney perfusion system with bypass loop.

[0066] FIGS. 9A-9B. Ex vivo biobag. FIG. 9A shows a side view and FIG. 9B shows a view of a cross-section of the ex vivo biobag.

[0067] FIGS. 10A-10B. Magnetic tubing connector. FIG. 10A shows a side view and FIG. 10B shows a view of a cross-section of the magnetic tubing connector.

[0068] FIGS. 11A-11B. Endoscopic method to deliver a gene therapy vector (e.g. lipid nanoparticle) into the nephron. FIG. 11A shows a schematic of the kidney with an expanded view of the nephron. FIG. 11B shows water-tight cannulation of the ureter/renal pelvis to allow pressurized delivery of a gene therapy vector (e.g., encapsulated in a lipid nanoparticle) into the nephron. Delivery can be targeted using direct visualization.

[0069] FIGS. 12A-12C show cannulas (FIG. 12A), a pump (FIG. 12B), and an oxygenator (FIG. 12C) that can be used in the normothermic ex vivo kidney perfusion system.

[0070] FIGS. 13A-13B. Blood potassium levels (FIG. 13A) and urine output (FIG. 13B) during normothermic ex vivo perfusion of porcine and human kidneys. 20 porcine kidneys underwent perfusion for a maximum of 36 hours. The porcine kidneys were able to produce urine, maintain electrolyte homeostasis, and were responsive to Lasix. 2 human kidneys (discarded donor organs) also underwent perfusion for a maximum of 24 hours. The human kidneys were also able to produce urine, maintain electrolyte homeostasis, and were responsive to Lasix.

[0071] FIG. 14. Photograph of the normothermic ex vivo perfusion system with bypass loop.

[0072] FIG. 15. Evidence of osmotic tubular changes resulting from ex vivo perfusion.

[0073] FIG. 16. Photograph of perfused and unperfused regions of a kidney shows that the perfused region had no ischemic necrosis after ex vivo perfusion, whereas the unperfused region has ischemic necrosis.

DETAILED DESCRIPTION

[0074] Methods and devices are provided for perfusion of an organ. The perfusion device uses a perfusion circuit comprising a bypass loop with a flow valve or pinch valve that can be adjusted to control the flow rate of the perfusate to and from an organ while maintaining flow to an oxygenator for oxygenating the perfusate. Also provided is a sterile bag for holding the organ, which is connected to the perfusion circuit. The bag can be customized for any size organ and provides three-dimensional compression to reduce organ edema. The perfusion device can be used for normothermic perfusion as well as sub-normothermic and hypothermic perfusion. Methods of organ gene therapy and gene editing during ex vivo perfusion are also provided.

[0075] Before exemplary embodiments of the present invention are described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0076] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0077] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and exemplary methods and materials may now be described. Any and all publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0078] It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a magnet" includes a plurality of such magnets and reference to "the therapeutic agent" includes reference to one or more therapeutic agents and equivalents thereof, e.g., drugs, therapeutic proteins, therapeutic RNAs, gene therapy vectors, gene editing systems, and the like, known to those skilled in the art, and so forth.

[0079] It is further noted that the claims may be drafted to exclude any element which may be optional. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely”, “only” and the like in connection with the recitation of claim elements, or the use of a “negative” limitation.

[0080] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. To the extent such publications may set out definitions of a term that conflicts with the explicit or implicit definition of the present disclosure, the definition of the present disclosure controls.

[0081] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. DEFINITIONS

[0082] The term "about," particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

[0083] The terms “individual”, “subject”, and “patient” are used interchangeably herein and refer to any mammalian subject, particularly humans. Mammalian subjects include human and non- human mammals such as non -human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; and farm animals such as sheep, goats, pigs, horses, and cows.

[0084] The term “user” as used herein refers to a person that interacts with a device and/or system disclosed herein for performing one or more steps of the presently disclosed methods. The user may be a physician or perfusionist operating the perfusion device to circulate perfusate to and from a donor organ, as described herein. For example, the user may be a nephrologist or renal technologist if the donor organ is a kidney.

[0085] The terms "protein", "peptide", and "polypeptide" refer to any compound comprising naturally occurring or synthetic amino acid polymers or amino acid-like molecules including but not limited to compounds comprising amino and/or imino molecules. No particular size is implied by use of the terms "protein", "peptide", and "polypeptide", and these terms are used interchangeably. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic). Thus, synthetic oligopeptides, dimers, multimers (e.g., tandem repeats, linearly-linked peptides), cyclized, branched molecules and the like, are included within the definition. The terms also include molecules comprising one or more peptoids (e.g., N-substituted glycine residues) and other synthetic amino acids or peptides. (See, e.g., U.S. Patent Nos. 5,831,005; 5,877,278; and 5,977,301; Nguyen et al. (2000) Chem Biol. 7(7):463-473; and Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89(20):9367-9371 for descriptions of peptoids). Non-limiting lengths of peptides suitable for use in the present invention includes peptides of 3 to 5 residues in length, 6 to 10 residues in length (or any integer therebetween), 11 to 20 residues in length (or any integer therebetween), 21 to 75 residues in length (or any integer therebetween), 75 to 100 (or any integer therebetween), or polypeptides of greater than 100 residues in length. Typically, polypeptides useful in this invention can have a maximum length suitable for the intended application. Preferably, the polypeptide is between about 3 and 100 residues in length. Generally, one skilled in art can easily select the maximum length in view of the teachings herein. Further, peptides and polypeptides, as described herein, for example synthetic peptides, may include additional molecules such as labels or other chemical moieties.

[0086] Thus, references to polypeptides or peptides also include derivatives of the amino acid sequences of the invention including one or more non-naturally occurring amino acids. A first polypeptide or peptide is "derived from" a second polypeptide or peptide if it is (i) encoded by a first polynucleotide derived from a second polynucleotide encoding the second polypeptide or peptide, or (ii) displays sequence identity to the second polypeptide or peptide as described herein. Sequence (or percent) identity can be determined as described below. Preferably, derivatives exhibit at least about 50% percent identity, more preferably at least about 80%, and even more preferably between about 85% and 99% (or any value therebetween) to the sequence from which they were derived. Such derivatives can include postexpression modifications of the polypeptide or peptide, for example, glycosylation, acetylation, phosphorylation, and the like.

[0087] The terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule” are used herein to include a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded DNA, as well as triple-, double- and single-stranded RNA. It also includes modifications, such as by methylation and/or by capping, and unmodified forms of the polynucleotide. More particularly, the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule” include poly deoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D- ribose), any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing nonnucleotidic backbones, for example, peptide nucleic acids (PNAs), morpholino nucleic acids, locked nucleic acids (LNAs), glycol nucleic acids (GNAs), threose nucleic acids (TNAs) and hexitol nucleic acids (HNAs). and other synthetic sequencespecific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. There is no intended distinction in length between the terms “polynucleotide,” “oligonucleotide,” “nucleic acid” and “nucleic acid molecule,” and these terms will be used interchangeably. Thus, these terms include, for example, 3'-deoxy-2',5'-DNA, oligodeoxyribonucleotide N3' P5' phosphoramidates, 2'-O-alkyl-substituted RNA, double- and single-stranded DNA, as well as double- and singlestranded RNA, DNA:RNA hybrids, and hybrids between PNAs and DNA or RNA, and also include known types of modifications, for example, labels which are known in the art, methylation, “caps,” substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), with negatively charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), and with positively charged linkages (e.g., aminoalklyphosphoramidates, aminoalkylphosphotriesters), those containing pendant moieties, such as, for example, proteins (including nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms of the polynucleotide or oligonucleotide.

[0088] "Substantially purified" generally refers to isolation of a substance (e.g., compound, drug, nucleic acid, polynucleotide, oligonucleotide, protein, polypeptide, peptide composition) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises 50%, preferably 8O%-85%, more preferably 90-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0089] “Isolated” refers to an entity of interest that is in an environment different from that in which it may naturally occur. “Isolated” is meant to include entities that are within samples that are substantially enriched for the entity of interest and/or in which the entity of interest is partially or substantially purified.

[0090] The term “derived from” is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which can be, for example, by chemical synthesis or recombinant means.

[0091] By ‘ ‘derivative” is intended any suitable modification of the native polypeptide of interest, of a fragment of the native polypeptide, or of their respective analogs, such as glycosylation, phosphorylation, polymer conjugation (such as with polyethylene glycol), or other addition of foreign moieties, as long as the desired biological activity of the native polypeptide is retained. Methods for making polypeptide fragments, analogs, and derivatives are generally available in the art.

[0092] "Homology" refers to the percent identity between two polynucleotide or two polypeptide molecules. Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% 98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence.

[0093] In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353 358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman Advances in Appl. Math. 2:482489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

[0094] Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the Smith Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs are readily available.

[0095] Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single stranded specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra', DNA Cloning, supra', Nucleic Acid Hybridization, supra.

[0096] Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms or host cells of organs, as described further below. The host organism or host cell of an organ expresses the foreign gene to produce the protein under expression conditions.

[0097] The term "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included. The exogenous polynucleotide may be maintained as a nonintegrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome. [0098] A "coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0099] Typical "control elements," include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.

[00100] "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[00101] "Encoded by" refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, more preferably at least 8 to 10 amino acids, and even more preferably at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence. [00102] "Expression cassette" or "expression construct" refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest. An expression cassette generally includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the expression cassette described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include, one or more selectable markers, a signal which allows the plasmid construct to exist as single stranded DNA (e.g., a M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).

[00103] "Purified polynucleotide" refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about at least 90%, of the protein with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well-known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[00104] The term "transfection" is used to refer to the uptake of foreign DNA by a cell. A cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.

[00105] A "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non- viral vectors, particulate carriers, and liposomes). Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[00106] "Gene transfer" or "gene delivery" refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non-viral vectors, adenoviruses, lentiviruses, alphaviruses, pox viruses, and vaccinia viruses.

[00107] A polynucleotide "derived from" a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence. The derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.

[00108] A “CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.

[00109] The term "Cas9" as used herein encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks).

[00110] A Cas9 endonuclease binds to and cleaves DNA at a site comprising a sequence complementary to its bound guide RNA (gRNA). For purposes of Cas9 targeting, a gRNA may comprise a sequence "complementary" to a target sequence (e.g., in an exon or an intron of a gene), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.

[00111] The Cas 9 protein naturally contains DNA endonuclease activity that depends on association of the protein with two naturally occurring or synthetic RNA molecules called crRNA and tracrRNA (also called guide RNAs). In some cases, the two molecules are covalently linked to form a single molecule (also called a single guide RNA (“sgRNA”)). Thus, the Cas9 associates with a DNA-targeting RNA (which term encompasses both the two-molecule guide RNA configuration and the single-molecule guide RNA configuration), which activates the Cas9 or Cas9-like protein and guides the protein to a target nucleic acid sequence. If the Cas9 protein retains its natural enzymatic function, it will cleave target DNA to create a double-strand break, which can lead to genome alteration (i.e., editing: deletion, insertion (when a donor polynucleotide is present), replacement, etc.), thereby altering gene expression.

[00112] The term “CRISPR agent” as used herein encompasses any agent (or nucleic acid encoding such an agent), comprising naturally occurring and/or synthetic sequences, that can be used in a Cas9-based system (e.g., a Cas9 or Cas9-like protein; any component of a DNA-targeting RNA, e.g., a crRNA-like RNA, a tracrRNA-like RNA, a single guide RNA, etc.; a donor polynucleotide; and the like).

[00113] A Cas9 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP_002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP_002344900), Campylobacter coli (WP_060786116); Campylobacter fetus (WP_059434633); Corynebacterium ulcerans (NC_015683, NC_017317);

Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP_033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychrojlexus torquisl (NC_018721); Streptococcus thermophilus (YP_820832), Streptococcus mutans (WP_061046374, WP_024786433); Listeria innocua (NP_472073); Listeria monocytogenes (WP_061665472); Legionella pneumophila (WP_062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP_033919308); Lactobacillus rhamnosus (WP_048482595, WP_032965177); and Neisseria meningitidis (WP_061704949,

YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein, wherein the variant retains biological activity, such as Cas9 site- directed endonuclease activity. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol. 198(5):797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385- 397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091-6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[00114] By "selectively binds" with reference to a guide RNA is meant that the guide RNA binds preferentially to a target sequence of interest or binds with greater affinity to the target sequence than to other genomic sequences. For example, a gRNA will bind to a substantially complementary sequence and not to unrelated sequences. A gRNA that selectively binds to a particular target DNA sequence will selectively direct binding of Cas9 to a substantially complementary sequence at the target site and not to unrelated sequences.

[00115] The term "donor polynucleotide" refers to a polynucleotide that provides a sequence of an intended edit to be integrated into the genome at a target locus by homology directed repair (HDR).

[00116] A "target site" or "target sequence" is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide. The target site may be in an exon or an intron or a specific allele.

[00117] By "homology arm" is meant a portion of a donor polynucleotide that is responsible for targeting the donor polynucleotide to the genomic sequence to be edited in a cell. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence comprising the intended edit to the genomic DNA. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively. The nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR or recombineering at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.

[00118] As used herein, the terms "complementary" or "complementarity" refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine. However, when a uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated. "Complementarity" may exist between two RNA strands, two DNA strands, or between a RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be "complementary" and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are "perfectly complementary" or "100% complementary" if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region. Two or more sequences are considered "perfectly complementary" or "100% complementary" even if either or both polynucleotides contain additional non-complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other. "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art. For purposes of Cas9 targeting, a gRNA may comprise a sequence "complementary" to a target sequence (e.g., in an intron), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.

[00119] A “zinc-finger nuclease” or “ZFN” is an artificial DNA endonuclease generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target desired DNA sequences and this enables zinc-finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on the use of ZFNs, see, for example: Asuri et al., Mol Ther. 2012 February; 20(2):329-38; Bibikova et al. Science. 2003 May 2; 300(5620):764; Wood et al. Science. 2011 Jul. 15; 333(60401:307; Ochiai et al. Genes Cells. 2010 August; 15(8) :875-85 ; Takasu et. al., Insect Biochem Mol Biol. 2010 October; 40(10) :759-65 ; Ekker et al, Zebrafish 2008 Summer; 5(2): 121-3; Young et al, Proc Natl Acad Sci USA. 2011 Apr. 26; 108(17):7052-7; Goldberg et al, Cell. 2010 Mar. 5; 140(5):678-91 ; Geurts et al, Science. 2009 Jul. 24; 325(5939):433; Flisikowska et al, PLoS One. 2011; 6(6):e21045. doi: 10.1371/journal.pone.0021045. Epub 2011 Jun. 13; Hauschild et al, Proc Natl Acad Sci USA. 2011 Jul. 19; 108(29): 12013-7; and Yu et al, Cell Res. 2011 November; 21(11): 1638-40; all of which are herein incorporated by reference for their teachings related to ZFNs. The term “ZFN agent” encompasses a zinc finger nuclease and/or a polynucleotide comprising a nucleotide sequence encoding a zinc finger nuclease.

[00120] A “transcription activator-like effector nuclease” or “TALEN” is an artificial DNA endonuclease generated by fusing a TAL (Transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALENS can be engineered to bind practically any desired DNA sequence and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on the use of TALENs, see, for example: Hockemeyer et al. Nat Biotechnol. 2011 Jul. 7; 29(8):731-4; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Tesson et al. Nat Biotechnol. 2011 Aug. 5; 29(8):695- 6; and Huang et. al., Nat Biotechnol. 2011 Aug. 5; 29(8):699-700; all of which are herein incorporated by reference for their teachings related to TALENs. The term “TALEN agent” encompasses a TALEN and/or a polynucleotide comprising a nucleotide sequence encoding a TALEN.

Perfusion device

[00121] In one aspect, a perfusion device for circulating perfusate to and from a donor organ is provided. Schematics of exemplary perfusion devices are shown in FIGS. 1 A-1C. The perfusion devices comprise a sterile container, designed to hold a donor organ, connected to a perfusion circuit loop. The perfusion devices shown in FIGS. IB and 1C show perfusion circuits with a bypass loop, which integrates a pressure regulation system to accommodate organs that require lower perfusion flow rates while simultaneously satisfying the higher flow rate requirements of an oxygenator.

[00122] In the exemplary device shown in FIG. IB, the perfusion circuit comprises: a sterile container to hold the donor organ; a perfusate reservoir that collects the perfusate flowing out from the donor organ; a venous tube, wherein the venous tube can be connected to a vein of the donor organ and the perfusate reservoir, wherein the venous tube carries the perfusate from the vein to the perfusate reservoir; an oxygenator that oxygenates the perfusate, wherein the oxygenator comprises an inlet for receiving oxygen from an oxygen reservoir (e.g., a tank comprising compressed oxygen or carbogen), an inlet for receiving the perfusate circulating through the perfusion circuit, and an outlet for delivering oxygenated perfusate to the perfusion circuit; a pump that propels the perfusate circulating through the perfusion circuit, wherein the pump is connected to the perfusate reservoir by a first tubing, and wherein the pump is connected to the inlet of the oxygenator by a second tubing; an arterial tube, wherein the arterial tube can be connected to an artery of the donor organ and the outlet of the oxygenator, wherein the arterial tube carries the oxygenated perfusate from the outlet of the oxygenator to the artery; a sterile gravity bag; a first bypass tube wherein the first bypass tube is connected to the sterile gravity bag and the outlet of the oxygenator; and a second bypass tube wherein the second bypass tube is connected to the sterile gravity bag and the perfusate reservoir.

[00123] In the exemplary device shown in FIG. 1C, the perfusion circuit comprises: a sterile container to hold the donor organ; a perfusate reservoir that collects the perfusate flowing out from the donor organ; a venous tube, wherein the venous tube can be connected to a vein of the donor organ and the perfusate reservoir, wherein the venous tube carries the perfusate from the vein to the perfusate reservoir; an oxygenator that oxygenates the perfusate, wherein the oxygenator comprises an inlet for receiving oxygen from an oxygen reservoir (e.g., a tank comprising compressed oxygen or carbogen), an inlet for receiving the perfusate circulating through the perfusion circuit, and an outlet for delivering oxygenated perfusate to the perfusion circuit; a pump that propels the perfusate circulating through the perfusion circuit, wherein the pump is connected to the perfusate reservoir by a first tubing, and wherein the pump is connected to the inlet of the oxygenator by a second tubing; an arterial tube, wherein the arterial tube can be connected to an artery of the donor organ and the outlet of the oxygenator, wherein the arterial tube carries the oxygenated perfusate from the outlet of the oxygenator to the artery; a bypass tube wherein the first bypass tube is connected to the outlet of the oxygenator and the perfusate reservoir; and a pinch valve, wherein the pinch valve is positioned between a first portion of the bypass tube and a second portion of the bypass tube. In certain embodiments, the perfusion device further comprises a connector, wherein the perfusate reservoir is connected to the sterile container by the connector.

[00124] A sample port can be connected to the arterial tube to allow monitoring of parameters of interest. In some embodiments, one or more sensors are connected to the sample port to measure, for example, pressure, temperature, flow-rate, pH, concentrations of oxygen, glucose, and/or lactate, or a biomarker of interest. In certain embodiments, the perfusion device further comprises an air bubble sensor that can detect air bubbles in the perfusate.

[00125] An infusion container can be connected to the perfusion circuit with infusion line tubing to allow an infusion comprising, for example, a therapeutic agent or nutrient to be introduced into the perfusate circulating through the organ, wherein the infusion line tubing carries the infusion from the infusion container to the perfusate in the perfusion circuit. A syringe pump or infusion pump can be used to propel an infusion from the infusion container through the infusion line tubing into the perfusate.

[00126] The perfusion device can be used for perfusion of any type of organ of any size, including, without limitation, a kidney, heart, liver, lung, stomach, small intestine, large intestine, pancreas, or gonad. The organ can be obtained from an individual of any age, such as an adult, child, or infant. In some embodiments, an entire organ may undergo perfusion with the perfusion device. In other embodiments, a portion of an organ may undergo perfusion with the perfusion device. In certain embodiments, the organ is obtained from a live organ donor or an organ donor after circulatory death.

[00127] Ex vivo perfusion can be used, for example, to maintain viability of an organ for transplantation or pre-clinical research, genetically modify an organ, perform gene therapy on an organ, or surgically repair an organ (e.g., with minor defects) prior to transplantation into a recipient. The ability to evaluate an organ and, if necessary, provide a treatment to the organ prior to transplantation improves the likelihood that a transplant will be successful and increases the number of organs available for transplant. In some cases, the ability of ex vivo perfusion to extend the time donor organs are viable allows donor organs to be transported further distances to reach a recipient for a transplant.

[00128] In certain embodiments, the perfusion device is used to maintain an organ ex vivo for extended periods of time, such as, for example, 3 hours to 48 hours or more, 12 hours to 36 hours or more, or 24 hours to 36 hours or more, including any amount of time within these ranges such as 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, or 48 hours or more.

[00129] The container that holds the organ should be capable of providing a humidified, sterile environment, which prevents infection of the organ and allows other components of the perfusion system to be connected to the organ. In some cases, specialized components are included to support a particular type of organ. For a kidney, for example, a ureteral catheter or cannula may be included, which can be connected to a ureter of the kidney. In addition, a urine collection container may be connected to the ureteral catheter or cannula by a urine drainage line tubing to allow collection of any urine produced by the kidney during perfusion. In certain embodiments, the arterial tube is connected to the renal artery and the venous tube is connected to a renal vein of the kidney. For a lung, a ventilator may be included for ventilating the lung. For a heart, temporary pacing wires may be inserted into the ventricular muscle, and a defibrillator may also be included. In certain embodiments, the container further comprises a drainage port, which allows blood or perfusate to drain from the container. The drainage port can be connected to the inlet of the perfusate reservoir by a drainage line tubing to allow blood or perfusate in the container to enter the perfusion circuit. For a description of exemplary containers for holding different types of organs during perfusion, see, e.g., U.S. Patent Application Publication No. 2009/0197241, Organ Transport Systems (2012) Technology (organtransportsystems.com/OurTechnology.html); Bryner et al. (2021) JTCVS Open 8:123-127; Kanani et al. (2023) Cureus 15(2): e34804; Michelotto et al. (2021) Langenbecks Arch. Surg. 406( 1): 39-54; Krezdorn et al. (2017) Innovative Surgical Sciences. 2(4): 171-187; Birnbaum et al. (2004) Eur. J. Cardio-Thoracic Surg. Suppl. 26(l):S82-85; herein incorporated by reference in their entireties). The container may be hung or placed on any suitable support. For example, a container may be placed on a table, shelf, or stand or hung from a pole.

[00130] An exemplary bag for holding an organ is shown in FIGS. 1C, 3 A, 9A, and 9B. The bag is designed to maintain a moist, sterile, temperature-controlled environment for housing the organ. The bag may include a drainage port, which allows the organ to bleed freely and for blood from the organ or perfusate to drain from the bag and be added to the perfusion circuit and recycled. As shown in FIGS. 9A and 9B, the bag contains magnets, which provide gentle compression to minimize edema of the organ. The size of the bag can be adjusted to fit any organ size or configuration. In addition, the bag allows endoscopic manipulation of an organ without removing it from the bag.

[00131] The perfusate may be a whole blood perfusate, a blood cell-based perfusate, or a non-blood cell-based perfusate. A commonly used blood cell-based perfusate comprises leukocyte-depleted, packed red blood cells suspended in Ringer’s lactate solution with mannitol, dexamethasone, heparin, sodium bicarbonate supplemented with insulin, glucose, multivitamins and vasodilators (see, e.g., Fard et al. (2022) Transpl. Int. 35:10236, herein incorporated by reference). Non-blood cell-based perfusates may include human albumin-based STEEN solution, synthetic hemoglobin -based oxygen carriers, naturally derived oxygen-carrying annelid globin molecules or polymers, and acellular perfusates with supraphysiological carbogen mixtures that support oxygenation (see, e.g., Selzner et al. (2016) Liver Transpl. 22(11): 1501- 1508, Aburawi et al. (2019) Am. J. Transplant 19( 10):2814-2824; herein incorporated by reference). The perfusate may contain nutrients and oxygen to help the organ function and maintain organ metabolism under physiological or near physiological conditions of temperature, pressure, and pH during perfusion. The perfusate may also include therapeutic agents to help maintain the organ and provide protection against ischemia, edema, reperfusion injury and other adverse effects of perfusion.

[00132] A heating or cooling element can be used to maintain the container holding the organ, the perfusate reservoir, and/or perfusate in the perfusion circuit at a desired temperature. In some embodiments, the perfusion device comprises: a first heating element or cooling element, wherein the first heating element or cooling element maintains the container holding the organ at a desired temperature; and a second heating element or cooling element, wherein the second heating element or cooling element maintains the perfusate reservoir at a desired temperature. Any suitable heating elements or cooling elements may be used. In some embodiments, a heating element is used to maintain the temperature such as, but not limited to, a water bath, a heater with a heat exchanger, a thermal regulating system, or a warming blanket. In other embodiments, a cooling element is used to maintain the temperature such as, but not limited to, an ice bath or a thermoelectric temperature controller.

[00133] For normothermic or subnormothermic perfusion, a temperature range is used that is close to or the same as the physiological temperature of the organ in vivo. In some embodiments, the temperature ranges from 20 °C to 40 °C, 20 °C to 32 °C, 30 °C to 40 °C, 35.5 °C to 40 °C, or

35°C to 37°C, including any temperature within these ranges such as 20 °C, 20.5 °C, 21 °C,

21.5 24.5 °C, 25 °C, 25.5 °C, 26 °C, 26.5 °C, 27 °C,

27.5 °C, 28 °C, 28.5 °C, 29 °C, 29.5

33.5 °C, 34 °C, 34.5 °C, 35 °C, 35.5 °C, 36 °C, 36.5 °C, 37 °C, 37.5 °C, 38 °C, 38.5 °C, 39 °C,

39.5 °C, or 40 °C. In some embodiments, the temperature is 37 °C.

[00134] For hypothermic perfusion, the temperature ranges from 1 °C to 20 °C. In some embodiments, the temperature ranges from 1 °C to 20 °C, 2 °C to 12 °C, or 4 °C to 10 °C, including any temperature within these ranges such as 1 °C, 2 °C, 3 °C, 4 °C, 4.5 °C, 5.0 °C, 5.5 °C, 6.0 °C,

6.5 °C, 7.0 °C, 7.5 °C, 8.0 °C, 8.5 °C, 9.0 °C, 9.5 °C, 10.0 °C, 10.5 °C, 11.0 °C, 11.5 °C, 12.0 °C,

12.5 °C, 13.0 °C, 13.5 °C, 14.0 °C, 14.5 °C, 15.0 °C, 15.5 °C, 16.0 °C, 16.5 °C, 17.0 °C, 17.5 °C,

18.0 °C, 18.5 °C, 19.0 °C, 19.5 °C, 20.0 °C. In some embodiments, the temperature is 4 °C. In other embodiments, the temperature is 10 °C.

[00135] Sub-normothermic perfusion is performed in a temperature range lower than normal body temperature (about 37 °C) but higher than hypothermic perfusion (typically below 20°C). For sub-normothermic perfusion, the temperature typically ranges from about 20 °C to 34 °C. In some embodiments, the temperature ranges from 20 °C to 34 °C, 25 °C to 32 °C, or 20 °C to 24 °C including any temperature within these ranges such as 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, or 34 °C.

[00136] Any suitable pump may be used to propel perfusate through the perfusion circuit. Exemplary pumps include, without limitation, centrifugal pumps, roller pumps, peristaltic pumps, non-pulsatile gear pumps, and atraumatic blood pumps. In some embodiments, the pump is adjusted to provide the lowest effective flow rate of the perfusate that is sufficient for delivery of oxygen and nutrients to the organ while minimizing damage to the vascular endothelium of the organ. In some embodiments, the pump is adjusted to perform organ perfusion at a pressure in a range of 60 mm Hg to 120 mm Hg, 60 mm Hg to 90 mm Hg, or 60 to 75 mm Hg, or any pressure within these ranges such as 60 mm Hg, 65 mm Hg, 70 mm Hg, 75 mm Hg, 80 mm Hg, 85 mm Hg, 90 mm Hg, 95 mm Hg, 100 mm Hg, 105 mm Hg, 110 mm Hg, 115 mm Hg, or 120 mm Hg.

[00137] An advantage of this perfusion device is that the overall flow of the perfusate through the perfusion circuit can be maintained within a range of 60 mL/min to 1000 mL/min or more while the flow of the perfusate within the organ can be lower, for example, as low as 5 mL/min to protect the vasculature. That is, two different flow rates can used with a higher overall flow rate through the perfusion circuit and a lower flow rate through the organ.

[00138] In some embodiments, the overall flow rate of the perfusate through the perfusion circuit or the organ is maintained in a range from 5 ml/minute to 1000 ml/minute, 60 ml/minute to 350 ml/minute, or 60 ml/minute to 100 ml/minute, or any flow rate within these ranges such as 60 ml/minute, 65 ml/minute, 70 ml/minute, 75 ml/minute, 80 ml/minute, 85 ml/minute, 90 ml/minute, 95 ml/minute, 100 ml/minute, 110 ml/minute, 120 ml/minute, 130 ml/minute, 140 ml/minute, 150 ml/minute, 160 ml/minute, 170 ml/minute, 180 ml/minute, 190 ml/minute, 200 ml/minute, 210 ml/minute, 220 ml/minute, 230 ml/minute, 240 ml/minute, 250 ml/minute, 260 ml/minute, 270 ml/minute, 280 ml/minute, 290 ml/minute, 300 ml/minute, 310 ml/minute, 320 ml/minute, 330 ml/minute, 340 ml/minute, 350 ml/minute, 360 ml/minute, 370 ml/minute, 380 ml/minute, 390 ml/minute, 400 ml/minute, 410 ml/minute, 420 ml/minute, 430 ml/minute, 440 ml/minute, 450 ml/minute, 460 ml/minute, 470 ml/minute, 480 ml/minute, 490 ml/minute, 500 ml/minute, 510 ml/minute, 520 ml/minute, 530 ml/minute, 540 ml/minute, 550 ml/minute, 560 ml/minute, 570 ml/minute, 580 ml/minute, 590 ml/minute, 600 ml/minute, 610 ml/minute, 620 ml/minute, 630 ml/minute, 640 ml/minute, 650 ml/minute, 660 ml/minute, 670 ml/minute, 680 ml/minute, 690 ml/minute, 700 ml/minute, 720 ml/minute, 740 ml/minute, 760 ml/minute, 780 ml/minute, 800 ml/minute, 820 ml/minute, 840 ml/minute, 860 ml/minute, 880 ml/minute, 900 ml/minute, 920 ml/minute, 940 ml/minute, 960 ml/minute, 980 ml/minute, or 1000 ml/minute.

[00139] In certain embodiments, magnetic tubing connectors are used for connecting the tubing of the perfusion circuit. As shown in FIGS. 10A-10B, the magnetic tubing connectors can be used to magnetically join two ends of tubing in the perfusion circuit, a first magnetic tubing connector is attached to a first tubing and a second magnetic tubing connector is attached to a second tubing, wherein the first tubing and the second tubing are connected to each other by magnetically joining the first magnetic tubing connector to the second magnetic tubing connector. In some embodiments, the first magnetic tubing connector and the second magnetic tubing connector, when magnetically joined, are rotated to reduce kinking of the first tubing and the second tubing. In some embodiments, each magnetic tubing connector comprises a rubber gasket, wherein tubing connections made with the plurality of magnetic tubing connector are water-tight up to a pressure of at least 250 millimeter of mercury (mm Hg). The use of magnetic tubing connectors allows rapid reconfiguration of the perfusion circuit.

[00140] An infusion container can be used to add any agent to the perfusate, including nutrients, therapeutic agents, recombinant nucleic acids, gene therapy vectors, gene editing agents, and the like. For example, heparin, prostacycline, glucose, insulin, a bile salt, or an amino acid, or any combination thereof may be added to the perfusate. In some embodiments, therapeutics that would otherwise be toxic in vivo such as gene therapy agents, chemotherapeutic agents, or radiotherapeutic agents can be delivered to the organ ex vivo while the organ undergoes perfusion. Therapeutics may be delivered through the infusion container to the perfusion circuit or administered locally to a site on the organ. After treatment, the organ may be reimplanted in the subject from whom the organ was obtained or transplanted to a different subject who needs the organ.

[00141] In certain embodiments, a recombinant nucleic acid, vector, or gene editing system is added to the perfusate or administered locally to the organ ex vivo while the organ is undergoing perfusion. For example, a recombinant DNA or RNA, encoding a therapeutic protein or RNA, may be added to the perfusate. In some embodiments, the recombinant nucleic acid is contained in a viral vector or plasmid. In some embodiments, a messenger RNA (mRNA) is added to the perfusate, wherein translation of the mRNA in a host cell of the organ results in production of a therapeutic protein. In some embodiments, a gene editing system is used to genetically modify the organ. Exemplary gene editing systems include, without limitation, those comprising a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease, a meganuclease, a zinc-finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN).

[00142] In an exemplary embodiment, ex vivo organ gene therapy and gene editing is performed on a kidney undergoing perfusion. Gene therapy can be used to treat patients with genetic kidney diseases such as polycystic kidney disease, Dent’s disease, cystinuria, or primary hyperoxaluria, or cancer. Gene therapy can also be used to modify donor organs to become more immunologically compatible with the receipient. Delivery of gene therapy could also benefit those with non-genetic causes of chronic kidney disease. In some embodiments, a recombinant nucleic acid, vector, or gene editing system is delivered into the perfusate, which circulates through the perfusion circuit to a nephron, as shown in FIGS. 11A-1 IB. Methods of genetically modifying an organ or performing gene therapy are described in further detail below.

Genetically Modifying the Organ

[00143] The genome of the organ may be genetically modified prior to transplantation. For example, the genome may be modified to delete or inactivate or reduce expression of a disease- associated allele, introduce or insert an engineered wild-type allele, or convert a disease associated allele to a normal wild-type allele. As another example, the genome may be modified to delete or inactivate a immunologically incompatible allele or introduce an allele that promotes immune tolerance. Various gene editing approaches can be used for this purpose, including, without limitation, the use of genome editing systems comprising clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) nucleases, meganucleases, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). See, e.g., CRISPR Gene Editing: Methods and Protocols (edited by Luo, Humana, 2019), Genome Editing and Engineering: From TALENs, ZFNs and CRISPRs to Molecular Surgery (edited by Appasani and Church, Cambridge University Press, 2018); herein incorporated by reference in their entireties. These gene editing techniques involve creating a double-strand break (DSB) in the DNA at a target site of the intended gene edit. In some embodiments, the DSB is repaired by homology-directed repair (HDR) using a donor DNA template that is inserted into the genome at the target locus using homologous recombination to replace a portion of the genomic sequence with a modified sequence.

[00144] In some embodiments, the donor polynucleotide comprises a nucleotide sequence encoding the intended gene edit, which is flanked by a pair of homology arms responsible for targeting the donor polynucleotide to a genomic locus (e.g., intron or exon) where the nucleotide sequence encoding the intended gene edit is integrated into the genome. The donor polynucleotide typically comprises a 5’ homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence encoding the intended gene edit within the donor polynucleotide. The 5’ and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively.

[00145] The homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus. For example, a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence encoding the intended gene edit is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.

[00146] In certain embodiments, the corresponding homologous nucleotide sequences in the genomic target sequence (i.e., the "5’ target sequence" and "3' target sequence") flank a specific site for cleavage and/or a specific site for introducing the nucleotide sequence encoding the intended gene edit. The distance between the specific cleavage site and the homologous nucleotide sequences (e.g., each homology arm) can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher gene targeting rate. In a preferred embodiment, the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.

[00147] A homology arm can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc. In some instances, the 5' and 3’ homology arms are substantially equal in length to one another, e.g. one may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology arm, or only a few nucleotides less than the other homology arm. In other instances, the 5' and 3' homology arms are substantially different in length from one another, e.g., one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.

[00148] An RNA-guided nuclease can be targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by altering its guide RNA sequence. A target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site. For example, the gRNA can be designed with a sequence complementary to a sequence of the genomic target locus to target the nuclease-gRNA complex to a target site.

[00149] In certain embodiments, the RNA-guided nuclease used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease. Any RNA-guided Cas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system type I, type II, or type III Cas nucleases. Examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8al, Cas8a2, Cas8b, Cas8c, Cas9 (Csnl or Csxl2), CaslO, CaslOd, CasF, CasG, CasH, Csyl, Csy2, Csy3, Csel (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, and Cul966, and homologs or modified versions thereof.

[00150] In certain embodiments, a type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to perform genome modification as described herein. The Cas9 need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP_002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP_002344900), Campylobacter coli (WP_060786116); Campylobacter fetus (WP_059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP_033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisl (NC_018721); Streptococcus thermophilus (YP_820832), Streptococcus mutans (WP_061046374, WP_024786433); Listeria innocua (NP_472073); Listeria monocytogenes (WP_061665472); Legionella pneumophila (WP_062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP 032729892, WP_014548420), Enterococcus faecalis (WP_033919308); Lactobacillus rhamnosus (WP_048482595, WP_032965177); and Neisseria meningitidis (WP_061704949, YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol. 198(5):797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42(101:6091-6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[00151] The CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA. The bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzes cleavage to produce a double-stranded break. Targeting of Cas9 typically further relies on the presence of a 5' protospacer-adjacent motif (PAM) in the DNA at or near the gRNA-binding site.

[00152] The genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA, and may further comprise a protospacer adjacent motif (PAM). In certain embodiments, the target site comprises 20-30 base pairs in addition to a 3 base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide. In certain embodiments, the intron sequence of the TCR gene targeted by a gRNA comprises a mutation that creates a PAM within the intron, wherein the PAM promotes binding of the Cas9-gRNA complex to the intron.

[00153] In certain embodiments, the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. The guide RNA may be a single guide RNA comprising crRNA and CracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.

[00154] In another embodiment, the CR1SPR nuclease from Prevotella and Francisella 1 (Cpfl) also referred to as CRISPR associated protein 12a (Casl2a) may be used. Casl2a is another class II CRISPR/Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Casl2a does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Casl2a for targeting than Cas9. Casl2a is capable of cleaving either DNA or RNA. The PAM sites recognized by Casl2a have the sequences 5'-YTN-3' (where "Y" is a pyrimidine and "N" is any nucleobase) or 5’-TTN-3', in contrast to the G-rich PAM site recognized by Cas9. Casl2a cleavage of DNA produces double-stranded breaks with sticky-ends having a 4 or 5 nucleotide overhang. For a discussion of Casl2a, see, e.g., Ledford et al. (2015) Nature. 526 (7571): 17-17 , Zetsche et al. (2015) Cell. 163 (3):759-771, Murovec et al. (2017) Plant Biotechnol. J. 15(8):917-926, Zhang et al. (2017) Front. Plant Sci. 8: 177, Fernandes et al. (2016) Postepy Biochem. 62(3):315-326; herein incorporated by reference.

[00155] C2clis another class II CRISPR/Cas system RNA-guided nuclease that may be used. C2cl, similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites. For a description of C2cl, see, e.g., Shmakov et al. (2015) Mol Cell. 60(3):385-397, Zhang et al. (2017) Front Plant Sci. 8:177; herein incorporated by reference.

[00156] In yet another embodiment, an engineered RNA-guided FokI nuclease may be used. RNA-guided FokI nucleases comprise fusions of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on FokI. For a description of engineered RNA-guided FokI nucleases, see, e.g., Havlicek et al. (2017) Mol. Ther. 25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32(6):569-576; herein incorporated by reference.

[00157] The RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a gRNA, or provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector such as a plasmid or viral vector). Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell, organoid, or organism. For example, a nucleic acid encoding an RNA-guided nuclease can be modified to substitute codons having a higher frequency of usage in a human cell or a non-human mammalian cell, such as a non-human primate cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the gRNA and/or RNA-guided nuclease is introduced into cells, the gRNA and/or RNA-guided nuclease can be transiently, conditionally, or constitutively expressed in the cell. Recombinant nucleic acids encoding the gRNA, RNA-guided nuclease, and/or donor polynucleotide can be introduced into a cell using any suitable transfection technique such as, but not limited to electroporation, nucleofection, or lipofection. Alternatively, a ribonucleoprotein complex of the gRNA and the RNA-guided nuclease may be introduced into a cell by microinjection into the cytoplasm or nucleus.

[00158] In some embodiments, the CRISPR system is introduced into cells with a viral vector that encodes the RNA-guided nuclease and guide RNA (gRNA). Viral delivery of CRISPR components has been demonstrated using lentiviral, retroviral, adenovirus, and adeno-associated virus (AAV) vectors. For a description of methods of introducing a CRISPR system into cells with various viral vectors, see, e.g., Shalem et al. (2014) Science 343:84-87, Williams et al. (2016) Sci Rep. 6:25611, Ran et al. (2015) Nature 520:186-191, Swiech et al. (2015) Nat Biotechnol. 33:102- 106; herein incorporated by reference.

[00159] Alternatively, a gRNA and a messenger RNA encoding the RNA-guided nuclease can be introduced into cells, wherein the RNA-guided nuclease is produced by translation of the mRNA in the cytoplasm. The gRNA and RNA-guided nuclease then form a complex in the cytoplasm and enter the nucleus. RNA transfection of cells can be performed using electroporation, cationic-lipid-mediated transfection, or using liposomes or lipid nanoparticles (LNPs) encapsulating the gRNA and mRNA. See, e.g., Billingsley et al. (2022) Nano Lett 22(l):533-542, Tchou et al. (2017) Cancer Immunol Res. 5(12): 1152- 1161 , Ye et al. (2022) ACS Biomater Sci Eng. 8(2):722-733, Guevara et al. (2020) Front. Chem. 8:589959; herein incorporated by reference.

[00160] Donor polynucleotides and gRNAs are readily synthesized by standard techniques, e.g., solid phase synthesis via phosphoramidite chemistry, as disclosed in U.S. Patent Nos. 4,458,066 and 4,415,732, incorporated herein by reference; Beaucage et al., Tetrahedron (1992) 48:2223-2311; and Applied Biosystems User Bulletin No. 13 (1 April 1987). Other chemical synthesis methods include, for example, the phosphotriester method described by Narang et al., Meth. Enzymol. (1979) 68:90 and the phosphodiester method disclosed by Brown et al., Meth. Enzymol. (1979) 68: 109. In view of the short lengths of gRNAs (typically about 20 nucleotides in length) and donor polynucleotides (typically about 100-150 nucleotides), gRNA-donor polynucleotide cassettes can be produced by standard oligonucleotide synthesis techniques and subsequently ligated into vectors.

[00161] Zinc-finger nucleases (ZFNs) are artificial DNA endonucleases generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. ZFNs can be engineered to target desired DNA sequences, which enables zinc-finger nucleases to cleave unique target sequences. When introduced into a cell, ZFNs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on the use of ZFNs, see, for example: Asuri et al., Mol Ther. 2012 February; 20(2):329-38; Bibikova et al. Science. 2003 May 2; 300(5620):764; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Ochiai et al. Genes Cells. 2010 August; 15(8) :875-85 ; Takasu et. al., Insect Biochem Mol Biol. 2010 October; 40(10) :759-65 ; Ekker et al, Zebrafish 2008 Summer; 5(2): 121-3; Young et al, Proc Natl Acad Sci USA. 2011 Apr. 26; 108(17):7052-7; Goldberg et al, Cell. 2010 Mar. 5; 140(5):678-91; Geurts et al, Science. 2009 Jul. 24; 325(5939):433; Flisikowska et al, PLoS One. 2011; 6(6):e21045. doi: 10.1371/journal.pone.0021045. Epub 2011 Jun. 13; Hauschild et al, Proc Natl Acad Sci USA. 2011 Jul. 19; 108(29): 12013-7; and Yu et al, Cell Res. 2011 November; 21(11): 1638-40; all of which are herein incorporated by reference for their teachings related to ZFNs. The term “ZFN agent” encompasses a zinc finger nuclease and/or a polynucleotide comprising a nucleotide sequence encoding a zinc finger nuclease.

[00162] Transcription activator-like effector nucleases (TALENs) are artificial DNA endonucleases generated by fusing a TAL (Transcription activator-like) effector DNA binding domain to a DNA cleavage domain. TALENS can be quickly engineered to bind practically any desired DNA sequence and when introduced into a cell, TALENs can be used to edit target DNA in the cell (e.g., the cell's genome) by inducing double strand breaks. For more information on the use of TALENs, see, for example: Hockemeyer et al. Nat Biotechnol. 2011 Jul. 7; 29(8):731-4; Wood et al. Science. 2011 Jul. 15; 333(6040):307; Tesson et al. Nat Biotechnol. 2011 Aug. 5; 29(8):695-6; and Huang et. al., Nat Biotechnol. 2011 Aug. 5; 29(8):699-700; all of which are herein incorporated by reference for their teachings related to TALENs. The term “TALEN agent” encompasses a TALEN and/or a polynucleotide comprising a nucleotide sequence encoding a TALEN.

Performing Gene Therapy on the Organ

[00163] In some embodiments, an organ is administered gene therapy prior to reimplantation in a subject from whom the organ was obtained or transplantation to a subject who needs a donor organ. Gene therapy may comprise administering a recombinant nucleic acid such as a DNA or RNA encoding a therapeutic protein or RNA to the organ. Methods of introducing a nucleic acid (e.g., DNA or RNA) such as a recombinant nucleic acid comprising a coding sequence encoding a therapeutic protein or RNA or a recombinant expression vector comprising a coding sequence encoding a therapeutic protein or RNA into a host cell are known in the art, and any convenient method can be used to introduce a nucleic acid (e.g., an expression construct) into a cell of the organ. Suitable methods include e.g., viral infection, transfection, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct microinjection, nanoparticle- mediated nucleic acid delivery, and the like.

[00164] In some embodiments, a nucleic acid encoding a therapeutic protein can be provided as RNA, such as a messenger RNA (mRNA), wherein translation of the mRNA results in production of the therapeutic protein in the organ. The RNA can be provided by direct chemical synthesis or may be transcribed in vitro from a DNA (e.g., encoding the therapeutic protein). Once synthesized, the RNA may be introduced into a cell by any of the well-known techniques for introducing nucleic acids into cells (e.g., microinjection, electroporation, transfection, etc.). Nucleic acids may be provided to the cells using well -developed transfection techniques; see, e.g., Angel and Yanik (2010) PLoS One 5(7): el 1756, and the commercially available TransMessenger® reagents from Qiagen, Stemfect™ RNA Transfection Kit from Stemgent, and TransIT®-mRNA Transfection Kit from Minis Bio LLC. See also Beumer et al. (2008) Proc. Natl. Acad. Sci. USA 105(50): 19821- 19826.

[00165] A vector may be provided directly to a target host cell, for example, by contacting the organ with the vector (e.g., a recombinant expression vector comprising a coding sequence encoding a therapeutic protein or RNA) such that the vector is taken up by the cells. Methods of transfecting cells are well known in the art, and include, without limitation, electroporation, calcium chloride transfection, microinjection, and lipofection. For viral vector delivery, cells of the organ can be contacted with viral particles comprising viral expression vectors.

[00166] Nucleic acids encoding a therapeutic protein or RNA can be inserted into an expression vector to create an expression cassette capable of producing the therapeutic protein or RNA in a suitable host cell of the organ. The ability of constructs to produce the therapeutic protein or RNA can be empirically determined. Expression cassettes typically include control elements operably linked to a coding sequence, which allow for the expression of a gene in vivo in the subject species. Depending on the host/vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector.

[00167] Promoters can be used to drive expression by an RNA polymerase (e.g., pol I, pol II, pol III). Suitable promoters can be derived from viruses (i.e., viral promoters) or an organism, including prokaryotic or eukaryotic organisms. Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); herpes simplex virus (HSV) promoter, cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), Rous sarcoma virus (RSV) promoter, human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497-500 (2002)), enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep. 1; 31(17)), and human Hl promoter (Hl), and the like.

[00168] The promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active/“ON” state) or an inducible promoter (i.e., a promoter whose state, active/“ON” or inactive/“OFF” is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein). In some cases, a promoter is a spatially restricted promoter (e.g., tissue-specific promoter or cell type-specific promoter controlled by a transcriptional control element, enhancer, etc.). In some cases, a promoter is a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process).

[00169] Inducible promoters suitable for use include any inducible promoter described herein or known to one of ordinary skill in the art. Examples of inducible promoters include, without limitation, chemically/biochemically-regulated and physically-regulated promoters such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)- responsive promoters and other tetracycline-responsive promoter systems, which include a tetracycline repressor protein (tetR), a tetracycline operator sequence (tetO) and a tetracycline trans activator fusion protein (tTA)), steroid-regulated promoters (e.g., promoters based on the rat glucocorticoid receptor, human estrogen receptor, moth ecdysone receptors, and promoters from the steroid/retinoid/thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (proteins that bind and sequester metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature/heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

[00170] In some cases, the promoter is a spatially restricted promoter (i.e., cell type-specific promoter, tissue- specific promoter, organ-specific, etc.) such that the promoter is active (i.e., “ON”) in a subset of specific cells. Spatially restricted promoters may be regulated by enhancers, transcriptional control elements, control sequences, etc. Any convenient spatially restricted promoter may be used as long as the promoter is functional in the targeted host cell. In some cases, the promoter is a tissue- specific promoter. In some cases, the promoter is a cell type-specific promoter. In some cases, the transcriptional control element (e.g., the promoter) is functional in a targeted cell type or targeted cell population. For example, in some cases, the transcriptional control element can be functional in a muscle cell (e.g., a cardiac muscle cell (cardiomyocyte), a skeletal muscle cell (skeletal myofiber), or a smooth muscle cell), a neuron, a retinal cell, a T cell, a B cell, a hematopoietic stem cell, a liver cell, a lung cell, or other targeted cell. In some cases, the transcriptional control element is functional in a postmitotic cell or non-dividing cell such as, but not limited to, a neuron, a cardiomyocyte, a skeletal muscle myofiber, a retinal ganglion cell, a cochlear hair cell, an osteocyte, or an adipocyte.

[00171] In some cases, the promoter is a reversible promoter. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from any of a variety of organisms. Modification of reversible promoters derived from a first organism for use in a second (different) organism is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like. A suitable promoter can include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tetracycline-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547; Oligino et al., 1998, Gene Ther., 5:491-496; Wang et al., 1997, Gene Then, 4:432-441; Neering et al., 1996, Blood, 88:1147-55; and Rendahl et al., 1998, Nat. Biotechno]., 16:757-761 ). [00172] For illustration purposes, examples of spatially restricted promoters include, but are not limited to, neuron- specific promoters, cardiomyocyte-specific promoters, skeletal musclespecific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, retinal ganglion cell-specific promoters, adipocyte- specific promoters, etc.

[00173] In some embodiments, the promoter is a neuron-specific promoter. Examples of neuron-specific promoters include, but are not limited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSENO2, X51956; see also, e.g., U.S. Pat. No. 6,649,811, U.S. Pat. No. 5,387,742); an aromatic amino acid decarboxylase (AADC) promoter; a neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1 promoter (see, e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn et al. (2010) Nat. Med. 16:1161); a serotonin receptor promoter (see, e.g., GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Nucl. Acids. Res. 15:2363-2384 (1987) and Neuron 6:583-594 (1991)); a GnRH promoter (see, e.g., Radovick et al., Proc. Natl. Acad. Sci. USA 88:3402-3406 (1991)); an L7 promoter (see, e.g., Oberdick et al., Science 248:223-226 (1990)); a DNMT promoter (see, e.g., Bartge et al., Proc. Natl. Acad. Sci. USA 85:3648-3652 (1988)); an enkephalin promoter (see, e.g., Comb et al., EMBO J. 17:3793-3805 (1988)); a myelin basic protein (MBP) promoter; a CMV enhancer/platelet-derived growth factor-. beta, promoter (see, e.g., Liu et al. (2620) Gene Therapy 11:52-60); a motor neuron-specific gene Hb9 promoter (see, e.g., U.S. Pat. No. 7,632,679; and Lee et al. (2620) Development 131:3295-3306); an alpha subunit of Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250), and a retinal ganglion cell Nefli promoter (see, e.g., Hanlon et al. (2017) Front Neurosci. 11:521). Other suitable promoters include elongation factor (EF) 1 and dopamine transporter (DAT) promoters, and the like.

[00174] In some embodiments, the promoter is a cardiomyocyte-specific promoter. Examples of cardiomyocyte-specific promoters include, but are not limited to, a cardiac musclespecific alpha myosin heavy chain (MHC) gene promoter (see, e.g., Gulick et al. (1991) I. Biol. Chem. 266:9180-9185, Aikawa et al. (2002) J. Biol. Chem. 277(21): 18979-18985). a ventriclespecific cardiac myosin light chain 2 (MLC-2v) promoter (see, e.g., Boecker et al. (2004) Mol. Imaging 3(2):69-75, Griscelli et al. (1997) C R Acad. Sci. Ill 320(2):103-12), a cardiac troponin T (cTNT) promoter (see, e.g., Ai et al. (2018) Cell Physiol. Biochem. 48(5):1894-1900), a troponin 2 (TNNT2) promoter (see, e.g., Fiedorowicz et al. (2020) Sci. Rep.10(1): 1895), an alpha cardiac actin (ACTC) promoter (see, e.g., Fiedorowicz et al., supra), and a cardiac ankyrin repeat protein gene (Carp/Ankrdl) promoter (see, e.g., Briegel et al. (2005) Development 132(14):3305- 16). [00175] In some embodiments, the promoter is a skeletal muscle-specific promoter. Examples of skeletal muscle-specific promoters include, but are not limited to, a skeletal muscle a-actin promoter, creatine kinase promoter, desmin promoter, troponin promoter, myosin light chain promoter, myosin heavy chain promoter, dystrophin promoter, and Pitx3 promoter (see, e.g., (see, e.g., Skopenkova et al. (2021) Acta Naturae 13(1): 47-58, Coulon et al. (2007) J. Biol. Chem. 282(45):33192-33200, Sartorelli et al. (1993) Circ. Res. 72(5):925-931).

[00176] In some embodiments, cell subtype- specific expression of a therapeutic protein or RNA is achieved by using a recombination system, e.g., Cre-Lox recombination, Flp-FRT recombination, etc. Cell type-specific expression of genes using recombination has been described in, e.g., Fenno et al., Nat Methods, 2014 July; 11(7):763; Gompf et al., Front. Behav. Neurosci. 2015 Jul. 2;9: 152, and McCarthy et al. (2012) Skelet. Muscle. 2(1):8; which are herein incorporated by reference.

[00177] Typically, transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon. Preferably, a sequence for optimization of initiation of translation, located 5' to the coding sequence, is also present. Examples of transcription terminator/polyadenylation signals include those derived from SV40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence.

[00178] Enhancer elements may also be used herein to increase expression levels of the mammalian constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al., EMPO J. (1985) 4:761, the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777 and elements derived from human CMV, as described in Boshart et al., Cell (1985) 41:521, such as elements included in the CMV intron A sequence.

[00179] Additionally, 5'- UTR sequences can be placed adjacent to the coding sequence in order to enhance expression of the same. Such sequences may include UTRs comprising an internal ribosome entry site (IRES). Inclusion of an IRES permits the translation of one or more open reading frames from a vector. For example, a therapeutic protein or RNA can be coexpressed from a multicistronic vector including an IRES element. The IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman et al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu et al., Biochem. Biophys. Res. Comm. (1996) 229:295-298; Rees et al., BioTechniques (1996) 20:102-110; Kobayashi et al., BioTechniques (1996) 21:399-402; and Mosser et al., BioTechniques (1997) 22 150-161. A multitude of IRES sequences are known and include sequences derived from a wide variety of viruses, such as from leader sequences of picomaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 63: 1651-1660), the polio leader sequence, the hepatitis A virus leader, the hepatitis C virus IRES, human rhinovirus type 2 IRES (Dobrikova et al., Proc. Natl. Acad. Sci. (2003) 100(25): 15125- 15130), an IRES element from the foot and mouth disease virus (Ramesh et al., Nucl. Acid Res. (1996) 24:2697-2700), a giardiavirus IRES (Garlapati et al., J. Biol. Chem. (2004) 279(51:3389-3397), and the like. A variety of nonviral IRES sequences will also find use herein, including, but not limited to IRES sequences from yeast, as well as the human angiotensin II type 1 receptor IRES (Martin et al., Mol. Cell Endocrinol. (2003) 212:51-61), fibroblast growth factor IRESs (FGF-1 IRES and FGF-2 IRES, Martineau et al. (2004) Mol. Cell. Biol. 24(17):7622-7635), vascular endothelial growth factor IRES (Baranick et al. (2008) Proc. Natl. Acad. Sci. U.S.A. 105(12):4733-4738, Stein et al. (1998) Mol. Cell. Biol. 18(6):3112-3119, Bert et al. (2006) RNA 12(6): 1074- 1083), and insulin-like growth factor 2 IRES (Pedersen et al. (2002) Biochem. J. 363 (Pt l):37-44). These elements are readily commercially available in plasmids sold, e.g., by Clontech (Mountain View, CA), Invivogen (San Diego, CA), Addgene (Cambridge, MA) and GeneCopoeia (Rockville, MD). See also IRESite: The database of experimentally verified IRES structures (iresite.org). An IRES sequence may be included in a vector, for example, to express multiple protein products in combination.

[00180] Alternatively, a polynucleotide encoding a viral T2A peptide can be used to allow production of multiple protein products (e.g., therapeutic proteins) from a single vector. 2A linker peptides are inserted between the coding sequences in the multicistronic construct. The 2A peptide, which is self-cleaving, allows co-expressed proteins from the multicistronic construct to be produced at equimolar levels. 2A peptides from various viruses may be used, including, but not limited to 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus- 1. See, e.g., Kim et al. (2011) PLoS One 6(4):el8556, Trichas et al. (2008) BMC Biol. 6:40, Provost et al. (2007) Genesis 45(10):625-629, Furler et al. (2001) Gene Ther. 8(11): 864-873; herein incorporated by reference in their entireties.

[00181] In certain embodiments, cells containing a construct encoding a therapeutic protein or RNA are identified in vitro or in vivo by including a selection marker expression cassette in the construct. Selection markers confer an identifiable change to the cell permitting positive selection of cells having the construct. For example, fluorescent or bioluminescent markers (e.g., green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein, blue fluorescent protein, mCherry, mOrange, mPlum, Venus, YPet, phycoerythrin, or luciferase), cell surface markers, expression of a reporter gene (e.g., GFP, dsRed, GUS, lacZ, CAT), drug selection markers such as genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin, or histidinol may be used to identify cells. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed. Any selectable marker may be used as long as it is capable of being expressed in the cell to allow identification of cells containing the construct. Further examples of selectable markers are well known to one of skill in the art.

[00182] In certain embodiments, the selection marker expression cassette encodes two or more selection markers. Selection markers may be used in combination, for example, a cell surface marker may be used with a fluorescent marker, or a drug resistance gene may be used with a suicide gene. In certain embodiments, the selection marker expression cassette is multicistronic to allow expression of multiple selection markers in combination. The multicistronic vector may include an IRES or viral 2A peptide to allow expression of more than one selection marker from a single vector.

[00183] In certain embodiments, a suicide marker is included as a negative selection marker to facilitate negative selection of cells. Suicide genes can be used to selectively kill cells by inducing apoptosis or converting a nontoxic drug to a toxic compound in genetically modified cells. Examples include suicide genes encoding thymidine kinases, cytosine deaminases, intracellular antibodies, telomerases, caspases, and DNases. In certain embodiments, a suicide gene is used in combination with one or more other selection markers, such as those described above for use in positive selection of cells. In addition, a suicide gene may be used in cells containing constructs expressing the therapeutic protein or RNA, for example, to improve their safety by allowing their destruction at will. See, e.g., lones et al. (2014) Front. Pharmocol. 5:254, Mitsui et al. (2017) Mol. Ther. Methods Clin. Dev. 5:51-58, Greco et al. (2015) Front. Pharmacol. 6:95; herein incorporated by reference.

[00184] Once complete, the constructs encoding a therapeutic protein or RNA can be administered to an organ using standard gene delivery protocols. Methods for gene delivery are known in the art. See, e.g., U.S. Pat. Nos. 5,399,346, 5,580,859, 5,589,466. Genes can be delivered to an organ ex vivo, which is reimplanted in the subject or a transplant recipient.

[00185] A number of viral based systems have been developed for gene transfer into mammalian cells. Suitable expression vectors include viral expression vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921 , 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:38223828; Mendelson et al., Virol. (1988) 166:154165; and Flotte et al., PNAS (1993) 90:1061310617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:78127816, 1999); a retroviral vector (e.g., a lentivirus, a y-retrovirus such as murine leukemia virus and feline leukemia virus, an avian retrovirus such as spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. See also, e.g., Warnock et al. (2011) Methods Mol. Biol. 737:1-25; Walther et al. (2000) Drugs 60(2):249-271 ; and Lundstrom (2003) Trends Biotechnol. 21(3): 117- 122; herein incorporated by reference).

[00186] For example, retroviruses provide a convenient platform for gene delivery systems.

Selected sequences can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109; and Ferry et al. (2011) Curr Pharm Des. 17(24):2516-2527). Lentiviruses are a class of retroviruses that are particularly useful for delivering polynucleotides to mammalian cells because they are able to infect both dividing and nondividing cells (see e.g., Lois et al (2002) Science 295:868-872; Durand et al. (2011) Viruses 3(2):132-159; herein incorporated by reference).

[00187] Commonly used retroviral vectors are “defective”, i.e., unable to produce viral proteins required for productive infection. Rather, replication of the vector requires growth in a packaging cell line. To generate viral particles comprising nucleic acids of interest, the retroviral nucleic acids comprising the nucleic acid are packaged into viral capsids by a packaging cell line. Different packaging cell lines provide a different envelope protein (ecotropic, amphotropic or xenotropic) to be incorporated into the capsid, this envelope protein determining the specificity of the viral particle for the cells (ecotropic for murine and rat; amphotropic for most mammalian cell types including human, dog and mouse; and xenotropic for most mammalian cell types except murine cells). The appropriate packaging cell line may be used to ensure that the cells are targeted by the packaged viral particles. Methods of introducing subject vector expression vectors into packaging cell lines and of collecting the viral particles that are generated by the packaging lines are well known in the art (see, e.g., Kafri et al. (2004) Methods Mol Biol. 246:367-390, herein incorporated by reference).

[00188] A number of adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Belt et al., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51- 58; Berkner, K. L. BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476). Additionally, various adeno-associated virus (AAV) vector systems have been developed for gene delivery. AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996; Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press); Carter, B. J. Current Opinion in Biotechnology (1992) 3:533-539; Muzyczka, N. Current Topics in Microbiol, and Immunol. (1992) 158:97-129; Kotin, R. M. Human Gene Therapy (1994) 5:793-801; Shelling and Smith, Gene Therapy (1994) 1:165-169; and Zhou et al., J. Exp. Med. (1994) 179: 1867-1875.

[00189] Another vector system useful for delivering nucleic acids encoding a therapeutic protein or RNA is the enterically administered recombinant poxvirus vaccines described by Small, Jr., P. A., et al. (U.S. Pat. No. 5,676,950, issued Oct. 14, 1997, herein incorporated by reference). [00190] Additional viral vectors which will find use for delivering the nucleic acid molecules encoding the therapeutic protein or RNA include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus. By way of example, vaccinia virus recombinants expressing the therapeutic protein or RNA can be constructed as follows. The DNA encoding the particular therapeutic protein or RNA is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the coding sequences of interest into the viral genome.

[00191] Alternatively, avipoxviruses, such as the fowlpox and canarypox viruses, can also be used to deliver the genes. Recombinant avipox viruses, expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species. The use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells. Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with, respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.

[00192] Molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.

[00193] Members of the Alphavirus genus, such as, but not limited to, vectors derived from the Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan Equine Encephalitis virus (VEE), will also find use as viral vectors for delivering the polynucleotides of the present invention. For a description of Sindbis-virus derived vectors useful for the practice of the instant methods, see, Dubensky et al. (1996) J. Virol. 70:508-519; and International Publication Nos. WO 95/07995, WO 96/17072; as well as Dubensky, Jr., T. W., et al., U.S. Pat. No. 5,843,723, issued Dec. 1, 1998, and Dubensky, Jr., T. W., U.S. Patent No. 5,789,245, issued Aug. 4, 1998, both herein incorporated by reference. Particularly preferred are chimeric alphavirus vectors comprised of sequences derived from Sindbis virus and Venezuelan equine encephalitis virus. See, e.g., Perri et al. (2003) J. Virol. 77: 10394-10403 and International Publication Nos. WO 02/099035, WO 02/080982, WO 01/81609, and WO 00/61772; herein incorporated by reference in their entireties. [00194] A vaccinia-based infection/transfection system can be conveniently used to provide for inducible, transient expression of the coding sequences of interest (for example, an expression cassette encoding a therapeutic protein or RNA) in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays exquisite specificity in that it only transcribes templates bearing T7 promoters. Following infection, cells are transfected with the polynucleotide of interest, driven by a T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into protein by the host translational machinery. The method provides for high level, transient, cytoplasmic production oflarge quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

[00195] As an alternative approach to infection with vaccinia or avipox virus recombinants, or to the delivery of genes using other viral vectors, an amplification system can be used that will lead to high level expression following introduction into host cells. Specifically, a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more template. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene. Because some of the T7 RNA polymerase is required to initiate the amplification, T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction. The polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase. For a further discussion of T7 systems and their use for transforming cells, see, e.g., International Publication No. WO 94/26911; Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130; Deng and Wolff, Gene (1994) 143:245-249; Gao et al., Biochem. Biophys. Res. Commun. (1994) 200: 1201- 1206; Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872; Chen et al., Nuc. Acids Res. (1994) 22:2114-2120; and U.S. Pat. No. 5,135,855.

[00196] The synthetic expression cassette of interest can also be delivered without a viral vector. For example, the synthetic expression cassette can be packaged as DNA or RNA in liposomes prior to delivery to the subject or to cells derived therefrom. Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid. The ratio of condensed DNA to lipid preparation can vary but will generally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991) 1097: 1-17; Straubinger et al., in Methods of Enzymology (1983), Vol. 101, pp. 512-527.

[00197] Liposomal preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416); mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081); and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192), in functional form.

[00198] Cationic liposomes are readily available. For example, N[ 1-2,3- dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, N.Y. (See, also, Feigner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416). Other commercially available lipids include (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; PCT Publication No. WO 90/1 1092 for a description of the synthesis of DOTAP (l,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. [00199] Similarly, anionic and neutral liposomes are readily available, such as, from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials. Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

[00200] The liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et al., in Methods of Immunology (1983), Vol. 101, pp- 512-527; Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilson et al., Cell (1979) 17:77); Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys. Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA (1979) 76:3348); Enoch and Strittmatter, Proc. Natl. Acad. Sci. USA (1979) 76:145); Fraley et ak, J. Biol. Chem. (1980) 255:10431; Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145; and Schaefer-Ridder et al., Science (1982) 215:166.

[00201] The DNA and/or peptide(s) can also be delivered in cochleate lipid compositions similar to those described by Papahadjopoulos et al., Biochem. Biophys. Acta (1975) 394:483- 491. See, also, U.S. Pat. Nos. 4,663,161 and 4,871,488.

[00202] The expression cassette of interest may also be encapsulated, adsorbed to, or associated with, particulate carriers. Examples of particulate carriers include those derived from polymethyl methacrylate polymers, as well as microparticles derived from poly(lactides) and poly(lactide-co-glycolides), known as PLG. See, e.g., Jeffery et al., Pharm. Res. (1993) 10:362- 368; McGee J. P., et al., J Microencapsul. 14(2): 197-210, 1997; O'Hagan D. T., et al., Vaccine 11(2): 149-54, 1993.

[00203] Furthermore, other particulate systems and polymers can be used for ex vivo delivery of the nucleic acid of interest. For example, polymers such as polylysine, polyarginine, polyomithine, spermine, spermidine, as well as conjugates of these molecules, are useful for transferring a nucleic acid of interest. Similarly, DEAE dextran-mediated transfection, calcium phosphate precipitation or precipitation using other insoluble inorganic salts, such as strontium phosphate, aluminum silicates including bentonite and kaolin, chromic oxide, magnesium silicate, talc, and the like, will find use with the present methods. See, e.g., Feigner, P. L., Advanced Drug Delivery Reviews (1990) 5:163-187, for a review of delivery systems useful for gene transfer. Peptoids (Zuckerman, R. N., et al., U.S. Pat. No. 5,831,005, issued Nov. 3, 1998, herein incorporated by reference) may also be used for delivery of a construct of the present invention.

[00204] Additionally, biolistic delivery systems employing particulate carriers such as gold and tungsten, are especially useful for delivering synthetic expression cassettes encoding a therapeutic protein or RNA. The particles are coated with the synthetic expression cassette(s) to be delivered and accelerated to high velocity, generally under a reduced atmosphere, using a gun powder discharge from a "gene gun." For a description of such techniques, and apparatuses useful therefore, see, e.g., U.S. Pat. Nos. 4,945,050; 5,036,006; 5,100,792; 5,179,022; 5,371,015; and 5,478,744. Also, needle-less injection systems can be used (Davis, H. L., et al, Vaccine 12:1503- 1509, 1994; Bioject, Inc., Portland, Oreg.).

[00205] Recombinant vectors carrying a synthetic expression cassette encoding a therapeutic protein or RNA are formulated into compositions for delivery to the organ. These compositions may either be prophylactic or therapeutic. The compositions will comprise a "therapeutically effective amount" of the nucleic acid of interest such that an amount of the therapeutic protein or RNA can be produced ex vivo to treat the organ to which it is administered. The exact amount necessary will vary depending on the organ being treated; general condition of the organ to be treated; the severity of the condition being treated; the particular therapeutic protein or RNA produced, among other factors. An appropriate effective amount can be readily determined by one of skill in the art. Thus, a "therapeutically effective amount" will fall in a relatively broad range that can be determined through routine trials.

[00206] The compositions will generally include one or more "pharmaceutically acceptable excipients or vehicles" such as water, saline, glycerol, polyethyleneglycol, hyaluronic acid, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, surfactants and the like, may be present in such vehicles. Certain facilitators of nucleic acid uptake and/or expression can also be included in the compositions or coadministered.

[00207] Methods for the delivery of nucleic cells to cells are known in the art and can include, e.g., dextran- mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, lipof ectamine and LT-1 mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

[00208] Direct delivery of synthetic expression cassette compositions ex vivo may be accomplished with or without viral vectors, as described above, by injection using either a conventional syringe, needless devices such as Bioject™ or a gene gun, such as the Accell™ gene delivery system (PowderMed Ltd, Oxford, England). Alternatively, the compositions may be added to the perfusate for delivery by the perfusion device to the organ through the perfusion circuit. In addition, these compositions may be delivered by pressurizing any hollow spaces within the organ, such as the renal pelvis or collecting system of the kidney.

Examples of Non-Limiting Aspects of the Disclosure

[00209] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-81 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

1. A perfusion device for circulating perfusate to and from a donor organ, wherein the perfusion device comprises a perfusion circuit comprising: a) a sterile container to hold the donor organ; b) a perfusate reservoir that collects the perfusate flowing out from the donor organ; c) a venous tube, wherein the venous tube can be connected to a vein of the donor organ and the perfusate reservoir, wherein the venous tube carries the perfusate from the vein to the perfusate reservoir; d) an oxygenator that oxygenates the perfusate, wherein the oxygenator comprises an inlet for receiving oxygen from an oxygen reservoir, an inlet for receiving the perfusate circulating through the perfusion circuit, and an outlet for delivering oxygenated perfusate to the perfusion circuit; e) a pump that propels the perfusate circulating through the perfusion circuit, wherein the pump is connected to the perfusate reservoir by a first tubing, and wherein the pump is connected to the inlet of the oxygenator by a second tubing; f) an arterial tube, wherein the arterial tube can be connected to an artery of the donor organ and the outlet of the oxygenator, wherein the arterial tube carries the oxygenated perfusate from the outlet of the oxygenator to the artery; g) a bypass tube wherein the first bypass tube is connected to the outlet of the oxygenator and the perfusate reservoir; and h) a pinch valve, wherein the pinch valve is positioned between a first portion of the bypass tube and a second portion of the bypass tube.

2. A perfusion device for circulating perfusate to and from a donor organ, wherein the perfusion device comprises a perfusion circuit comprising: a) a sterile container to hold the donor organ; b) a perfusate reservoir that collects the perfusate flowing out from the donor organ; c) a venous tube, wherein the venous tube can be connected to a vein of the donor organ and the perfusate reservoir, wherein the venous tube carries the perfusate from the vein to the perfusate reservoir; d) an oxygenator that oxygenates the perfusate, wherein the oxygenator comprises an inlet for receiving oxygen from an oxygen reservoir, an inlet for receiving the perfusate circulating through the perfusion circuit, and an outlet for delivering oxygenated perfusate to the perfusion circuit; e) a pump that propels the perfusate circulating through the perfusion circuit, wherein the pump is connected to the perfusate reservoir by a first tubing, and wherein the pump is connected to the inlet of the oxygenator by a second tubing; f) an arterial tube, wherein the arterial tube can be connected to an artery of the donor organ and the outlet of the oxygenator, wherein the arterial tube carries the oxygenated perfusate from the outlet of the oxygenator to the artery; g) a sterile gravity bag; h) a first bypass tube wherein the first bypass tube is connected to the sterile gravity bag and the outlet of the oxygenator; and i) a second bypass tube wherein the second bypass tube is connected to the sterile gravity bag and the perfusate reservoir.

3. The perfusion device of aspect 1 or 2, further comprising one or more heating elements or cooling elements.

4. The perfusion device of aspect 3, wherein the one or more heating elements comprise a water bath, a warming plate, or a combination thereof.

5. The perfusion device of aspect 3, wherein the one or more cooling elements comprise an ice bath, a thermoelectric temperature controller, or a combination thereof. 6. The perfusion device of any one of aspects 3-5, wherein the perfusion device comprises: a first heating element or cooling element, wherein the first heating element or cooling element maintains the sterile container at a desired temperature; and a second heating element or cooling element, wherein the second heating element or cooling element maintains the perfusate reservoir at a desired temperature.

7. The perfusion device of any one of aspects 1-6, further comprising an infusion container, wherein the infusion container is connected to the perfusion circuit by infusion line tubing.

8. The perfusion device of aspect 7, wherein the infusion container is a syringe, wherein the syringe is connected to the infusion line tubing by a luer lock.

9. The perfusion device of aspect 7 or 8, wherein the infusion line tubing is connected to the outlet of the oxygenator and the infusion container.

10. The perfusion device of any one of aspects 7-9, wherein the infusion container contains an infusion comprising a therapeutic agent or nutrient.

11. The perfusion device of aspect 10, wherein the infusion comprises heparin, prostacycline, glucose, insulin, a bile salt, an amino acid, a fatty acid, a steroid, a diuretic, a recombinant nucleic acid, a vector, a gene editing system, or any combination thereof.

12. The perfusion device of any one of aspects 7-11, further comprising an infusion pump or syringe pump to propel an infusion contained in the infusion container through the infusion line tubing into the perfusate.

13. The perfusion device of any one of aspects 1-12, further comprising a sample port, wherein the sample port is connected to the arterial tube.

14. The perfusion device of aspect 13, further comprising a pressure sensor, wherein the pressure sensor is connected to the sample port. 15. The perfusion device of any one of aspects 1-14, wherein the oxygenator is a pediatric oxygenator.

16. The perfusion device of any one of aspects 1-15, further comprising a connector, wherein the perfusate reservoir is connected to the sterile container by the connector.

17. The perfusion device of any one of aspects 1-16, wherein the sterile container further comprises a drainage port.

18. The perfusion device of aspect 17, wherein the drainage port is connected to the perfusate reservoir by a drainage line tubing.

19. The perfusion device of any one of aspects 1-18, wherein the pump is a centrifugal pump or a peristaltic pump.

20. The perfusion device of any one of aspects 1-19, wherein the pump maintains an artery pressure between in a range between 60 mm Hg to 75 mm Hg.

21. The perfusion device of any one of aspects 1-20, wherein the donor organ is a kidney, a heart, a liver, a lung, a stomach, a small intestine, a large intestine, a pancreas, or a gonad, or a portion thereof.

22. The perfusion device of aspect 21 , wherein the donor organ is a kidney or a portion thereof.

23. The perfusion device of aspect 22, wherein the artery is a renal artery and the vein is a renal vein.

24. The perfusion device of aspect 22 or 23, further comprising a ureteral catheter or cannula that can be connected to a ureter of the kidney and a urine collection container that can be connected to the ureteral catheter or cannula by a urine drainage line tubing. 25. The perfusion device of any one of aspects 22-24, wherein flow rate of the perfusate through the renal artery is maintained in a range from 5 ml/minute to 1000 ml/minute.

26. The perfusion device of any one of aspects 1-25, further comprising an air bubble sensor that can detect air bubbles in the perfusate.

27. The perfusion device of any one of aspects 1-26, further comprising a stand, wherein the sterile container is placed on the stand.

28. The perfusion device of any one of aspects 1-27, wherein the sterile container is a bag.

29. The perfusion device of aspect 28, further comprising a pole, wherein the pole is used to hang the bag.

30. The perfusion device of any one of aspects 1-29, wherein the sterile container further comprises a plurality of magnets, wherein the plurality of magnets applies compression to reduce edema of the donor organ.

31. The perfusion device of any one of aspects 1-30, wherein the donor organ is obtained from a live organ donor or an organ donor after circulatory death.

32. The perfusion device of any one of aspects 1-31, wherein a venous cannula is used to connect the venous tube to a vein of the donor organ, and wherein an arterial cannula is used to connect the arterial tube to an artery of the donor organ.

33. The perfusion device of any one of aspects 1-32, further comprising a plurality of magnetic tubing connectors for connecting tubing of the perfusion circuit, wherein a first magnetic tubing connector is attached to a first tubing and a second magnetic tubing connector is attached to a second tubing, wherein the first tubing and the second tubing are connected to each other by magnetically joining the first magnetic tubing connector to the second magnetic tubing connector. 34. The perfusion device of aspect 33, wherein the first magnetic tubing connector and the second magnetic tubing connector, when magnetically joined, can be rotated to reduce kinking of the first tubing and the second tubing.

35. The perfusion device of aspect 33 or 34, wherein each magnetic tubing connector comprises a rubber gasket, wherein tubing connections made with the plurality of magnetic tubing connector are water-tight up to a pressure of at least 250 millimeter of mercury (mm Hg).

36. The perfusion device of any one of aspects 1-35, further comprising a sensor for measuring a level of a biomarker in a sample of the perfusate to determine fitness of the organ for transplant.

37. The perfusion device of any one of aspects 1-36, further comprising a sensor for measuring temperature of the perfusate, flow-rate of the perfusate, pH of the perfusate, concentration of oxygen in the perfusate, concentration of glucose in the perfusate, concentration of hemoglobin in the perfusate, concentration of sodium in the perfusate, concentration of potassium in the perfusate, concentration of calcium in the perfusate, concentration of carbon dioxide in the perfusate, percent saturation of oxygen in the perfusion, or concentration of lactate in the perfusate, or any combination thereof.

38. A method of using the perfusion device of any one of aspects 1-37 for perfusion of an organ, the method comprising: placing the organ in the sterile container; adding perfusate to the perfusate reservoir; connecting the venous tube to a vein of the organ and the perfusate reservoir; connecting the arterial tube to an artery of the organ and the outlet of the oxygenator; connecting the oxygen reservoir to the inlet of the oxygenator; and turning on the pump to circulate the perfusate through the perfusion circuit and to and from the donor organ.

39. The method of aspect 38, further comprising: turning on a first heating element or cooling element, wherein the first heating element or cooling element maintains the sterile container at a first desired temperature; and turning on a second heating element or cooling element, wherein the second heating element or cooling element maintains the perfusate reservoir at a second desired temperature.

40. The method of aspect 39, wherein the first heating element or cooling element and the second heating element or cooling element are adjusted to maintain the temperature of the sterile container and the perfusate reservoir in a range from 4 °C to 40 °C during perfusion.

41. The method of aspect 40, wherein the temperature is maintained in a range from 20 °C to 40 °C, 35 °C to 37 °C, 20 °C to 32 °C, 13 °C to 24 °C, or 4 °C to 10 °C during perfusion.

42. The method of any one of aspects 38-41, wherein the sterile container further comprises a drainage port, wherein the method further comprises connecting the drainage port to the perfusate reservoir with a drainage line tubing.

43. The method of any one of aspects 38-42, further comprising: connecting a sample port to the arterial tube; and connecting a pressure sensor to the sample port.

44. The method of any one of aspects 38-43, wherein the donor organ is a kidney, a heart, a liver, a lung, a stomach, a small intestine, a large intestine, a pancreas, or a gonad, or a portion thereof.

45. The method of aspect 44, wherein the donor organ is a kidney.

46. The method of aspect 45, wherein said connecting the arterial tube comprises connecting the arterial tube to a renal artery, and wherein said connecting the venous tube comprises connecting the venous tube to a renal vein.

47. The method of aspect 45 or 46, further comprising connecting a ureteral catheter or cannula to a ureter of the kidney; and connecting a urine drainage line tubing to the ureteral catheter or cannula and a urine collection container, wherein urine is collected in the urine collection container. 48. The method of any one of aspects 45-47, further comprising adjusting the pinch valve such that flow rate of the perfusate through the renal artery is maintained in a range from 5 ml/minute to 1000 ml/minute.

49. The method of any one of aspects 38-48, further comprising: connecting infusion line tubing to an infusion container and the perfusion circuit; adding an infusion to the infusion container; and using a syringe pump or infusion pump to propel the infusion into the perfusate.

50. The method of aspect 49, wherein the infusion comprises a therapeutic agent or a nutrient.

51. The method of aspect 50, wherein the infusion comprises heparin, prostacycline, glucose, insulin, a bile salt, an amino acid, a fatty acid, a steroid, a diuretic, a recombinant nucleic acid, a vector, a gene editing system, or any combination thereof.

52. The method of aspect 51, wherein the organ is a kidney, wherein the recombinant nucleic acid, vector, or gene editing system is delivered in the perfusate to a nephron.

53. The method of any one of aspects 38-52, further comprising administering a therapeutic agent locally to a site on the organ during ex vivo perfusion of the organ.

54. The method of any one of aspects 50-53, wherein the therapeutic agent is toxic when administered to a subject in vivo.

55. The method of aspect 54, wherein the therapeutic agent is a gene therapy agent, a chemotherapeutic agent, or a radiotherapeutic agent.

56. The method of any one of aspects 38-55, further comprising pressurizing hollow spaces within the organ.

57. The method of aspect 56, wherein the organ is a kidney, and said pressurizing comprises pressurizing a renal pelvis or collecting system of the kidney. 58. The method of any one of aspects 38-57, further comprising placing the sterile container on a stand.

59. The method of any one of aspects 38-58, wherein the sterile container is a bag.

60. The method of aspect 59, further comprising hanging the bag on a pole.

61. The method of any one of aspects 38-60, further comprising adding a plurality of magnets to the sterile container, wherein the plurality of magnets applies compression to reduce edema of the donor organ during the perfusion.

62. The method of any one of aspects 38-61, wherein the donor organ is obtained from a live organ donor or an organ donor after circulatory death.

63. The method of any one of aspects 38-62, wherein a venous cannula is used to connect the venous tube to a vein of the donor organ, and wherein an arterial cannula is used to connect the arterial tube to an artery of the donor organ.

64. The method of any one of aspects 38-63, wherein a plurality of magnetic tubing connectors are used for connecting tubing of the perfusion circuit, wherein a first magnetic tubing connector is attached to a first tubing and a second magnetic tubing connector is attached to a second tubing, wherein the first tubing and the second tubing are connected to each other by magnetically joining the first magnetic tubing connector to the second magnetic tubing connector.

65. The method of aspect 64, rotating the first magnetic tubing connector relative to the second magnetic tubing connector, when the first magnetic tubing connector and the second magnetic tubing connector are magnetically joined to reduce kinking of the first tubing and the second tubing.

66. The method of any one of aspects 38-65, further comprising measuring a level of a biomarker in a sample of the perfusate to determine fitness of the organ for transplant. 67. The method of any one of aspects 38-66, further comprising genetically modifying the organ during perfusion.

68. The method of aspect 67, wherein said genetically modifying the organ comprises converting a disease-associated allele to a wild-type allele or converting an immunologically non-compatible allele to a compatible allele.

69. The method of any one of aspects 38-68, further comprising surgically repairing the organ prior to transplantation into a recipient.

70. The method of any one of aspects 38-69, further comprising measuring temperature of the perfusate, flow-rate of the perfusate, pH of the perfusate, concentration of oxygen in the perfusate, concentration of glucose in the perfusate, concentration of hemoglobin in the perfusate, concentration of sodium in the perfusate, concentration of potassium in the perfusate, concentration of calcium in the perfusate, concentration of carbon dioxide in the perfusate, percent saturation of oxygen in the perfusion, or concentration of lactate in the perfusate, or any combination thereof.

71. The method of any one of aspects 38-70, wherein a higher overall flow rate of the perfusate is maintained through the perfusion circuit and a lower flow rate of the perfusate is maintained through the organ.

72. The method of any one of aspects 38-71, wherein the perfusion of the organ is normothermic organ perfusion, sub-normothermic organ perfusion, or hypothermic organ perfusion.

73. A bag to hold an organ, wherein the bag comprises: a plurality of magnets, wherein the plurality of magnets applies compression to reduce edema of the donor organ during perfusion of the organ; and a drainage port, wherein the drainage port can be connected to a perfusion circuit such that blood from bleeding of the organ or perfusate in the bag flows through the drainage port into the perfusion circuit.

74. The bag of aspect 73, wherein the bag has a size designed to fit the organ. 75. The bag of aspect 74, wherein the organ is from an adult, child, or infant.

76. The bag of any one of aspects 73-75, wherein the organ is a kidney, a heart, a liver, a lung, a stomach, a small intestine, a large intestine, a pancreas, or a gonad, or a portion thereof.

77. The bag of any one of aspects 73-76, wherein the bag is sterilized, humidified, heated to a desired temperature, or any combination thereof.

78. The bag of aspect 77, wherein temperature of the bag is maintained in a range from 4 °C to 40 °C.

79. The bag of aspect 78, wherein temperature of the bag is maintained in a range from 20 °C to 40 °C, 35 °C to 37 °C, 20 °C to 32 °C, 13 °C to 24 °C, or 4 °C to 10 °C.

80. The bag of any one of aspects 73-79, wherein the bag further comprises perfusate.

81. The bag of any one of aspects 73-80, wherein the bag further comprises an organ.

EXAMPLES

[00210] As can be appreciated from the disclosure provided above, the present disclosure has a wide variety of applications. Accordingly, the following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, dimensions, etc.) but some experimental errors and deviations should be accounted for. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results. EXAMPLE 1: A LOW-COST STRATEGY FOR ESTABLISHING A HIGH-PERFORMANCE

NORMOTHERMIC EX VIVO KIDNEY PERFUSION SYSTEM

INTRODUCTION

[00211] Normothermic ex vivo organ perfusion, also known as normothermic machine perfusion, is a burgeoning field of biomedical exploration.^1-5 The ability to sustain solid organs in a physiologic state outside the body using the principles of extracorporeal membranous oxygenation (ECMO) and cardiopulmonary bypass (CPB) promises to unlock numerous possibilities for organ preservation, organ-directed therapy, and disease modeling.^6,7 This technology has already been used clinically in organ transplantation to prolong the viability of hearts, livers, and lungs, helping more donor organs reach suitable recipients.^8-12 Recent studies have further demonstrated the therapeutic potential of this technology as an opportunity to therapeutically modify the biology of an organ.^{13 16}

[00212] Despite the immense enthusiasm around normothermic ex vivo organ perfusion, few laboratories worldwide conduct this research. There are numerous barriers to entry. The cost of normothermic organ perfusion devices, the basic requirement for performing organ perfusion experiments, currently ranges between tens to hundreds of thousands of US dollars per unit.¹⁷ The proprietary nature of these devices also limits their availability for academic research. Acquiring suitable reagents can also be challenging for inexperienced investigators. Not only do organ perfusion experiments often rely on specialized reagents such as University of Wisconsin Solution, HTK Solution, or STEEN Solution, but they also require sourcing blood and organs from large laboratory animals or human donors. The steep technical learning curve also compounds the costs and logistical challenges associated with each experiment. Given these barriers, the activation energy required to enter the field of organ perfusion research and to maintain this line of inquiry can appear to be insurmountable.

[00213] To solve these problems, we developed a practical, low-cost approach to building an organ perfusion system and attaining proficiency in normothermic ex vivo organ perfusion. We focused on kidney perfusion to address the need for a device tailored to perform normothermic ex vivo kidney perfusion (NEVKP).⁷ To achieve this, we engineered an NEVKP circuit that included a simple pressure regulation mechanism and a magnetized containment bag. We utilized inexpensive, recycled, and repurposed materials to reduce costs while overcoming the learning curve. Importantly, we developed a strategy to perfuse nonheparinized porcine organs, which helped to diversify potential sources of porcine blood and organs for perfusion research. Finally, we adapted a porcine renal autotransplantation protocol to test in vivo kidney viability following prolonged NEVKP. Ultimately, our experience led to the development of an NEVKP system capable of sustaining human and porcine kidneys for over 24 hours. Here we provide a blueprint for establishing a low-cost, modifiable organ perfusion research model using off-the-shelf components.

METHODS

Circuit design

[00214] Three circuit design iterations were used in this study (FIG. 1A). Each circuit consisted of a peristaltic roller pump (Maquet or Cole-Parmer), a Capiox FX05 neonatal oxygenator (Terumo), and 0.25-inch Tygon tubing (Masterflex L/S 17) with polycarbonate straight and T-connectors (McMaster-Carr). Microclave needleless valves (ICU Medical Inc) were used for sample ports. Carbogen (95% O2, 5% CO2, Airgas) was delivered to the oxygenator using a rotameter (Snowate LZB-4W(B)). A digital blood pressure analyzer was used to monitor perfusion pressure (Digi-Med)

[00215] Prototype A: This initial circuit was designed with a pump, oxygenator, and kidney in series. A plastic 1 L bottle was used as the reservoir; warming was provided by placing the reservoir in a water bath and placing the kidney container over a hot plate set at 40 °C. The kidney was housed in an open polytetrafluoroethylene (PTFE) container filled with Plasma-Lyte (Baxter). [00216] Prototype B: This subsequent design was like prototype A except for the addition of a bypass segment between the arterial recirculation port on the oxygenator to the reservoir. An empty, open-to-atmosphere 1 L bag was suspended -80-120 cm above the kidney to assist in controlling perfusion pressure.

[00217] Prototype C: In this design, the gravity bag mechanism was replaced with a short segment of silicone Penrose tubing (0.25-inch diameter, Medline) that could be variably occluded to provide pressure control. A custom reservoir was created from a neonatal suction canister that was modified to include a drainage port at the bottom, four ports in the lid (venous, bypass, kidney bag, urine). Warming was accomplished by running warmed water through the oxygenator using either a standard peristaltic pump (Cole-Parmer) or an ECMO warmer (Cincinnati Sub-Zero). The cannulated kidney was housed in a plastic 3 L bag (Baxter) modified to include a drainage port using either the infusion or spike port. The kidney was secured in place within the bag with a series of 9mm x 3mm neodymium disc magnets (MIN CI). A small opening in the side was created to allow the ureteral catheter to exit. After securing the cannulas and ureter within the bag, the apparatus was suspended above the reservoir.

[00218] Of note, for prolonged NEVKP experiments (>12 hours), the peristaltic pump was replaced with a centrifugal pump (Terumo) to reduce hemolysis.¹⁸

Reuse of circuit components

[00219] During the circuit development phase, diposable items (oxygenators, tubing, connectors, reservoirs) were cleaned between experiments and reused to reduce cost. Each component was rinsed liberally with deionized water, 10% bleach, Tergazyme (Alconox), and water again until clear. Prior to storage, oxygenators were dried overnight using compressed air.

Nonheparinized porcine kidneys and autologous porcine blood

[00220] Nonheparinized porcine kidneys and autologous blood were primarily obtained from two sources: during necropsy for other investigators’ porcine experiments within our animal facilities or from a local slaughterhouse. In each case, organs and blood were obtained without charge.

[00221] In the case of necropsy animals, 1 L autologous whole blood was obtained upon exsanguination for euthanasia. CPDA-1 (150 mL per L) and heparin (5000 u per L) were added immediately to prevent clot formation. After cardiac death, kidneys were extracted from bilateral flank incisions and flushed immediately with at least 100 mL cold heparinized saline or Viaspan solution (Belzer) until effluent ran clear.

[00222] In the case of slaughterhouse animals, animals were electrically stunned and then immediately exsanguinated from the jugular vein. Similar to above, CPDA- 1 (150 mL per L) and heparin (5000 u per L) were added immediately to the collected blood to prevent clot formation. Animals were then eviscerated within 10 minutes of exsanguination. The kidneys were identified within the entrails and dissected free. Once dissected, kidneys were flushed immediately with at least 100 mL cold heparinized saline or Viaspan solution (Belzer) until effluent ran clear.

[00223] After flushing, all organs were immersed in cold Viaspan solution for transportation back to the laboratory prior to NEVKP. Blood was subsequently leukoreduced using a Sepacell RS-2000 filter (Fenwal; 500 mL per filter) prior to use.

Human donor kidneys and allogenic blood products

[00224] Human donor kidneys unsuitable for transplantation were obtained via the UCSF Viable Tissue Acquisition Lab (VITAL) Core (IRB 20-31618). Kidneys were maintained on a Lifeport Kidney Transporter (Organ Recovery Systems) with circulating Viaspan solution for up to 36 hours after procurement. Kidneys were delivered to our laboratory in static cold storage. Expired human allogenic packed red blood cells (RBCs) and fresh frozen plasma (FFP) were provided as a gift from the UCSF blood bank.

Kidney preparation and cannulation

[00225] In both porcine and human kidneys, excess perirenal tissue was removed using suture ligatures or Ligasure bipolar cautery (Covidien). The renal artery was identified and cannulated using an appropriately sized Luer lock tubing connector (FIG. 6). The cannula was secured using a single 2-0 silk tie (Ethicon). The arterial cannula was flushed with UW solution to help identify the renal vein. The vein was then similarly cannulated and secured with a silk tie. The artery was flushed a final time to ensure no additional vessels were missed. In the case of multiple arteries or veins, all vessels were cannulated and joined into a single inflow (arterial) and outflow (venous) line using Y connectors. The ureter was cannulated using a 5 Fr ureteral catheter and secured using a silk tie.

Perfusate composition

[00226] An autologous leuko-reduced blood preparation was used for porcine perfusion experiments as described above. For human experiments, packed RBCs were mixed with thawed FFP in a 1 : 1 ratio. All RBCs, FFP, and human donor organ were selected for serocompatibility.

[00227] Both porcine and human blood mixtures were supplemented with a perfusate additive consisting of Dextran 40 (Thermo Fisher), AlbuMAX lipid-rich bovine serum albumin (Gibco), sodium bicarbonate (7.5%), and calcium gluconate (Fresenius Kabi) osmotically balanced with Plasma- Lyte (Baxter) and sterile water. Nutrient supplementation (RPMI 1640 (Gibco) 4- GlutaMAX (Gibco) 4- dextrose (Duravet) 4- HumulinR (Lilly)) was infused via a syringe pump for perfusion experiments exceeding 6 hours duration.

Physiologic monitoring during normothermic perfusion

[00228] Arterial pressure was monitored using a digital blood pressure analyzer (Digi- Med). Temperature was monitored using a digital infrared thermometer (ThermoBio). Arterial flow rate was measured using a clamp-on ultrasonic flow sensor (Sonotec). Blood gases and electrolytes were assessed using an i-STAT 1 blood gas analyzer (Abbott) with CG84- cartridges. Porcine nephrectomy and renal autotransplantation protocol

[00229] This large animal protocol was developed with University of California San Francisco and University of Colorado IACUC approval (AN203191, 01437, respectively). This protocol was adapted from methodology described by Kaths et al.¹⁹ Female juvenile Yorkshire pigs (-40-50 kg) were brought to the facility and allowed to acclimate for at least 72 hours. A unilateral nephrectomy was performed under anesthesia through a midline incision. 5000 u heparin and 10 mg Lasix were administered prior to ligating the renal vessels. Upon removal, the kidney was quickly flushed with cold storage solution through the artery until the venous effluent turned clear.

[00230] In the immediate autotransplantation condition, autotransplantation of the removed kidney was performed without delay. The kidney was prepared for autotransplantation on a back table. The infrarenal aorta and inferior vena cava (IVC) were exposed. Once the vessels were adequately mobilized and a suitable location for vascular anastomosis was identified, 5000 u heparin was given, and the IVC was occluded with a Satinsky vascular clamp. The IVC was incised and flushed with heparinized saline. The venous anastomosis was performed in a running fashion with 6-0 prolene suture. The aorta was then clamped and an aortotomy was made using a 4.0mm aortic punch (Medtronic). The arterial anastomosis was then performed also with 6-0 prolene suture. Upon completion, a second dose of 5000 u heparin was given and the vascular clamps were released. The ureter was then spatulated and anastomosed to the bladder with a running 5-0 polydioxanone (PDS) suture. Contralateral nephrectomy was then performed, and the bladder was emptied with a syringe. Abdominal closure was performed in three layers.

[00231] For autotransplantation experiments involving NEVKP, the animal was closed and allowed to recover for two days prior to autotransplantation. The removed kidney was perfused on our NEVKP device with leukoreduced autologous blood (in these cases, 500 mL autologous blood was collected during the nephrectomy) for 12 or 24 hours. Upon conclusion of NEVKP, the kidney was flushed with cold storage solution and placed in cold storage until autotransplantation (procedure as described above) on Day 0.

[00232] Following autotransplantation, the animal was recovered for two days. During this period, urine production was qualitatively assessed by placing absorbent pads beneath the cage away from the animal’s water source; these pads were checked twice daily for visible urine spots. Ultrasonic assessment of the autograft was performed on Day 2 prior to euthanasia. Bladder urine and the autotransplanted kidney were harvested during necropsy and preserved for downstream analysis Serum and urine chemistries

[00233] Serum and perfusate chemistries were performed on an iStat device (Abbott Medical) with GC8 and Chem8 cartridges. Urinalysis was performed on a urine analyzer (McKesson).

Histology

[00234] Tissue samples were fixed in 10% formalin for 24-48 hours, stored in 70% ethanol, and then embedded in paraffin. Tissue sections sliced to 4 pm and mounted on positively charged Superfrost microscope slides (Fisher Scientific). Hematoxylin and eosin (H&E) staining was performed using a standard method.

Statistical analysis

[00235] All quantitative data were analyzed using R Studio. Data are shown as mean ± standard deviation (if normally distributed) or mean and interquartile range (IQR) (25* - 75^th percentile; if not normally distributed). Group comparisons were performed using Student’s t-test or Wilcoxon rank-sum. Statistical significance was set at p < 0.05.

RESULTS

A normothermic ex vivo kidney perfusion circuit can be constructed using commercially available components

[00236] We first constructed a simple organ perfusion circuit (Prototype A) using a peristaltic pump, a neonatal hollow fiber oxygenator, and Tygon tubing (FIG. 1A). All circuit components were sourced from commercial vendors for a total cost of less than 1500 US dollars. A pressure sensor was connected to the arterial tubing to monitor the inflow pressure (Prototype A, segment I), and an ultrasonic flow sensor was attached to either the arterial (Prototype A, segment I) or venous tubing (Prototype A, segment B).

[00237] The three prototypes shown in FIGS. 1A-1C represent the chronological evolution of our circuit design. In Prototype A, the circuit was designed according to previously described normothermic organ perfusion devices with the organ and oxygenator in series.²⁰ A perfusate reservoir was fashioned from a clean plastic 1 L bottle, and an open plastic container was used to house the kidney. Normothermic (37 °C) warming was provided by a hot plate beneath the kidney and a water bath around the reservoir. This prototype allowed us to perform initial short-term NEVKP experiments. [00238] In Prototype B, a bypass segment was added to help regulate perfusion pressure to accommodate kidneys that could not tolerate the minimum 100 mL/min of flow required by the oxygenator. The bypass segment thus served as an adjustable pressure release valve and the parallel design of the circuit allowed the overall flow through the oxygenator to be maintained. In Prototype B, pressure regulation via the bypass segment was accomplished by gravity. Flow was directed to a height of -100 cm above the kidney to achieve a pressure limit of -74 mmHg. Importantly, the fluid bag at the top of the bypass segment was left open to atmosphere to create this pressure differential.

[00239] Finally, Prototype C was designed to facilitate longer periods of perfusion (-24 hrs). An infusion drip was added to the system to replenish nutrients and heparin. To replace the open-to-air mechanism used in Prototype B, we created a closed bypass mechanism using a segment of silicone tubing that could be compressed to varying degrees, essentially serving as an adjustable flow resistor. Perfusion pressures increased as the angle tightened (FIG. 1C, Prototype C, inset).

[00240] A sterile containment bag for the kidney was also added. This was fashioned from a 3 L fluid bag (Baxter), utilizing the drainage port at the bottom to allow for collection and recycling of venous hemorrhage. The kidney was stabilized within the bag using a series of neodymium magnets. Additional detail on bag design is provided below.

[00241] A new reservoir was customized from a 300 mL neonatal suction canister. This was favored over the prefabricated Capiox FX05 reservoir due to simplicity of design and ease of cleaning for reuse. Multiple openings were created in the lid to allow all fluids to drain into the reservoir, including a port for urine recycling. Rather than warm the reservoir directly, perfusate warming was achieved by cycling -37 °C water through the warming ports of the oxygenator.

Cost reduction during circuit development and protocol optimization can be achieved by practicing with non-heparinized porcine kidneys and reusing circuit components.

[00242] To mitigate the cost associated with circuit construction during the early phases of the learning curve, disposable circuit components were cleaned and reused multiple times. After each experiment, all components were air dried after being flushed with Tergazyme, 10% bleach, and deionized water. The ability to reuse oxygenators, the costliest disposable component, was an impactful cost-saving measure. We found that drying out the oxygenator chamber between uses was the key to preserving oxygenator efficiency (FIG. 6). This was accomplished by blowing dry air through both the gas and perfusate channels overnight. Using this approach, oxygenators could be used 3-5 times before they showed discoloration or wear. [00243] To reduce the cost of organs and blood, fresh porcine kidneys and autologous blood were obtained as unutilized byproducts from a local slaughterhouse or during necropsy of other porcine experiments in our animal facility. In both cases, kidneys and blood were obtained free of charge. Autologous blood was heparinized immediately and stored in CPDA (citrate-phosphate- dextrose-adenine) buffer. To optimize organ viability, cold storage of kidneys was typically achieved within 20 minutes of circulatory arrest. These materials were then transported to our laboratory and prepared for NEVKP (FIG. ID, see methods).

Non-heparinized kidneys can be perfused ex vivo by maintaining a constant perfusion pressure.

[00244] High intrarenal resistance was a frequently encountered challenge when attempting to perfuse porcine kidneys that were harvested without systemic heparinization prior to cardiac death. Despite liberally flushing each kidney with heparinized solution upon harvest, diffuse mottling of the renal parenchyma was observed, consistent the presence of microvascular thrombi (FIG. 2A). When perfusing these kidneys at the oxygenator’s minimum flow rate (100 mL/min), the arterial pressure would rise above 300 mmHg and cause hemorrhage around the hilum and into the collecting system (FIG. 2B). Histologic examination of these tissues after 1 hour of NEVKP under high pressures revealed distorted glomerular and tubular architecture and diffuse extravasation of RBCs (FIG. 2D).

[00245] To overcome this problem, we incorporated the pressure-regulating bypass mechanisms described above (Prototype B and C) to permit lower flow rates to the kidney while delivering a higher flow rate through the oxygenator. After an initial period of lower flow rates, we observed a gradual increase in flow to the kidney (FIG. 4C) and a resolution in the mottled appearance of the kidney, likely consistent with the dissolution of microvascular clots. After 60 minutes of perfusion, these kidneys were histologically indistinguishable from heparinized kidneys (FIG. 2D). From a functional perspective, primary consequence of using non-heparinized kidneys was a delayed onset of urine production (non-heparinized 22.9 (10-30) mins, heparinized 7.4 (3- 11) mins) (FIG. 2E).

[00246] The ability to vary the flow rate to the kidney while holding pressure and flow rate to the oxygenator constant was a key innovation that accelerated our learning and mitigated damage to the organs (FIG. 7).

A containment bag with adjustable magnets provides stability and perfusate recycling during prolonged NEVKP. [00247] Most normothermic ex vivo organ perfusion systems house the organ in a rigid container. Using this approach in prototypes A and B, we found that frequent adjustment of the vascular cannulas was required to maintain patency. The venous tubing was particularly prone to kinking and twisting due to the flimsy nature of the porcine renal vein. The need for constant manipulation was labor-intensive and raised concerns for maintaining sterility during prolonged NEVKP experiments. Separately, we also recognized the need to recycle persistent leakage of perfusate. Venous hemorrhage around the hilum was unavoidable despite our best efforts to ligate small vessels using suture ligatures or bipolar cautery,

[00248] To address these needs, we housed the kidney in an empty 3L saline bag (FIG. 3A). Within the bag, the kidney and vascular cannulas were fixed in space by a series of neodymium magnets. The vascular tubing exited at the top and was further secured using stand clamps. This configuration minimized the frequency of kinking or twisting at the hilum, and any further adjustment could be performed without entering the bag.

[00249] To enable perfusate recycling, the magnets were spaced 1-2 cm apart to allow effluent to drain around the kidney without pooling. The drainage port at the bottom was connected to the reservoir, allowing the fluid to be promptly recycled. We found that the native drainage port could handle a maximum flow rate of 600.5 + 42 mL/min, which was useful in cases where the renal vein was not cannulated and instead allowed to bleed freely into the bag. As the bag was not directly warmed by a heat source, we assessed whether normothermia could be maintained by warming the perfusate alone. We observed that the temperature of kidneys reached 37 °C within 20 minutes of perfusion initiation and could be maintained throughout the duration of NEVKP (FIG. 3B).

[00250] Finally, we noted that having an airtight contact between the bag and the kidney provided the additional benefit of facilitating ultrasonography during NEVKP without impacting sterility (FIG. 3C). Perfusion of different regions could be assessed in real-time using color Doppler (FIG. 3D).

NEVKP of porcine and human kidneys can be achieved for up to 24 hours using prototype C.

[00251] Having overcome many of the initial challenges associated with NEVKP, we next sought to explore whether prolonged NEVKP (> 24 hours) could be achieved using prototype C. For these experiments, the peristaltic pump was replaced with a centrifugal pump (Terumo-Sarns Delphin IT) to reduce hemolysis within the perfusate.¹⁸ [00252] Five porcine kidneys were perfused: three for f2 hours (P1-P3), one for 24 hours (P4), and one for 36 hours (P5). In each experiment there was a pattern of initial alkalosis peaking at pH 7.75 between hour 4-5 of NEVKP; this was typically resolved by reducing the flow rate of carbogen to the oxygenator (FIG. 4A). Throughout all experiments, pO2 remained above 400 mmHg, indicating adequate oxygenation, while pCO2 gradually increased over time. Urine production varied widely within and across experiments (FIG. 4A). To explore renal potassium handling during NEVKP, urine recycling was not performed for P5. In this experiment, the concentration of potassium was initially high (likely due to hemolysis during blood collection and processing) but reached homeostasis between 3.2 and 4.7 mmol/L at hour 5 of NEVKP through the remainder of NEVKP (FIG. 4B). This trend corresponded to a peak in urinary potassium concentration at hour 5 followed by a gradual reduction over time (FIG. 4B). Histologic examination of P5 at hour 36 of NEVKP revealed evidence of congestion and hyaline deposits in the glomerulus along with evidence of osmotic tubular changes when compared with the 4-hour mark.

[00253] Prolonged NEVKP was also attempted in four untransplantable human donor kidneys from three donors. Donor demographics and clinical profiles are shown in supplementary table 1. These kidneys were perfused for 18 hrs (Hl), 24 hrs (H2), and 16 hrs (H3-4). Hl was terminated early for inadequate oxygenation due excess condensation in the oxygenator; pO2 and pCO2 were otherwise maintained throughout perfusion for the others (FIG. 6D). Physiologically, the pH stabilized around 7.3-7.4 at around the five-hour mark in each experiment (FIG. 6D). Urine output for Hl and H2 ranged between 5-20 mL/min throughout NEVKP and increased with administration of furosemide. H3 and H4 exhibited brisk gross hematuria from trauma within the collecting system; as a result, urine output could not be accurately measured. Although urine was not recycled in these experiments, potassium homeostasis in the perfusate was not observed, likely due to poor RBC quality from prolonged storage, ongoing hemolytic release of potassium, and poor inherent kidney quality as untransplantable organs.

[00254] Hl had an unperfused segment due to an accessory artery that we ligated prior to NEVKP. The histologic architecture of the perfused segment was preserved compared to the unperfused segment, which exhibited signs of necrosis (FIG. 4F). Similar to porcine kidneys, the perfused portion of the human kidney exhibited histologic evidence of hyaline deposits, glomerular congestion, and osmotic tubular changes after prolonged NEVKP (FIG. 4F). Weight gain during NEVKP in the human kidney cohort was not statistically different from that observed in the porcine kidney cohort (FIG. 4G). [00255] Taken together, these data indicate that while further protocol optimization is needed, human and porcine kidneys exhibit evidence of viability and function during prolonged NEVKP with prototype C.

Supplementary Table 1. Donor profile for untransplanted human kidneys. Cr = Creatinine. KDPI

= Kidney Donor Profile Index. PMH = Past medical history.

Porcine kidneys demonstrate viability and urine production in vivo after prolonged NEVKP.

[00256] To test the in vivo performance of kidneys after NEVKP, we adapted a porcine renal autotransplantation protocol using juvenile Yorkshire pigs (40-50 kg) based on previously described methodology.²¹ We compared autotransplantation outcomes among three NEVKP conditions: no NEVKP (i.e. nephrectomy with immediate autotransplantation), after 12 hours of NEVKP, and after 24 hours of NEVKP. In each condition, the contralateral kidney was removed concurrently to render the animal dependent on the autograft for urine production (FIG. 5A).

[00257] In vivo urine production was observed in each case. Urine production was noted upon completion of the vascular anastomosis in the case of the immediately autotransplanted kidney and the 12-hour NEVKP kidney. These animals urinated overnight between Day 0 and Day 1. Although immediate urine production was not observed for the 24-hour NEVKP kidney, the animal began to urinate between Days 1 and 2 (FIG. 5B). Doppler assessment of the autograft demonstrated adequate flow with a resistive index of -0.60 (FIG. 5C). All animals were noted to have at least 100 mL of urine in the bladder upon necropsy. In each case, the bladder urine contained elevated potassium and creatinine concentrations and had a specific gravity consistent with ongoing electrolyte clearance and urine concentration (FIG. 5A). Histologic examination of the kidney perfused for 24 hours demonstrated preserved architecture in both cortex and medulla and no evidence of osmotic tubular changes (FIG. 5D). While more studies are needed to optimize the results, these experiments confirm that kidney viability and urine production are maintained during prolonged NEVKP.

Discussion

[00258] Here we describe creation of an open-source NEVKP platform for the prolonged perfusion of porcine and human kidneys. We employed a low-cost engineering approach and achieved proficiency in NEVKP using nonstandard sources of porcine kidneys and blood to overcome the learning curve. We then adapted a porcine renal autotransplantation model to confirm in vivo that kidney viability and function can be maintained on our platform for up to 24 hours. Through this experience, we introduced several innovations to facilitate the technical aspects of NEVKP, and we outlined a reproducible strategy to perform NEVKP experiments sustainably, humanely, and at scale.

[00259] Our approach makes NEVKP more accessible to the broader scientific community. Many published NEVKP studies rely on commercial organ perfusion systems, often facilitated by preexisting industry partnerships, or on cardiopulmonary bypass systems, options that are not readily accessible. The cost of acquiring or leasing these systems without institutional or corporate affiliations can be prohibitive. Our perfusion circuit represents an economically favorable alternative as it can be assembled for approximately $1,000-$ 1,500 using widely available materials. The modular design also allows for customization and iterative improvements based on specific research needs, an advantage not easily achieved with proprietary systems. By lowering the financial and technical barriers to NEVKP, this approach has the potential to expand access to organ perfusion research and accelerate innovation in the field.

[00260] Importantly, our approach also lowers the technical barrier to performing NEVKP. Our circuit is simple to construct, and its essential components do not require an extensive technological background to operate. The system also makes it more feasible to perform NEVKP without formal surgical training. The design of the bag to stabilize the vascular cannulas and recycle venous hemorrhage obviates the need for advanced suturing skills or painstaking attention to hemostasis during kidney preparation. The only surgical skills necessary to prepare a kidney for NEVKP are basic tissue dissection to harvest the kidney and simple knot-tying to secure the cannulas. These skills can be acquired easily with practice.

[00261] A key innovation of our system is the integration of a pressure regulation system to accommodate kidneys that require lower perfusion flow rates while simultaneously satisfying the higher flow requirements of the oxygenator. By facilitating the perfusion of nonheparinized kidneys for NEVKP studies, organs can be obtained from a variety of sources to lower the cost. In our case, we obtained kidneys and blood via necropsy within our animal facility and from a local slaughterhouse to overcome the early learning curve. We anticipate that our circuit design could also potentially facilitate the perfusion of other small and delicate organs such as pancreas, spleen, small intestine, or gonads.

[00262] Although we have demonstrated that our system can achieve prolonged NEVKP in both porcine and human kidneys, we must highlight several important limitations. The materials and components used in our circuit may not be optimized for hemo- or biocompatibility. Additionally, despite meticulous attention to maintaining a sterile, closed environment during prolonged perfusion experiments, contamination could pose a challenge for longer periods of NEVKP. These issues will be addressed in future evolutions of the model, and hopefully the organ perfusion community will expand, leading to further refinement of these systems. There were also a number of experimental variables that were difficult to control for such as blood product storage duration, warm ischemia time, and pre-existing kidney injury — these factors almost certainly influenced NEVKP performance and contributed to the variability in results. Finally, we acknowledge that there remain numerous gaps in understanding for how to optimally perform NEVKP. For example, there are currently no evidence-based strategies to manage nutrition, osmotic balance, blood product exchange timing, and waste clearance ex vivo. The reproducibility and sustainability of our NEVKP system should enable rigorous large-scale experiments to investigate these important questions.

[00263] Ultimately, organ perfusion is an emerging technology that promises to unlock new avenues of discovery. While its most salient application lies in organ preservation for transplantation, additional uses include the development of therapeutics using ex vivo models and the ex vivo administration of organ-targeted therapies. The ability to perform ex vivo organ perfusion sustainably and at scale represents powerful new research paradigm and should be made more accessible to the scientific community. As we continue to advance the organ perfusion research programs at our respective institutions, our experience can be used as a blueprint to attract expand the scope of this work and its accessibility for the broader research community.

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Extended Criteria Donor Hearts for Transplantation: The OCS Heart EXPAND Trial. JACC Heart Fail. Mar 2024; 12(3):438-447. doi:10.1016/j.jchf.2023.11.015

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Normothermic Machine Perfusion on Complications and Costs: A Multicenter, Real-World Risk- Matched Analysis. Ann Surg. Aug 1 2024;280(2):300-310. doi: 10.1097/SLA.0000000000006291 [00274] 11. Isath A, Ohira S, Levine A, et al. Ex Vivo Heart Perfusion for Cardiac

Transplantation Allowing for Prolonged Perfusion Time and Extension of Distance Traveled for Procurement of Donor Hearts: An Initial Experience in the United States. Transplant Direct. Mar 2023;9(3):el 455. doi: 10.1097/TXD.0000000000001455 [00275] 12. Hosgood SA, Callaghan CJ, Wilson CH, et al. Normothermic machine perfusion versus static cold storage in donation after circulatory death kidney transplantation: a randomized controlled trial. Nat Med. Jun 2023 ;29(6): 1511- 1519. doi:10.1038/s41591-023- 02376-7

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Autotransplantation in a Porcine Model: A Step-by-Step Protocol. J Vis Exp. Feb 21 2016;(108):53765. doi:10.3791/53765

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Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation. J Vis Exp. Jul 15 2015;(101):e52909. doi:10.3791/52909

EXAMPLE 2: NORMOTHERMIC EX VIVO KIDNEY PERFUSION SYSTEM WITH BYPASS LOOP

[00285] We assembled a normothermic ex vivo kidney perfusion (NEVKP) circuit using recycled, repurposed, and low-cost components. Our goal was to sustain a viable kidney for over 24 hours and allow for the delivery of therapeutics that would otherwise be toxic in vivo (e.g., gene therapy). Extracorporeal membrane oxygenation (ECMO) is used to provide oxygenation and perfusion to the organ. Pressure and flow to and from the organ is carefully monitored and controlled. The organ itself is contained in a warm, sterile environment while allowing easy access to the intrarenal collecting system. Therapeutics can be delivered via the collecting system or via the circulation.

[00286] We applied a phased strategy for the learning curve, focusing first on kidney handling and circuit initiation before progressing to longer durations of NEVKP with physiologic adjustments. Over a period of six months, we obtained 20 porcine kidneys from a local abattoir to optimize our NEVKP system and technique, ultimately achieving a maximum of 36 hours of ex vivo perfusion with constant urine production (FIG. 13B). We then perfused three discarded human donor kidneys for 12-24 hours after cold storage, also with constant urine production. Functional assessment of porcine and human kidneys demonstrated electrolyte homeostasis (FIG. 13A) and responsiveness to furosemide, and histologic assessment demonstrated preserved tubular and glomerular architecture with late changes related to osmotic tubular injury (FIG. 15). These results are on par with previous NEVKP studies and indicate that proficiency in NEVKP can be achieved quickly and at low cost. While much remains to be understood and improved in NEVKP, our experience provides a road map for investigators seeking entry into this field.

[00287] An exemplary NEVKP device with a bypass loop is shown in FIG. 8. A key feature of the NEVKP device is that it maintains constant arterial pressure and oxygenator flow via a bypass loop with an adjustable flow valve. The flow rate to the kidney is variable to allow kidney vasculature to adjust to ex vivo physiology. Urine recycling is not necessary with our system compared to others, but urine can be recycled into the perfusate to help maintain solute or oxygen carrier levels in certain contexts. The bag configuration with a drainage port permits a small amount of expected bleeding from the kidney. The entire system fits on bench top. As a manufactured device, this can be even more compact and ergonomic for investigation and therapeutic applications.

EXAMPLE 3: EX VIVO BIOBAG FOR NORMOTHERMIC EX VIVO KIDNEY PERFUSION SYSTEM

[00288] An exemplary ex vivo biobag is shown in FIGS. 9A-9B. Key features of the ex vivo biobag include that the biobag maintains a moist, sterile, temperature-controlled environment, allows the kidney capsule to bleed freely and for blood/perfusate to be recycled (this fluid flows in between the magnet contact points), provides gentle compression to minimize edema, can be adjustable to fit any organ size or configuration, and allows for endoscopic manipulation of the kidney without removing it from the sterile environment.

EXAMPLE 4: MAGNETIC TUBING CONNECTOR FOR NORMOTHERMIC EX VIVO KIDNEY

PERFUSION SYSTEM

[00289] An exemplary magnetic tubing connector is shown in FIGS. 10A-10B. Key features of the magnetic tubing connectors include that they can be magnetically joined and once joined, the two ends can freely rotate, which reduces the kinking of the tubing; the magnetic tubing connectors are water tight up to 250 mm Hg, hemocompatible, and allow for rapid reconfiguration of the perfusion circuit.

EXAMPLE 5; ENDOSCOPIC METHOD TO DELIVER GENE THERAPY VECTOR (e.g., LIPID NANOPARTICLE) INTO THE NEPHRON

[00290] As shown in FIGS. 11 A- 1 IB, water-tight cannulation of the ureter/renal pelvis allows pressurization of collecting and controlled pressurized delivery of LNP or other gene therapy vector into nephron. Delivery can be targeted using direct visualization and is compatible with lipid nanoparticles and adeno-associated viruses.

[00291] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[00292] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

CLAIMS WHAT IS CLAIMED IS:

3. The perfusion device of claim 1 or 2, further comprising one or more heating elements or cooling elements.

4. The perfusion device of claim 3, wherein the one or more heating elements comprise a water bath, a warming plate, or a combination thereof.

5. The perfusion device of claim 3, wherein the one or more cooling elements comprise an ice bath, a thermoelectric temperature controller, or a combination thereof.

6. The perfusion device of any one of claims 3-5, wherein the perfusion device comprises: a first heating element or cooling element, wherein the first heating element or cooling element maintains the sterile container at a desired temperature; and a second heating element or cooling element, wherein the second heating element or cooling element maintains the perfusate reservoir at a desired temperature.

7. The perfusion device of any one of claims 1-6, further comprising an infusion container, wherein the infusion container is connected to the perfusion circuit by infusion line tubing.

8. The perfusion device of claim 7, wherein the infusion container is a syringe, wherein the syringe is connected to the infusion line tubing by a luer lock.

9. The perfusion device of claim 7 or 8, wherein the infusion line tubing is connected to the outlet of the oxygenator and the infusion container.

10. The perfusion device of any one of claims 7-9, wherein the infusion container contains an infusion comprising a therapeutic agent or nutrient.

11. The perfusion device of claim 10, wherein the infusion comprises heparin, prostacycline, glucose, insulin, a bile salt, an amino acid, a fatty acid, a steroid, a diuretic, a recombinant nucleic acid, a vector, a gene editing system, or any combination thereof.

12. The perfusion device of any one of claims 7-11, further comprising an infusion pump or syringe pump to propel an infusion contained in the infusion container through the infusion line tubing into the perfusate.

13. The perfusion device of any one of claims 1-12, further comprising a sample port, wherein the sample port is connected to the arterial tube.

14. The perfusion device of claim 13, further comprising a pressure sensor, wherein the pressure sensor is connected to the sample port.

15. The perfusion device of any one of claims 1-14, wherein the oxygenator is a pediatric oxygenator.

16. The perfusion device of any one of claims 1-15, further comprising a connector, wherein the perfusate reservoir is connected to the sterile container by the connector.

17. The perfusion device of any one of claims 1-16, wherein the sterile container further comprises a drainage port.

18. The perfusion device of claim 17, wherein the drainage port is connected to the perfusate reservoir by a drainage line tubing.

19. The perfusion device of any one of claims 1-18, wherein the pump is a centrifugal pump or a peristaltic pump.

20. The perfusion device of any one of claims 1-19, wherein the pump maintains an artery pressure between in a range between 60 mm Hg to 75 mm Hg.

21. The perfusion device of any one of claims 1-20, wherein the donor organ is a kidney, a heart, a liver, a lung, a stomach, a small intestine, a large intestine, a pancreas, or a gonad, or a portion thereof.

22. The perfusion device of claim 21, wherein the donor organ is a kidney or a portion thereof.

23. The perfusion device of claim 22, wherein the artery is a renal artery and the vein is a renal vein.

24. The perfusion device of claim 22 or 23, further comprising a ureteral catheter or cannula that can be connected to a ureter of the kidney and a urine collection container that can be connected to the ureteral catheter or cannula by a urine drainage line tubing.

25. The perfusion device of any one of claims 22-24, wherein flow rate of the perfusate through the renal artery is maintained in a range from 5 ml/minute to 1000 ml/minute.

26. The perfusion device of any one of claims 1-25, further comprising an air bubble sensor that can detect air bubbles in the perfusate.

27. The perfusion device of any one of claims 1-26, further comprising a stand, wherein the sterile container is placed on the stand.

28. The perfusion device of any one of claims 1-27, wherein the sterile container is a bag.

29. The perfusion device of claim 28, further comprising a pole, wherein the pole is used to hang the bag.

30. The perfusion device of any one of claims 1-29, wherein the sterile container further comprises a plurality of magnets, wherein the plurality of magnets applies compression to reduce edema of the donor organ.

31. The perfusion device of any one of claims 1-30, wherein the donor organ is obtained from a live organ donor or an organ donor after circulatory death.

32. The perfusion device of any one of claims 1-31, wherein a venous cannula is used to connect the venous tube to a vein of the donor organ, and wherein an arterial cannula is used to connect the arterial tube to an artery of the donor organ.

33. The perfusion device of any one of claims 1-32, further comprising a plurality of magnetic tubing connectors for connecting tubing of the perfusion circuit, wherein a first magnetic tubing connector is attached to a first tubing and a second magnetic tubing connector is attached to a second tubing, wherein the first tubing and the second tubing are connected to each other by magnetically joining the first magnetic tubing connector to the second magnetic tubing connector.

34. The perfusion device of claim 33, wherein the first magnetic tubing connector and the second magnetic tubing connector, when magnetically joined, can be rotated to reduce kinking of the first tubing and the second tubing.

35. The perfusion device of claim 33 or 34, wherein each magnetic tubing connector comprises a rubber gasket, wherein tubing connections made with the plurality of magnetic tubing connector are water-tight up to a pressure of at least 250 millimeter of mercury (mm Hg).

36. The perfusion device of any one of claims 1-35, further comprising a sensor for measuring a level of a biomarker in a sample of the perfusate to determine fitness of the organ for transplant.

37. The perfusion device of any one of claims 1-36, further comprising a sensor for measuring temperature of the perfusate, flow-rate of the perfusate, pH of the perfusate, concentration of oxygen in the perfusate, concentration of glucose in the perfusate, concentration of hemoglobin in the perfusate, concentration of sodium in the perfusate, concentration of potassium in the perfusate, concentration of calcium in the perfusate, concentration of carbon dioxide in the perfusate, percent saturation of oxygen in the perfusion, or concentration of lactate in the perfusate, or any combination thereof.

38. A method of using the perfusion device of any one of claims 1-37 for perfusion of an organ, the method comprising: placing the organ in the sterile container; adding perfusate to the perfusate reservoir; connecting the venous tube to a vein of the organ and the perfusate reservoir; connecting the arterial tube to an artery of the organ and the outlet of the oxygenator; connecting the oxygen reservoir to the inlet of the oxygenator; and turning on the pump to circulate the perfusate through the perfusion circuit and to and from the donor organ.

39. The method of claim 38, further comprising: turning on a first heating element or cooling element, wherein the first heating element or cooling element maintains the sterile container at a first desired temperature; and turning on a second heating element or cooling element, wherein the second heating element or cooling element maintains the perfusate reservoir at a second desired temperature.

40. The method of claim 39, wherein the first heating element or cooling element and the second heating element or cooling element are adjusted to maintain the temperature of the sterile container and the perfusate reservoir in a range from 4 °C to 40 °C during perfusion.

41. The method of claim 40, wherein the temperature is maintained in a range from 20 °C to 40 °C, 35 °C to 37 °C, 20 °C to 32 °C, 13 °C to 24 °C, or 4 °C to 10 °C during perfusion.

42. The method of any one of claims 38-41, wherein the sterile container further comprises a drainage port, wherein the method further comprises connecting the drainage port to the perfusate reservoir with a drainage line tubing.

43. The method of any one of claims 38-42, further comprising: connecting a sample port to the arterial tube; and connecting a pressure sensor to the sample port.

44. The method of any one of claims 38-43, wherein the donor organ is a kidney, a heart, a liver, a lung, a stomach, a small intestine, a large intestine, a pancreas, or a gonad, or a portion thereof.

45. The method of claim 44, wherein the donor organ is a kidney.

46. The method of claim 45, wherein said connecting the arterial tube comprises connecting the arterial tube to a renal artery, and wherein said connecting the venous tube comprises connecting the venous tube to a renal vein.

47. The method of claim 45 or 46, further comprising connecting a ureteral catheter or cannula to a ureter of the kidney; and connecting a urine drainage line tubing to the ureteral catheter or cannula and a urine collection container, wherein urine is collected in the urine collection container.

48. The method of any one of claims 45-47, further comprising adjusting the pinch valve such that flow rate of the perfusate through the renal artery is maintained in a range from 5 ml/minute to 1000 ml/minute.

49. The method of any one of claims 38-48, further comprising: connecting infusion line tubing to an infusion container and the perfusion circuit; adding an infusion to the infusion container; and using a syringe pump or infusion pump to propel the infusion into the perfusate.

50. The method of claim 49, wherein the infusion comprises a therapeutic agent or a nutrient.

51. The method of claim 50, wherein the infusion comprises heparin, prostacycline, glucose, insulin, a bile salt, an amino acid, a fatty acid, a steroid, a diuretic, a recombinant nucleic acid, a vector, a gene editing system, or any combination thereof.

52. The method of claim 51, wherein the organ is a kidney, wherein the recombinant nucleic acid, vector, or gene editing system is delivered in the perfusate to a nephron.

53. The method of any one of claims 38-52, further comprising administering a therapeutic agent locally to a site on the organ during ex vivo perfusion of the organ.

54. The method of any one of claims 50-53, wherein the therapeutic agent is toxic when administered to a subject in vivo.

55. The method of claim 54, wherein the therapeutic agent is a gene therapy agent, a chemotherapeutic agent, or a radiotherapeutic agent.

56. The method of any one of claims 38-55, further comprising pressurizing hollow spaces within the organ.

57. The method of claim 56, wherein the organ is a kidney, and said pressurizing comprises pressurizing a renal pelvis or collecting system of the kidney.

58. The method of any one of claims 38-57, further comprising placing the sterile container on a stand.

59. The method of any one of claims 38-58, wherein the sterile container is a bag.

60. The method of claim 59, further comprising hanging the bag on a pole.

61. The method of any one of claims 38-60, further comprising adding a plurality of magnets to the sterile container, wherein the plurality of magnets applies compression to reduce edema of the donor organ during the perfusion.

62. The method of any one of claims 38-61, wherein the donor organ is obtained from a live organ donor or an organ donor after circulatory death.

63. The method of any one of claims 38-62, wherein a venous cannula is used to connect the venous tube to a vein of the donor organ, and wherein an arterial cannula is used to connect the arterial tube to an artery of the donor organ.

64. The method of any one of claims 38-63, wherein a plurality of magnetic tubing connectors are used for connecting tubing of the perfusion circuit, wherein a first magnetic tubing connector is attached to a first tubing and a second magnetic tubing connector is attached to a second tubing, wherein the first tubing and the second tubing are connected to each other by magnetically joining the first magnetic tubing connector to the second magnetic tubing connector.

65. The method of claim 64, rotating the first magnetic tubing connector relative to the second magnetic tubing connector, when the first magnetic tubing connector and the second magnetic tubing connector are magnetically joined to reduce kinking of the first tubing and the second tubing.

66. The method of any one of claims 38-65, further comprising measuring a level of a biomarker in a sample of the perfusate to determine fitness of the organ for transplant.

67. The method of any one of claims 38-66, further comprising genetically modifying the organ during perfusion.

68. The method of claim 67, wherein said genetically modifying the organ comprises converting a disease-associated allele to a wild-type allele or converting an immunologically non-compatible allele to a compatible allele.

69. The method of any one of claims 38-68, further comprising surgically repairing the organ prior to transplantation into a recipient.

70. The method of any one of claims 38-69, further comprising measuring temperature of the perfusate, flow-rate of the perfusate, pH of the perfusate, concentration of oxygen in the perfusate, concentration of glucose in the perfusate, concentration of hemoglobin in the perfusate, concentration of sodium in the perfusate, concentration of potassium in the perfusate, concentration of calcium in the perfusate, concentration of carbon dioxide in the perfusate, percent saturation of oxygen in the perfusion, or concentration of lactate in the perfusate, or any combination thereof.

71. The method of any one of claims 38-70, wherein a higher overall flow rate of the perfusate is maintained through the perfusion circuit and a lower flow rate of the perfusate is maintained through the organ.

72. The method of any one of claims 38-71, wherein the perfusion of the organ is normothermic organ perfusion, sub-normothermic organ perfusion, or hypothermic organ perfusion.

74. The bag of claim 73, wherein the bag has a size designed to fit the organ.

75. The bag of claim 74, wherein the organ is from an adult, child, or infant.

76. The bag of any one of claims 73-75, wherein the organ is a kidney, a heart, a liver, a lung, a stomach, a small intestine, a large intestine, a pancreas, or a gonad, or a portion thereof.

77. The bag of any one of claims 73-76, wherein the bag is sterilized, humidified, heated to a desired temperature, or any combination thereof.

78. The bag of claim 77, wherein temperature of the bag is maintained in a range from 4 °C to 40 °C.

79. The bag of claim 78, wherein temperature of the bag is maintained in a range from 20 °C to 40 °C, 35 °C to 37 °C, 20 °C to 32 °C, 13 °C to 24 °C, or 4 °C to 10 °C.

80. The bag of any one of claims 73-79, wherein the bag further comprises perfusate.

81. The bag of any one of claims 73-80, wherein the bag further comprises an organ.