Acellular blood alternative for transfusion and organ perfusion

Abstract

Compositions comprising Lumbricus terrestris erythrocruorin (LtEc) as an oxygen carrier for use in transfusion and organ perfusion are provided. In particular, methods of using acellular compositions comprising LtEc as a universal blood substitute for transfusion of patients are provided. The methods also include ex vivo perfusion of an organ or tissue with an oxygenated acellular perfusate comprising LtEc as an oxygen carrier. Additionally, methods are provided for using compositions comprising LtEc in treatment of acute ischemia and hyperbaric oxygen therapy.

Description

ACELLULAR BLOOD ALTERNATIVE FOR TRANSFUSION AND ORGANPERFUSIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit under 35 U.S.C. § 119(e) of provisional application 63 / 718,172, filed November 8, 2024, which application is hereby incorporated by reference in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML file, “UCSF- 825WO” created on November 4, 2025, and having a size of 39,317 bytes. The contents of the Sequence Listing XML file are incorporated by reference herein in their entireties.INTRODUCTION

[0003] The limitations of red blood cells (RBCs) and human blood products have generated a large market for red blood cell substitutes, which is estimated to grow to $24 billion by 2029. Several acellular hemoglobin-based oxygen carriers (HBOCs) have been developed. Polymerized bovine hemoglobin (polybHb; also known as HBOC-201) has shown promise in preclinical testing, but clinical trials have revealed significant disadvantages such as autoxidation, which leads to the generation of harmful reactive oxygen species (e.g., Ch and H2O2) that induce oxidative stress and tissue damage, and nitric oxide (NO) scavenging, which induces severe vasoconstriction. Newer products like Erythromer™ encapsulate Hb inside a liposome to block interactions with NO in the bloodstream, but its safety and stability has yet to be rigorously evaluated in vivo (Mittal, N. et al. Erythromer (EM), a Nanoscale Bio-Synthetic Artificial Red Cell. Blood Substitutes and Oxygen Biotherapeutics 253-265 (2022)). Naturally occurring acellular hemoglobins (i.e. erythrocruorins) derived from annelids represent a third approach. Since annelids do not express hemoglobin inside red blood cells, these molecules evolved to avoid the autoxidation, NO scavenging, and other issues that hinder HBOCs. Arenicola marina erythruocruorin (AmEc), or HEMO2Life®, derived from the marine lugworm, is one such molecule that has shown promise for organ perfusion. However, AmEc has only been used for hypothermic (not normothermic) organ perfusion (Varney, J. et al. Mini-review on the properties and possible applications of therapeutic oxygen carrier Hemarina-MIOL Transfusion and Apheresis Science 60, 103016 (2021)). AmEc also dissociates into smaller dodecamers in human plasma at 37°C (Rousselot, M. et al. Arenicola marina extracellular hemoglobin: A new promisingblood substitute. Biotechnol J 1, 333-345 (2006); Walpurgis, K. et al. Detection of extracellular hemoglobin from Arenicola marina in doping control serum samples by means of liquid chromatography and high-resolution tandem mass spectrometry. Drug Test Anal 15, 1430-1438 (2023)).

[0004] The ability to maintain the viability and function of organs outside the body (ex vivo) at body temperature (normothermia) is an emerging field of innovation. Normothermic ex vivo perfusion systems have been developed to maintain hearts, lungs, and livers in this fashion for up to several days and have been a valuable tool in transplantation medicine to extend the longevity of donor organs. The potential for these technologies to be utilized in other ways is now also being recognized. Normothermic ex vivo kidney perfusion (NEVKP), in comparison, has been more challenging to develop. The kidney is highly susceptible to damage from the products of red blood cell (RBC) degradation that are generated by machine perfusion. Because of this, existing NEVKP systems cannot reliably sustain the viability of kidneys beyond 24 hours.

[0005] Thus, there remains a need to find better alternatives to using red blood cells for transfusion and organ perfusion.SUMMARY

[0006] Compositions comprising Lumbricus terrestris erythrocruorin (LtEc) as an oxygen carrier for use in transfusion and organ perfusion are provided. In particular, methods of using acellular compositions comprising LtEc as a universal blood substitute for transfusion of patients are provided. The methods also include ex vivo perfusion of an organ or tissue with an oxygenated acellular perfusate comprising LtEc as an oxygen carrier. Additionally, methods are provided for using compositions comprising LtEc in treatment of acute ischemia and hyperbaric oxygen therapy.

[0007] In one aspect, an acellular oxygen-carrying perfusate comprising Lumbricus terrestris erythrocruorin (LtEc) is provided.

[0008] In certain embodiments, the LtEc is at a concentration in a range from 0.1 g / dL to 2.5 g / dL. In some embodiments, the LtEc is at a concentration of about 0.1 g / dL, about 0.5 g / dL, or about 2.5 g / dL.

[0009] In certain embodiments, the LtEc comprises: a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:1; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:3; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:5; a polypeptidecomprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:7; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 9; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 11 ; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 13; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 15; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 17; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:21; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 23; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:25: a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:27; and a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:29.

[0010] In certain embodiments, the LtEc is cross-linked with a cross-linking agent such that subunits of LtEc do not dissociate.

[0011] In certain embodiments, the acellular oxygen-carrying perfusate further comprises one or more of nutrients, electrolytes, reducing agents, antibiotics, anticoagulants, osmotic agents, colloids, diuretics, vasodilators, insulin, growth factors, immunosuppressive agents, or combinations thereof. In some embodiments, the nutrients comprise carbohydrates, amino acids, nucleotides, fatty acids, vitamins, or a combination thereof. In some embodiments, the amino acids comprise alanine, aspartate, asparagine, glycine, glutamate, glutamine, lysine, arginine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, isoleucine, leucine, valine, histidine, cysteine, proline, or a combination thereof. In some embodiments, the fatty acids comprise oleic acid, linoleic acid, palmitic acid, stearic acid, or a combination thereof. In some embodiments, the carbohydrates comprise glucose. In some embodiments, the electrolytes comprise sodium (Na+), chloride (O’), potassium (K+), calcium (Ca2+) magnesium (Mg2+), bicarbonate (HCO3 ), acetate, citrate, or a combination thereof. In some embodiments, the nucleotides comprise adenosine and its metabolites like adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP). Tn some embodiments, the reducing agents comprise ascorbic acid, glutathione, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), or N-acetyl-L-cysteine, or a combination thereof. In some embodiments, the osmotic agents comprise mannitol, glycerol, sorbitol, dextrose, or ribose. In some embodiments, the colloids comprise albumin, dextran, hydroxyethyl starch (HES), or a combination thereof. In some embodiments, the vasodilators comprise nitroglycerin, hydralazine, or sodium nitroprusside. In some embodiments, the antibiotics comprise ceftriaxone, gentamicin, penicillin, streptomycin, or a combination thereof. In some embodiments, the growth factors comprise one or more hematopoietic growth factors or interleukins, or a combination thereof. In some embodiments, the hematopoietic growth factors comprise erythropoietin, thrombopoietin, insulin-like growth factor (IGF)-l, granulocyte colony-stimulating factor (G- CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colonystimulating factor (M-CSF), stem cell factor (SCF), interleukin-3 (IL-3), or a combination thereof.

[0012] In certain embodiments, the acellular oxygen-carrying perfusate further comprises a crystalloid solution. In some embodiments, the crystalloid solution is saline or Ringer's lactate solution.

[0013] In certain embodiments, the LtEc is oxygenated.

[0014] In certain embodiments, the LtEc is deoxygenated during storage, for example, to prolong its shelf-life.

[0015] In certain embodiments, the acellular oxygen-carrying perfusate further comprises a therapeutic agent. In some embodiments, the therapeutic agent is a drug, a gene therapy agent, a gene editing agent, a chemotherapeutic agent, or a radiotherapeutic agent.

[0016] In another aspect, a method for perfusion of an organ is provided, the method comprising perfusing the organ with an acellular oxygen-carrying perfusate comprising LtEc, described herein.

[0017] In certain embodiments, the perfusion is in situ perfusion or ex vivo perfusion.

[0018] In certain embodiments, the perfusion is normothermic organ perfusion, mid- thermic organ perfusion, sub-normothermic organ perfusion, or hypothermic organ perfusion.

[0019] In certain embodiments, the temperature of the acellular oxygen-carrying perfusate is maintained in a range from 20 °C to 40 °C, 1 °C to 10 °C, 13 °C to 24 °C, or 25 °C to 34 °C during perfusion.

[0020] In certain embodiments, the temperature of the organ or a container holding the organ is maintained in a range from 20 °C to 40 °C, 1 °C to 10 °C, 13 °C to 24 °C, or 25 °C to 34 °C during perfusion.

[0021] In certain embodiments, the organ is a kidney, a heart, a liver, a lung, a stomach, a small intestine, a large intestine, a pancreas, bladder, spleen, a gonad, a limb, an extremity, or a tissue graft or a portion thereof.

[0022] In certain embodiments, the organ is obtained from a live organ donor or an organ donor after circulatory death.

[0023] In certain embodiments, the acellular oxygen-carrying perfusate is oxygenated with a gas mixture having 1% to 99% oxygen. In certain embodiments, the acellular oxygencarrying perfusate is oxygenated with a gas mixture having 95% to 99% oxygen.

[0024] In certain embodiments, the method further comprises adding a therapeutic agent or nutrient to the acellular oxygen-carrying perfusate.

[0025] In certain embodiments, the therapeutic agent or nutrient is heparin, prostacycline, glucose, insulin, a bile salt, an amino acid, a fatty acid, a lipid, a vitamin, a mineral, a hormone, a cytokine, a steroid, a diuretic, a vasoactive molecule, an antibiotic, an antibody, or a cell.

[0026] In certain embodiments, the therapeutic agent is a recombinant nucleic acid or gene editing system.

[0027] In certain embodiments, the recombinant nucleic acid or gene editing system is encapsulated in a lipid nanoparticlc (LNP).

[0028] In certain embodiments, the recombinant nucleic acid is a DNA or RNA encoding a therapeutic protein or regulatory RNA.

[0029] In certain embodiments, the recombinant nucleic acid comprises a viral vector or plasmid. Exemplary viral vectors include, without limitation, adeno-associated viral vectors, adenoviral vectors, lentiviral vectors, and retroviral vectors.

[0030] In certain embodiments, expression of the therapeutic protein or regulatory RNA is inducible.

[0031] In certain embodiments, the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of a therapeutic protein.

[0032] In certain 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).

[0033] In certain embodiments, the recombinant nucleic acid or gene editing system is delivered in the perfusate to the organ.

[0034] In certain embodiments, the therapeutic agent is administered locally to a site on the organ during ex vivo perfusion of the organ.

[0035] In certain embodiments, the therapeutic agent is toxic when administered to a subject in vivo.

[0036] In certain embodiments, the therapeutic agent is a drag, a gene therapy agent, a gene editing agent, a chemotherapeutic agent, or a radiothcrapcutic agent.

[0037] In certain embodiments, the method further comprises measuring a level of a biomarker in a sample of the perfusate to determine fitness of the organ for transplant.

[0038] In certain embodiments, the method further comprises genetically modifying the organ during perfusion. In some embodiments, genetically modifying the organ comprises converting a disease-associated allele to a wild-type allele.

[0039] In certain embodiments, the method further comprises surgically repairing the organ or performing a tumor resection prior to transplantation into a recipient.

[0040] In certain embodiments, the method further comprises measuring temperature of the acellular oxygen-carrying perfusate, flow-rate of the acellular oxygen-carrying perfusate, pH of the acellular oxygen-carrying perfusate, concentration of oxygen in the acellular oxygencarrying perfusate, concentration of glucose in the acellular oxygen-carrying perfusate, concentration of LtEc in the acellular oxygen-carrying perfusate, concentration of sodium in the acellular oxygcn-carrying perfusate, concentration of potassium in the acellular oxygcn-carrying perfusate, concentration of calcium in the acellular oxygen-carrying perfusate, concentration of carbon dioxide in the acellular oxygen-carrying perfusate, percent saturation of oxygen in the acellular oxygen-carrying perfusate, or concentration of lactate in the acellular oxygen-carrying perfusate, or any combination thereof.

[0041] In another aspect, a method for transfusion of a subject is provided, the method comprising transfusing the subject with an acellular oxygen-carrying perfusate comprising LtEc. This method can be performed on a subject of any blood type (i.e., the acellular oxygen-carrying perfusate comprising LtEc can be used as a universal blood substitute for transfusions).

[0042] In certain embodiments, the subject has acute blood loss from trauma, a surgical procedure, or a hemorrhage.

[0043] In certain embodiments, the subject has anemia such as, but not limited to, chronic anemia from a nutrient deficiency, a malignancy, or a genetic disorder.

[0044] In certain embodiments, the subject is unable or unwilling to receive a transfusion with a human blood product.

[0045] In another aspect, a method of performing liquid hyperbaric oxygen therapy is provided, the method comprising infusing an acellular oxygen-carrying perfusate comprising LtEcinto a tissue, limb, organ, or organism in need of the liquid hyperbaric oxygen therapy, wherein the acellular oxygen-carrying perfusate is oxygenated in a hyperbaric oxygen chamber.

[0046] In certain embodiments, the liquid hyperbaric oxygen therapy is used to treat a subject for necrotizing fasciitis, gas gangrene, hemorrhagic cystitis, poor wound healing, a skin graft, or a themial or radiation bum.

[0047] In another aspect, a method of treating ischemia in a subject is provided, the method comprising infusing the acellular oxygen-carrying perfusate comprising LtEc into an ischemic tissue in the subject. In some embodiments, the acellular oxygen-carrying perfusate is oxygenated with a gas mixture having 1 % to 99% oxygen. In some embodiments, the acellular oxygen-carrying perfusate is oxygenated with a gas mixture having 95% to 99% oxygen.

[0048] In certain embodiments, the ischemia is caused by a stroke, a transient ischemic attack, a myocardial infarction, acute limb ischemia, or ischemic bowel syndrome.

[0049] In another aspect, a method of supporting cellular respiration in a cell or tissue is provided, the method comprising delivering an acellular oxygen-carrying perfusate comprising LtEc to the cell or tissue.

[0050] In certain embodiments, the acellular oxygen-carrying perfusate is delivered to the cell or tissue in vitro, ex vivo, or in vivo.

[0051] In another aspect, a method of performing radiation therapy of a tumor is provided, the method comprising: delivering the acellular oxygen-carrying perfusate of any one of claims 1-23 to the tumor; and administering the radiation therapy to the tumor.

[0052] In certain embodiments, the tumor is hypoxic.

[0053] In certain embodiments, the tumor has previously been resistant to radiation therapy.

[0054] In certain embodiments, the acellular oxygen-carrying perfusate is delivered prior to or during radiation therapy.

[0055] In certain embodiments, the acellular oxygen-carrying perfusate is delivered by infusion, perfusion, or injection locally into the tumor.

[0056] In another aspect, a system is provided, the system comprising: a perfusion machine comprising a reservoir, wherein the reservoir comprises an acellular oxygen-carrying perfusate comprising LtEc, described herein; and an oxygenator, wherein the oxygenator oxygenates the acellular oxygen-carrying perfusate.

[0057] In certain embodiments, the system further comprises a container for holding an organ or tissue.

[0058] In certain embodiments, the perfusion machine performs normothermic organ perfusion, mid-thermic organ perfusion, sub-normothermic organ perfusion, or hypothermic organ perfusion.

[0059] In certain embodiments, the perfusion machine performs perfusion of an organ or tissue in situ or ex vivo.

[0060] In certain embodiments, the system further comprises a temperature controller, wherein the temperature controller maintains the acellular oxygen-carrying perfusate at a desired temperature.

[0061] In certain embodiments, the temperature controller maintains the temperature of the acellular oxygen-carrying perfusate or the container holding the organ or tissue in a range from 20 °C to 40 °C, 1 °C to 10 °C, 13 °C to 24 °C, or 25 °C to 34 °C during perfusion.

[0062] In certain embodiments, the temperature controller is a water bath, an ice bath, a thermoelectric temperature controller, or a heat exchanger.BRIEF DESCRIPTION OF THE DRAWINGS

[0063] FIG. 1. Consequences of RBC degradation include oxidative stress, NO scavenging, and heme toxicity.

[0064] FIG. 2. Assembly of the 180 subunits of LtEc into a hexagonal bilayer (HBL) structure along with tetrameric human hemoglobin (HbA) and monomeric myoglobin.

[0065] FIG. 3. LtEc will be directly compared to RBCs, polybHb, and Arenicola marina Ec in matched kidneys for 1-7 days.

[0066] FIGS. 4A-4C. Exchange transfusions of LtEc in hamsters show that LtEc can effectively oxygenate tissues (FIG. 4A) without inducing hypertension (FIG. 4B). LtEc also does not oxidize in vivo and its circulation half-life is 14 hrs (FIG. 4C).

[0067] FIGS. 5A-5B. (FIG. 5A) NEVKP circuit diagram. (FIG. 5B) Tandem circuits used for paired kidney experiments.

[0068] FIGS. 6A-6E. A porcine kidney perfused with LtEc functions in vivo for 7 days. FIG. 6A. Experimental schematic. FIG. 6B. Urine pads placed beneath cage during recovery demonstrated daily urine output after auto-transplantation. FIG. 6C. Color Doppler ultrasonography of the autotransplanted kidney on Day 7 demonstrated robust blood flow. FIG. 6D. Representative hematoxylin and eosin (H&E) staining of the glomerulus following necropsy on Day 7 revealed minimal architectural changes and minimal cell death. FIG. 6E. Serum potassium levels of the animal throughout experiment demonstrated normalization by Day 7.DETAILED DESCRIPTION

[0069] Compositions comprising Lumbricus terrestris erythrocruorin (LtEc) as an oxygen carrier for use in transfusion and organ perfusion are provided. In particular, methods of using acellular compositions comprising LtEc as a universal blood substitute for transfusion of patients are provided. The methods also include ex vivo perfusion of an organ or tissue with an oxygenated acellular perfusate comprising LtEc as an oxygen carrier. Additionally, methods are provided for using compositions comprising LtEc in treatment of acute ischemia and hyperbaric oxygen therapy.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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 "an erythrocruorin" includes a plurality of such erythrocruorins, andreference 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.

[0074] It is further noted that the claims may be drafted to exclude any clement 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.

[0075] 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.

[0076] 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

[0077] The term "about," particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

[0078] “Perfusion” refers to circulation of a fluid (also referred to as a perfusion solution or perfusate) through an organ or tissue to maintain function and viability of the organ or tissue. In some embodiments, the perfusate includes an LtEc oxygen carrier. A perfusate may be circulated to and from an organ by a mechanical device (i.e., machine perfusion). Such devices may include one or more chambers for holding an organ and the perfusate, one or more pumps for delivery of the perfusate to the organ, one or more means to regulate temperature of the perfusate, and one or more means to oxygenate the perfusate. In some embodiments, perfusion includes introduction of an acellular oxygen-carrying perfusate comprising LtEc into an organ and removal of oxygen depleted fluid from the organ by circulation of oxygenated perfusate through the organ. In some embodiments, the perfusion can be pulsatile, with periodic increases and decreases offlow, to mimic arterial blood flow from a beating heart. In other embodiments, the perfusion can be continuous, with a substantial absence of flow rate variations, to mimic venous blood flow under most physiologic conditions.

[0079] The term “organ” as used herein refers to a structure of tissue in a subject, wherein the tissue structure as a whole is specialized to perform a particular bodily function. Organs may include, for example, without limitation, a kidney, a heart, a liver, a lung, a stomach, a small intestine, a large intestine, a pancreas, a gonad, a bladder, a spleen, a limb (such as arm or leg, or portion thereof) or extremity (such as hand, foot, finger, toe, or a portion thereof), or a tissue graft or a portion thereof. The term “organ” may also encompass decellularized and recellularized organs, as well as engineered and artificial organs and tissues, including engineered organs (e.g., tissue engineered constructs), engineered organs comprising a bioscaffold, tissues, organ slices and partial organs. In some embodiments, the organ is a vascular composite tissue that can be cannulated for perfusion, including limbs, face, abdominal wall, among others.

[0080] The term “perfusion” as used herein refers to flowing fluid through a tissue or organ. In some cases a perfusion fluid (i.e., perfusate) is circulated through blood vessels (e.g., arteries or veins) or other natural channels.

[0081] The terms “individual”, “subject”, and “patient”, arc 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.

[0082] 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, nurse, phlebotomist, or perfusionist operating a perfusion device to circulate perfusate to and from an organ or performing a transfusion with an acellular oxygencarrying perfusate comprising Lumbricus terrestris erythrocruorin (LtEc), as described herein. If the organ undergoing perfusion is a kidney, the user may be a nephrologist, renal technologist, or other health care provider including urologists.

[0083] 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 containingone 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, multimcrs (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.

[0084] 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.

[0085] 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 deoxy ribonucleotides. 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 polydeoxyribonucleotides (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 nonnuclcotidic 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, phosphotricstcrs, 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-T-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.

[0086] "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 80%-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.

[0087] ‘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 thatare substantially enriched for the entity of interest and / or in which the entity of interest is partially or substantially purified.

[0088] 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.

[0089] 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.

[0090] "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.

[0091] 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. 2:482 489, 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 bedetermined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

[0092] 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.

[0093] 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.

[0094] "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.

[0095] 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.

[0096] 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) temiinus 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.

[0097] 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.

[0098] "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.

[0099] "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.

[0100] "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).

[0101] "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 arc 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.

[0102] 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.

[0103] 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.

[0104] "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, lenti viruses, alphaviruses, pox viruses, and vaccinia viruses.

[0105] 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 anymanner, 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.

[0106] 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.

[0107] 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).

[0108] 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.

[0109] 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.

[0110] 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 beused 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).

[0111] 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 colt (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, 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.

[0112] 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 aparticular target DNA sequence will selectively direct binding of Cas9 to a substantially complementary sequence at the target site and not to unrelated sequences.

[0113] 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).

[0114] 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.

[0115] 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 arc 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.

[0116] 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 an 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-complcmcntary 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.

[0117] 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(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.

[0118] 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 useof 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. 201 1 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.Perfusate Compositions

[0119] Perfusate compositions comprising Lumbricus terreslris erythrocruorin (LtEc) as an oxygen carrier are provided. In some embodiments, the disclosed perfusate compositions comprise acellular LtEc (i.e., LtEc that is not contained in a cell), which serves as an oxygen carrier for oxygenating an organ, tissue, or cell.

[0120] LtEc comprises an assembly of 144 globin subunits held together by a network of intra- and inter-subunit disulfide bonds, dozens of calcium binding sites, and 36 linker subunits (Royer et al. (2006) Structure 14(7): 1167-1177; herein incorporated by reference). The assembly includes four different types of globin subunits and 4 different types of linkers. Representative sequences of the globin subunits and nucleic acids encoding them arc presented in SEQ ID NOS : 1 - 16, and representative sequences of the linkers and nucleic acids encoding them are presented in SEQ ID NOS: 17-30. Any of these sequences or a variant thereof comprising a sequence having at least about 80-100% sequence identity thereto, including any percent identity within this range, such as 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 to produce an LtEc protein for use as an oxygen carrier in the compositions and methods described herein.

[0121] The perfusate will generally contain an effective amount of the LtEc oxygen carrier to provide sufficient oxygen carrying capacity for an intended purpose. For example, an effective amount of the LtEc in a perfusate may provide sufficient oxygenation for in situ or ex vivo organ perfusion, treatment of ischemia, or hyperbaric oxygen therapy, as discussed further below. In some embodiments, an acellular perfusate comprises about 0.1 g / dL to about 10 g / dL of LtEc, such as about 0.1 g / dL to about 5 g / dL, about 0.1 g / dL to about 2.5 g / dL, or about 0.5 g / dL to about 2.5 g / dL, or any amount within these ranges such as 0.1 g / dL, 0.2 g / dL, 0.3 g / dL, 0.4 g / dL,0.5 g / dL, 0.6 g / dL, 0.7 g / dL, 0.8 g / dL, 0.9 g / dL, 1.0 g / dL, 1.1 g / dL, 1.2 g / dL, 1.3 g / dL, 1.4 g / dL,1.5 g / dL, 1.6 g / dL, 1.7 g / dL, 1.8 g / dL, 1.9 g / dL, 2.0 g / dL, 2.1 g / dL, 2.2 g / dL, 2.3 g / dL, 2.4 g / dL,2.5 g / dL, 2.6 g / dL, 2.7 g / dL, 2.8 g / dL, 2.9 g / dL, 3 g / dL, 3.25 g / dL, 3.5 g / dL, 3.75 g / dL, 4 g / dL,4.25 g / dL, 4.5 g / dL, 4.75 g / dL, 5 g / dL, 5.25 g / dL, 5.5 g / dL, 5.75 g / dL, 6 g / dL, 6.25 g / dL, 6.5 g / dL,6.75 g / dL, 7 g / dL, 7.25 g / dL, 7.5 g / dL, 7.75 g / dL, 8 g / dL, 8.25 g / dL, 8.5 g / dL, 8.75 g / dL, 9 g / dL, 9.25 g / dL, 9.5 g / dL, 9.75 g / dL, or 10 g / dL of LtEc. In some exemplary embodiments, the LtEc is at a concentration of about 0.1 g / dL, about 0.5 g / dL, or about 2.5 g / dL.

[0122] In some embodiments, the acellular LtEc is cross-linked to prevent dissociation of subunits. Any suitable cross-linking agents can be used for this purpose. Exemplary crosslinking agents include, without limitation, glutaraldehyde, dimethyl suberimidate, N- hydroxysuccinimide, and formaldehyde. In addition, carboxyl-reactive chemical groups such as diazomethane, diazoacetyl, and carbodiimide can be included for crosslinking carboxylic acids to primary amines. In particular, the carbodiimide compounds, l-ethyl-3 -(-3 -dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N',N'-dicyclohexyl carbodiimide (DCC) can be used for conjugation with carboxylic acids. In order to improve the efficiency of crosslinking reactions, N- hydroxysuccinimide (NHS) or a water-soluble analog (e.g., Sulfo-NHS) may be used in combination with a carbodiimide compound. The carbodiimide compound (e.g., EDC or DCC) couples NHS to carboxyl groups to form an NHS ester intermediate, which readily reacts with primary amines at physiological pH. For a description of various crosslinking agents and techniques, see, e.g., Wong and Jameson Chemistry of Protein and Nucleic Acid Cross-Linking and Conjugation (CRC Press, 2ndedition, 2011), Hermanson Bioconjugatc Techniques (Academic Press, 3rdedition, 2013), herein incorporated by reference in their entireties. Methods of crosslinking LtEc with glutaraldehyde are described, for example, in Rajesh et al. (Biotechnol. Prog. (2018) 34(2):521 -528; herein incorporated by reference in its entirety).

[0123] In addition to the LtEc oxygen earner, perfusate compositions may also include one or more other components, such as, but not limited to, nutrients, electrolytes, reducing agents, antibiotics, anticoagulants, osmotic agents, colloids, diuretics, vasodilators, insulin, growth factors, immunosuppressive agents, buffers, and combinations thereof. Additional components may be added to the perfusate, for example, to simulate physiological conditions and better maintain the function and viability of an organ or tissue.

[0124] In some embodiments, a perfusate composition comprises one or more nutrients such as, but not limited to, carbohydrates, amino acids, nucleotides, fatty acids, vitamins, or combinations thereof. Carbohydrates include, for example: monosaccharides, such as glucose, fructose, maltose, galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like. Amino acids include, for example: alanine, aspartate, asparagine, glycine, glutamate, glutamine, lysine,arginine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, isoleucine, leucine, valine, histidine, cysteine, and proline. Nucleotides include, for example: adenosine and its metabolites such as adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP). Fatty acids include, for example: oleic acid, linoleic acid, palmitic acid, and stearic acid. Vitamins include, for example: vitamin A (all-trans-retinols, all-trans- retinyl-esters, all-trans-P-carotene, and other provitamin A carotenoids), vitamin Bl (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6 (pyridoxine), vitamin B7 (biotin), vitamin B9 (folic acid and folates), vitamin B12 (cobalamins), vitamin C (ascorbic acid and ascorbates), vitamin D (calciferols), vitamin E (tocopherols and tocotrienols), and vitamin K (phylloquinones, menaquinones, and menadiones), and combinations thereof.

[0125] In some embodiments, a perfusate composition comprises a reducing agent. Exemplary reducing agents include, without limitation, ascorbic acid, glutathione, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), and N-acetyl-L-cysteine.

[0126] A perfusate composition can also include agents to adjust pH and osmolality such as buffers, electrolytes, colloids, crystalloids, and the like. Buffers may be used in a perfusate to maintain a physiological pH or other suitable pH to maintain the function and viability of an organ or tissue. Exemplary buffers include, for example: phosphate (e.g., phosphate buffered saline (PBS)), lactate (e.g., Ringer's lactate solution), bicarbonate, HEPES (N-2- hydroxyethylpiperazine-N'-ethanesulfonic acid), Tris (Tris(hydroxymethyl)aminomethane), and the like. In certain embodiments, the perfusate has a pH in a range of about 7.0 to about 8.0, such as about 7.2 to about? .9, about 7.4 to about 7.85, about 7 to about 7.45, or about 7.35 to about 7.4, including any pH within these ranges such as 7.0, 7.1, 7.15, 7.2, 7.25, 7.3, 7.35, 7.4, 7.45, 7.5, 7.55, 7.6, 7.65, 7.7, 7.75, 7.8,7.85, 7.9, 7.95, or 8.0. For most organs or tissues, the physiological pH is in a range from about 7 to about 7.45, more preferably in a range from 7.35 to 7.4. Electrolytes may include, but are not limited to, sodium (Na+), chloride (Cl ), potassium (K+), calcium (Ca2+) magnesium (Mg2+), bicarbonate (HCO3 ), acetate, citrate, and combinations thereof. Osmotic agents may include, but are not limited to, mannitol, glycerol, sorbitol, dextrose, and ribose, and combinations thereof. Colloids may include, but are not limited to, albumin, dextran, and hydroxyethyl starch (HES), and combinations thereof. In certain embodiments, the perfusate further comprises a crystalloid solution such as, but not limited to, saline or Ringer's lactate solution.

[0127] In some embodiments, the perfusate comprises Ca2+, which is a weak allosteric effector for LtEc that modulates the affinity of LtEc for oxygen. Accordingly, the concentration of Ca2+in the perfusate can be varied to modulate the affinity of LtEc for oxygen.

[0128] In some embodiments, the perfusate includes a vasodilator. Examples of vasodilators include nitrosovasodilators such as sodium nitroprusside and nitroglycerin; smooth muscle vasodilators such as hydralazine; a-adrenergic blockers, such as phentolamine and phenoxybenzamine; dihydroxypyridine calcium channel blockers such as nicardipine; cAMP- mediated vasodilators such as prostacyclin; and cGMP-mediated vasodilators such as nitric oxide. Such vasodilators may be used to increase flow of perfusate to organs or tissues, reduce vascular resistance, and / or improve regional perfusion.

[0129] In some embodiments, the perfusate includes an antibiotic. Examples of antibiotics include: broad spectrum, bactericidal, or bacteriostatic antibiotics such as penicillins including penicillin G, penicillin V, procaine penicillin, benzathine penicillin, veetids (Pen-Vee-K), piperacillin, pipracil, pfizerpen, temocillin, negaban, ticarcillin, and Ticar; penicillin combinations such as amoxicillin / clavulanate, augmentin, ampicillin / sulbactam, unasyn, piperacillin / tazobactam, zosyn, ticarcillin / clavulanate, and timentin; tetacyclines such as chlortetracycline, doxycycline, demeclocycline, eravacycline, lymecycline, meclocycline, methacycline, minocycline, omadacycline, oxytetracycline, rolitetracycline, sarecycline, tetracycline, and tigecycline; cephalosporins such as cefacetrile (cephacetrile), cefadroxil (cefadroxyl; duriccl), cefalexin (cephalexin; keflex), cefaloglycin (cephaloglycin), cefalonium (cephalonium), cefaloridine (cephaloradine), cefalotin (cephalothin; keflin), cefapirin (cephapirin; cefadryl), cefatrizine, cefazafhir, cefazedone, cefazolin (cephazolin; ancef, kefzol), cefradine (cephradine; velosef), cefroxadine, ceftezole, cefaclor (ceclor, distaclor, keflor, raniclor), cefonicid (monocid), cefprozil (cefproxil; cefzil), cefuroxime (zefu, zinnat, zinacef, ceftin, biofuroksym, xorimax), cefuzonam, loracarbef (lorabid) cefbuperazone, cefmetazole (zefazone), cefminox, cefotetan (cefotan), cefoxitin (mefoxin), cefotiam (pansporin), cefcapene, cefdaloxime, cefdinir (sefdin, zinir, omnicef, kefnir), cefditoren, cefetamet, cefixime (fixx, zifi, suprax), cefmenoxime, cefodizime, cefotaxime (claforan), cefovecin (convenia), cefpimizole, cefpodoxime (vantin, pecef, simplicef), cefteram, ceftamere (enshort), ceftibuten (cedax), ceftiofur (naxcel, excenel), ceftiolene, ceftizoxime (cefizox), ceftriaxone (rocephin), cefoperazone (cefobid), ceftazidime (meezat, fortum, fortaz), latamoxef (moxalactam), cefclidine, cefepime (maxipime), cefluprenam, cefoselis, cefozopran, cefpirome (cefrom), cefquinome, flomoxef, ceftobiprole, ceftaroline, ceftolozane, cefaloram, cefaparole, cefcanel, cefedrolor, cefempidone, cefetrizole, cefivitril, cefmatilen, cefmepidium, cefoxazole, cefrotil, cefsumide, ceftioxide,cefuracetime, and nitrocefin; quinolonesZ / fluoroquinolones such as flumequine (Flubactin), oxolinic acid (Uroxin), rosoxacin (Eradacil), cinoxacin (Cinobac), nalidixic acid (NegGam, Wintomylon), piromidic acid (Panacid), pipemidic acid (Dolcol), ciprofloxacin (Zoxan, Ciprobay, Cipro, Ciproxin), flcroxacin (Mcgalonc, Roquinol), lomcfloxacin (Maxaquin), nadifloxacin (Acuatim, Nadoxin, Nadixa), norfloxacin (Lexinor, Noroxin, Quinabic, Janacin), ofloxacin (Floxin, Oxaldin, Tarivid), pefloxacin (Peflacine), rufloxacin (Uroflox), enoxacin (Enroxil, Penetrex), balofloxacin (Baloxin), grepafloxacin (Raxar), levofloxacin (Cravit, Levaquin), pazufloxacin (Pasil, Pazucross), sparfloxacin (Zagam), temafloxacin (Omniflox), tosufloxacin (Ozex, Tosacin), clinafloxacin, gatifloxacin (Zigat, Tequin, Zymar-ophthalmic), moxifloxacin (Avelox.Vigamox), sitafloxacin (Gracevit), prulifloxacin (Quisnon), besifloxacin (Besivance), delafloxacin (Baxdela), gemifloxacin (Factive) and trovafloxacin (Trovan), ozenoxacin, danofloxacin (Advocin, Advocid), difloxacin (Dicural, Vetequinon), enrofloxacin (Baytril), ibafloxacin (Ibaflin), marbofloxacin (Marbocyl, Zenequin), orbifloxacin (Orbax, Vietas), and sarafloxacin (Floxasol, Saraflox, Sarafin); macrolides such as azithromycin, clarithromycin, erythromycin, fidaxomicin, telithromycin, carbomycin A, josamycin, kitasamycin, midecamycin / midecamycin acetate, oleandomycin, solithromycin, spiramycin, troleandomycin, tylosin / tylocinc, roxithromycin, telithromycin, ccthromycin, solithromycin, tacrolimus, pimecrolimus, sirolimus, amphotericin B, nystatin, and cruentaren; sulfonamides such as sulfonamide, sulfacetamide, sulfadiazine, sulfadimidine, sulfafurazole (sulfisoxazole), sulfisomidine (sulfaisodimidine), sulfamethoxazole, sulfamoxole, sulfanitran, sulfadimethoxine, sulfamethoxypyridazine, sulfametoxydiazine, sulfadoxine, sulfametopyrazine, and terephtyl; aminoglycosides such as kanamycin A, amikacin, tobramycin, dibekacin, gentamicin, sisomicin, netilmicin, neomycins B, C, neomycin E (paromomycin), streptomycin, plazomicin, amikin, garamycin, kantrex, neo-fradin, netromycin, nebcin, humatin, spectinomycin(Bs), and trobicin; carbapenems such as imipenem, meropenem, ertapenem, doripenem, panipenem / betamipron, biapenem, tebipenem, razupenem (PZ-601), lenapenem, tomopenem, and thienamycin (thienpenem); ansamycins such as geldanamycin, herbimycin, rifaximin, and xifaxan; carbacephems such as loracarbef and lorabid; carbapenems such as ertapenem, invanz, doripenem, doribax, imipenem / cilastatin, primaxin, meropenem, and merrem; glycopeptides such as teicoplanin, targocid, vancomycin, vancocin, telavancin, vibativ, dalbavancin, dalvance, oritavancin, and orbactiv; lincosamides such as clindamycin, cleocin, lincomycin, and lincocin; lipopeptides such as daptomycin and cubicin; macrolides such as azithromycin, zithromax, sumamed, xithrone, clarithromycin, biaxin, dirithromycin, dynabac, erythromycin, erythocin, erythroped, roxithromycin, troleandomycin, tao, telithromycin, ketek, spiramycin, androvamycine; monobactams such as aztreonam and azactam; nitrofurans such as furazolidone, furoxone, nitrofurantoin, macrodantin, and macrobid; oxazolidinones such as linezolid, zyvox, vrsa, posizolid, radezolid, and torezolid; polypeptides such as bacitracin, colistin, coly-mycin-S, and polymyxin B; drugs against mycobacteria such as clofazimine, lamprcnc, dapsonc, avlosulfon, capreomycin, capastat, cycloserine, seromycin, ethambutol, myambutol, ethionamide, trecator, isoniazid,pyrazinamide, aldinamide, rifampicin, rifadin, rimactane, rifabutin, mycobutin, rifapentine, priftin, and streptomycin; and other antibiotics such as arsphenamine, salvarsan, chloramphenicol, Chloromycetin, fosfomycin, monurol, monuril, fusidic acid, fucidin, metronidazole, flagyl, mupirocin, bactroban, platensimycin, quinupristin / dalfopristin, synercid, thiamphenicol, tigecycline, tigacyl, tinidazole, tindamax fasigyn, trimethoprim, proloprim, and trimpex; In some exemplary embodiments, the perfusate comprises ceftriaxone, gentamicin, penicillin, streptomycin, or a combination thereof.

[0130] In some embodiments, the perfusate comprises one or more hematopoietic growth factors. Examples of hematopoietic growth factors include: erythropoietin, thrombopoietin, insulin-like growth factor (IGF)-1, granulocyte colony-stimulating factor (G-CSF), granulocytemacrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M- CSF), stem cell factor (SCF), and intcrlcukin-3 (IL-3).

[0131] In some embodiments, the perfusate comprises an immunosuppressive agent. Examples of immunosuppressive agents include: glucocorticoids such as, but not limited to, prednisone, methylprednisolone, dexamethasone, and hydrocortisone; antimetabolite / proliferative agents such as, but not limited to, azathioprine, cyclophosphamide, mycophenolate mofetil, and mycophenolate sodium; and other immunosuppressive agents such as tacrolimus, everolimus, sirolimus, and cyclosporine.

[0132] Cytokines including interleukins such as IL- la, IL-2, IL-6, IL10, and IL- 18, and other cytokines such as granulocyte colony-stimulating factor (GCSF), MCP1, and TGF-P may accumulate in a perfusate during perfusion of an organ or tissue. Production of pro-inflammatory cytokines is linked to poor graft outcomes. Therefore, it may be beneficial to use a filter to adsorb cytokines in the perfusate during perfusion.

[0133] In some embodiments, the acellular oxygen-carrying perfusate is oxygenated with a gas mixture having 1% to 99% oxygen, 50% to 99% oxygen, 80% to 99% oxygen, or 95% to 99% oxygen, or any amount of oxygen in these ranges such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 32%, 34%, 36%, 38%, 40%, 42%, 44%, 46%, 48%, 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% oxygen. In some embodiments, the acellular oxygen-carrying perfusate is deoxygenated, for example, for storage to increase its shelf-life.Methods of Perfusion

[0134] An acellular oxygen-carrying perfusate comprising LtEc can be used for organ perfusion, including ex vivo organ perfusion or in situ organ perfusion. In some embodiments, the methods are used for ex vivo preservation of an organ or composite tissue allografts, for example, wherein an organ or tissue is removed from a donor and preserved during storage and / or transport prior to implantation in a recipient. In other embodiments, the methods are used for autotransplantation, wherein an organ or tissue is temporarily removed for ex vivo therapy (such as surgical repair, resection of a tumor, radiation therapy, chemotherapy, or gene therapy), followed by reimplantation. In further embodiments, the methods can be used to preserve, resuscitate, or enhance ex vivo organ function, for example, prior to transplantation, or during isolation of cells or tissue from an organ for further use (such as transplantation of cells or tissue into a recipient).

[0135] An acellular oxygen-carrying perfusate comprising LtEc 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, bladder, or gonad, or any portion thereof. In some embodiments the oxygen-carrying perfusate comprising LtEc is used for perfusion of more than one organ in combination such as, but not limited to, heart and lung, heart and kidney, heart and liver, liver and kidney, liver and heart, liver and lung, or liver, small bowel, and pancreas. As used herein, the term "organ" also includes tissues, such as tissue allografts or composite tissue allografts (such as a finger, hand, arm, toe, foot, leg, face, or portion thereof). The organ can be obtained from an individual of any age, such as an adult, child, or infant. In certain embodiments, the organ is obtained from a live organ donor or an organ donor after circulatory death. In some embodiments, an entire organ may undergo perfusion with the perfusate. In other embodiments, a portion of an organ or a tissue graft may undergo perfusion with the perfusate.

[0136] 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. Tn some cases, the ability of ex vivo perfusion to extendthe time donor organs are viable allows donor organs to be transported further distances to reach a recipient for a transplant.

[0137] Any suitable perfusion device may be used with an acellular oxygen-carrying perfusate comprising LtEc for perfusion of an organ or tissue. Exemplary perfusion devices arc available from Organ Assist, Groningen, Netherlands (such as Kidney Assist or Liver Assist), Organ Recovery Systems, Itasca, 111. (such as LifePort kidney transporter or liver transporter), Transmedics, Andover, Mass, (such as the heart or lung Organ Care System), OrganOx, Oxford, UK (such as OrganOx Metra), and XVIVO Perfusion Engelwood, Colo. Exemplary devices and systems are also described in U.S. Patent Nos. 6,994,954; 6,953,655; 6,977,1420; 7,678,563; 7,811,808; 7,897,357; 8,268,547; 8,268,612; 8,287,580; 8,535,934; 8,741,555, 8,802,425; 8,835,158; 9,301,519; 9,357,764; 9,426,979; 10,076,112; 11,963,526; 11,856,944; 11,576,371; and 11,528,903; all of which are incorporated herein by reference in their entireties. It will be understood by one of ordinary skill in the art that additional organ perfusion devices or systems may be used with an acellular oxygen-carrying perfusate comprising LtEc, as described herein.

[0138] Perfusion devices may include one or more chambers for holding an organ, one or more reservoirs for holding a perfusate (such as an acellular oxygen-carrying perfusate comprising LtEc, as disclosed herein), and one or more pumps (for example, one or more rotary pumps or peristaltic pumps) for delivery of the perfusate to the organ. Such devices also include one or more means to regulate temperature of the perfusate, such as one or more heat exchangers or thermoelectric temperature controllers, and one or more means to oxygenate the perfusate (such as an oxygenator in the perfusion circuit).

[0139] In some embodiments, the perfusion device includes one or more ports for introducing additional substances into the perfusion fluid and / or for sampling the perfusion fluid during perfusion of an organ. The device may also include one or more displays which provide information regarding perfusion pressure, flow, temperature, and other information. Thus, in some embodiments, the disclosed methods include monitoring characteristics of the perfusion fluid during machine perfusion of an organ (such as pH, pCL, pCCh, oxygen saturation, mitochondrial function, lactate production, enzyme function, bile production, urine production, and so on). The parameters measured may depend on the organ being perfused, for example, bile production and liver function indicators (such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin levels) can be monitored if the organ is a liver, while urine production can be measured if the organ is a kidney.

[0140] The perfusate may be delivered to an organ using one or more cannulas which are inserted in a vessel of the organ (such as an artery or vein). The choice of the appropriate vesselwill depend on the type of organ undergoing perfusion. For example, for a kidney, a cannula may be inserted in the renal artery. For a liver, a cannula may be inserted in the hepatic artery and / or the portal vein. For a heart, one or more cannulas may be inserted in coronary arteries. For a lung, one or more cannulas may be inserted in pulmonary arteries. For a tissue graft, a cannula may be inserted in an artery in the tissue graft. In some embodiments, the perfusate exits an organ from one or more veins. In some embodiments, perfusate is pumped through a perfusion circuit, wherein oxygenated perfusate flows into a cannulated artery of an organ, and deoxygenated perfusate, exiting the organ, flows out of a cannulated vein and is then reoxygenated by an oxygenator in the perfusion circuit before recirculating through the organ. In some embodiments, the methods include passive venous drainage into a perfusion reservoir. In some embodiments, a catheter is inserted in a vein, for example for selective collection of fluid samples. The method can also include sample collection through side ports in the perfusion device (for example, for random sample collection).

[0141] In some embodiments, the flow of the perfusate to the organ is a continuous flow, such as a flow without substantial variations of flow rate, for example, to mimic venous blood flow under most physiologic conditions. In other embodiments, the flow of the perfusate to the organ is a pulsatile flow, such as having flow rate variations that mimic arterial pulsatile blood flow. For example, flow of the perfusate through a cannula inserted in an artery of the organ or tissue graft may be pulsatile. In some examples, the pulsatile flow of the perfusate has a pulse of about 50-70 beats per minute (such as about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 beats per minute); however, one of ordinary skill in the art can select an alternative pulse rate based on the type and condition of organ that is being perfused.

[0142] In some examples, a dual perfusion technique is used, wherein the organ is perfused using simultaneous pulsatile and continuous flow. For example, the liver has two different blood supplies; the hepatic artery, which carries oxygenated blood from the circulatory system and the hepatic portal vein, which carries blood from the gut to the liver. Accordingly, pulsatile flow may be used for perfusion of a liver through the hepatic artery, and continuous (or non-pulsatile) flow may be used for perfusion of the same liver through the portal vein. In some embodiments, the perfusion pressure through the hepatic artery is about 10-25 mm Hg (such as about 12-25 or about 15-20 mm Hg) with a pulse of about 50-70 beats per minute (about 55-65 or about 60 beats per minute) and an amplitude of about 20%. The perfusion through the portal vein is about 2-5 mm Hg, such as about 2-4 or 3-4 mm Hg. In some embodiments, the hepatic artery flow is about 50- 150 ml / min (such as about 50-125, 60-1 10, 75-100, 85-95, or about 90 ml / min) and the portal vein flow is about 200-300 ml / min (such as about 225-275, 230-270, 240-265, or about 260 ml / min)One of ordinary skill in the art can select appropriate pressures and flows for perfusion of the liver or other organs.

[0143] In certain embodiments, ex vivo perfusion is performed 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.

[0144] 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. Tor 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. Alternatively, the urine may be recirculated back into the oxygen carrying fluid. In certain embodiments, the arterial line tubing is connected to the renal artery and the venous line tubing 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(l):39-54; Krezdom et al. (2017) Innovative Surgical Sciences. 2(4):171-187; Birnbaum et al. (2004) Eur. I. 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.

[0145] A temperature controller can be used to maintain the container holding the organ and / or the perfusate in the perfusion circuit at a desired temperature. The acellular oxygen-carrying perfusate comprising LtEc can be used to perform perfusion at any temperature, including hypothermic perfusion, mid-thermic perfusion, sub-normothermic perfusion, or normothermic machine perfusion. In some embodiments, a heating element is used to maintain the temperature such as, but not limited to, a water bath, 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. In some embodiments, the temperature of the organ and / or the chamber holding the perfused organ is maintained at the same temperature as the perfusate, for example by means of one or more temperature controller units connected to the organ chamber.

[0146] For normothermic 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, from 30 °C to 40 °C, or 35.5 °C to 40 °C, including any temperature within these ranges such as 20 °C, 20.5 °C, 21 °C, 21.5 °C, 22 °C, 22.5 °C, 23 °C, 23.5 °C, 34 °C,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 °C, 30 °C,30.5 °C, 31 °C, 31.5 °C, 32 °C, 32.5 °C, 33 °C, 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.

[0147] For hypothermic perfusion, an organ is kept at colder temperatures. In some embodiments, the temperature ranges from 1 °C to 12 °C, 4 °C to 12 °C, or 4 °C to 8 °C, including any temperature within these ranges such as 1 °C, 1 .5 °C, 2 °C, 2.5 °C, 3 °C, 3.5 °C, 4 °C, 4.5 °C, 5 °C, 5.5 °C, 6 °C, 6.5 °C, 7 °C, 7.5 °C, 8 °C, 8.5 °C, 9 °C, 9.5 °C, 10 °C, 10.5 °C, 11 °C, 11.5 °C, or 12 °C.

[0148] In some embodiments the temperature of the perfusate ranges from 12 °C to 37 °C, such as about 12 °C to 30 °C, 20 °C to 32 °C, 20 °C to 25 °C, 12 °C to 28 °C, 12 °C to 25 °C, 12 °C to 21 °C, 15 °C to 25 °C, 15 °C to 22 °C, 15 °C to 21 °C, 15 °C to 20 °C, or 20 °C to 22 °C. In some embodiments, a sub-normothermic temperature (such as about 20 °C to 32 °C is selected for the perfusate. Decreasing temperature may reduce the risk of infection by slowing or inhibiting bacterial growth. In some examples, the temperature of the perfusate is about 12 °C, 13 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 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, 34 °C, 35 °C, 36 °C, or 37 °C.

[0149] Any suitable pump may be used to pump perfusate through the perfusion circuit. Exemplary pumps include, without limitation, centrifugal pumps, roller pumps, peristaltic pumps, non-pulsatile gear pumps, diaphragm 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 fordelivery 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.

[0150] 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.

[0151] An infusion container can be used to add an agent to the perfusate, including nutrients, electrolytes, osmotic agents, therapeutic agents, 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 an 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.

[0152] In certain embodiments, a recombinant nucleic acid 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).

[0153] 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. 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. Methods of genetically modifying an organ or performing gene therapy are described in further detail below.Additional Applications for the Use of LtEc as a Blood Substitute

[0154] An acellular oxygen-carrying composition comprising LtEc can also be used as a blood substitute for transfusion, including transfusion of subjects of any blood type (i.e., an acellular oxygen-carrying composition comprising LtEc can be used as a universal blood substitute for transfusions). Transfusion with an acellular oxygen-carrying composition comprising LtEc can be used, for example, to treat subjects having acute blood loss from trauma, a surgical procedure, or a hemorrhage: subjects having anemia, including subject with chronic anemia from a nutrient deficiency, blood dyscrasias such as thalassemis, a malignancy, or a genetic disorder; or subjects unable or unwilling to receive a transfusion with a human blood product, for example, because of religious beliefs (e.g., Jehovah's Witnesses) or allergies or immune reactions to human blood products.

[0155] An acellular oxygen-carrying composition comprising LtEc can also be used for treating ischemia by infusion or perfusion of ischemic tissue. In certain embodiments, the ischemiais caused by a stroke, a transient ischemic attack, a myocardial infarction, acute limb ischemia, or ischemic bowel syndrome.

[0156] An acellular oxygen-carrying composition comprising LtEc can also be used for “liquid hyperbaric oxygen therapy.” The acellular oxygcn-carrying perfusate can be oxygenated in a hyperbaric oxygen chamber in order to deliver high pressures of oxygen to an affected tissue, limb, organ, or organism. In certain embodiments, liquid hyperbaric oxygen therapy is performed by infusing an acellular oxygen-carrying perfusate comprising LtEc into a tissue, limb, organ, or organism to treat a subject for necrotizing fasciitis, gas gangrene, hemorrhagic cystitis, poor wound healing, a skin graft, or a thermal or radiation burn.

[0157] In addition, an acellular oxygen-carrying composition comprising LtEc can be used to increase delivery of oxygen to a tumor to improve the effectiveness of radiation therapy. Resistance to radiation therapy is often a problem when tumors are hypoxic. Without being bound by theory, exposure of oxygen to radiation generates free radicals that kill cancer cells. When oxygen levels in a tumor are low, radiation therapy is less effective. Oxygen can be delivered to a tumor prior to or during radiation therapy to overcome resistance of a hypoxic tumor to radiation therapy.

[0158] In another aspect, a method of supporting cellular respiration in a cell or tissue is provided, the method comprising delivering an acellular oxygen-carrying perfusate comprising LtEc to the cell or tissue. In certain embodiments, the acellular oxygen-carrying perfusate is delivered to the cell or tissue in vitro, ex vivo, or in vivo.Genetically Modifying an Organ

[0159] 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 or convert a disease associated allele to a normal wild-type allele. 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 (CRISPRj / 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) usinga 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.

[0160] 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.

[0161] 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.

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] 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 orrecombinantly 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_03391 308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus lorquisl (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(10):6091-6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[0167] 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.

[0168] 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 nucleotideswill 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.

[0169] 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 tracrRNA 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.

[0170] In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpfl) also referred to as CRISPR associated protein 12a (Casl2a) may be used. Casl2ais 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. Cas 12a 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.

[0171] 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.

[0172] 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.

[0173] 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-guidcd 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.

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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 ct 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.

[0178] 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. 201 1 Jul. 15; 333(6040):307; Tesson et al. Nat Biotechnol. 201 1 Aug. 5; 29(8):695-6; and Huang et. al., Nat Biotechnol. 2011 Aug. 5; 29(8):699-700; all of which areherein 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

[0179] 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, nanoparticlc-mcdiatcd nucleic acid delivery, and the like.

[0180] 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) PLoSel 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. Sei. USA 105(50): 19821-19826.

[0181] 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.

[0182] 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.

[0183] 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.

[0184] 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).

[0185] 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 tetracyclinetransactivator 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 mctallothioncin (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 (B TH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light responsive promoters from plant cells).

[0186] 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 myofibcr), 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.

[0187] 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 arc 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 Ther., 4:432-441 ; Neering et al., 1996, Blood, 88:1147-55; and Rendahl et al., 1998, Nat. Biotechnol., 16:757-761).

[0188] 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.

[0189] 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 (sec, e.g., GcnBank HUMNFL, L04147); a synapsin promoter (sec, e.g., GcnBank HUMSYNIB, M55301); a thy-1 promoter (see, e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn et al. (2010) Nat. Med. 16:1 161); 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 Nefh 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.

[0190] 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 (sec, e.g., Ai et al. (2018) Cell Physiol. Biochcm. 48(5): 1894-1900), a troponin 2 (TNNT2) promoter (see, e.g., Fiedorowicz et al. (2020) Sci. Rep.lO(l): 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).

[0191] 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).

[0192] 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 typc-spccific 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.

[0193] 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 S V40, as described in Sambrook et al., supra, as well as a bovine growth hormone terminator sequence.

[0194] 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.

[0195] 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 moreopen reading frames from a vector. For example, a therapeutic protein or RNA can be coexpressed from a multici stronic vector including an IRES element. The IRES element attracts a eukaryotic ribosomal translation initiation complex and promotes translation initiation. See, e.g., Kaufman ct al., Nuc. Acids Res. (1991) 19:4485-4490; Gurtu ct 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 picorna viruses 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(5):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 ct 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.

[0196] 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 vims, Thosea asigna vims and porcine tescho vims- 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(1 1):864-873; herein incorporated by reference in their entireties.

[0197] 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 biolumincsccnt markers (c.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.

[0198] 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.

[0199] 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 destraction at will. See, e.g., .Tones 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.

[0200] 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 areknown 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.

[0201] 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 vims; human immunodeficiency vims (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 lentivims, ay-retrovirus such as murine leukemia vims and feline leukemia vims, an avian retrovims such as spleen necrosis vims, and vectors derived from retroviruses such as Rous Sarcoma Vims, Harvey Sarcoma Vims, avian leukosis vims, human immunodeficiency vims, myeloproliferative sarcoma vims, and mammary tumor vims); and the like. See also, e.g., Warnock et al. (2011) Methods Mol. Biol. 737: 1-25; Walther et al. (2000) Drags 60(2):249-271 ; and Lundstrom (2003) Trends Biotechnol. 21(3): 117- 122; herein incorporated by reference).

[0202] For example, retrovimses 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 vims 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). Lentivimses are a class of retrovimses 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) Vimses 3(2): 132- 159; herein incorporated by reference).

[0203] 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 arc 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).

[0204] 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; Bctt ct al., J. Virol. (1993) 67:5911-5921; Mittcrcdcr ct 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 vims (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.

[0205] 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).

[0206] 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 vims and avian poxvims. By way of example, vaccinia vimsrecombinants 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 arc 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.

[0207] 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.

[0208] Molecular conjugate vectors, such as the adenovirus chimeric vectors described in Michael ct aL, J. Biol. Chcm. (1993) 268:6866-6869 and Wagner ct al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.

[0209] 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.

[0210] 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 templatesbearing 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 of large 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.

[0211] 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 tcmplatc(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.

[0212] 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.

[0213] 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 ofplasmid 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.

[0214] Cationic liposomes arc readily available. For example, N[l-2,3- dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from G1BCO 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 / 11092 for a description of the synthesis of DOTAP (l,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

[0215] 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.

[0216] 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 al., 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.

[0217] 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.

[0218] 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) andpoly(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 ll(2):149-54, 1993.

[0219] 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, polyornithine, 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.

[0220] 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.).

[0221] 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 detemiined through routine trials.

[0222] 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, pHbuffering 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.

[0223] Methods for the delivery of nucleic cells to cells arc known in the art and can include, e.g., dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, lipofectamine and LT-1 mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.

[0224] 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.Examples of Non-Limiting Aspects of the Disclosure

[0225] 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 1-78 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. An acellular oxygen-carrying perfusate comprising Lumbricus terrestris erythrocruorin (LtEc).2. The acellular oxygen-carrying perfusate of aspect 1, wherein the LtEc is at a concentration in a range from 0.1 g / dL to 2.5 g / dL.3. The acellular oxygen-carrying perfusate of aspect 2, wherein the LtEc is at a concentration of about 0.1 g / dL, about 0.5 g / dL, or about 2.5 g / dL.4. The acellular oxygen-carrying perfusate of any one of aspects 1-3, wherein the LtEc comprises: a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:1; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:3: a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:5; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:7: a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:9; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 11 ; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:13; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:15; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 17; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:21; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:23; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:25; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:27; and a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:29.5. The acellular oxygen-carrying perfusate of any one of aspects 1-4, wherein the LtEc is cross-linked with a cross-linking agent such that subunits of LtEc do not dissociate.6. The acellular oxygcn-carrying perfusate of any one of aspects 1-5, further comprising one or more of nutrients, electrolytes, reducing agents, antibiotics, anticoagulants, osmotic agents, colloids, diuretics, vasodilators, insulin, growth factors, immunosuppressive agents, or a combination thereof.7. The acellular oxygen-carrying perfusate of aspect 6, wherein the nutrients comprise carbohydrates, amino acids, nucleotides, fatty acids, vitamins, or a combination thereof.8. The acellular oxygen-carrying perfusate of aspect 7, wherein the amino acids comprise alanine, aspartate, asparagine, glycine, glutamate, glutamine, lysine, arginine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, isoleucine, leucine, valine, histidine, cysteine, proline, or a combination thereof.9. The acellular oxygen-carrying perfusate of aspect 7, wherein the fatty acids comprise oleic acid, linoleic acid, palmitic acid, stearic acid, or a combination thereof.10. The acellular oxygen-carrying perfusate of aspect 7, wherein the carbohydrates comprise glucose.11. The acellular oxygen-carrying perfusate of aspect 7, wherein the nucleotides comprise adenosine and its metabolites like adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP).12. The acellular oxygen-carrying perfusate of aspect 6, wherein the electrolytes comprise sodium (Na+), chloride (Cl ), potassium (K+), calcium (Ca2+) magnesium (Mg2+), bicarbonate (HCCh ), acetate, citrate, or a combination thereof.13. The acellular oxygen-carrying perfusate of aspect 12, wherein the acellular oxygen-carrying perfusate comprises Ca2+.14. The acellular oxygen-carrying perfusate of aspect 6, wherein the reducing agents comprise ascorbic acid, glutathione, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine, or a combination thereof.15. The acellular oxygen-carrying perfusate of aspect 6, wherein the osmotic agents comprise mannitol, glycerol, sorbitol, dextrose, or ribose.16. The acellular oxy gen-carrying perfusate of aspect 6, wherein the colloids comprise albumin, dextran, hydroxyethyl starch (HES), or a combination thereof.17. The acellular oxy gen-carrying perfusate of aspect 6, wherein the vasodilators comprise nitroglycerin, hydralazine, or sodium nitroprusside.18. The acellular oxygen-carrying perfusate of aspect 6, wherein the antibiotics comprise ceftriaxone, gentamicin, penicillin, streptomycin, or a combination thereof.19. The acellular oxy gen-carrying perfusate of aspect 6, wherein the growth factors comprise one or more hematopoietic growth factors or interleukins, or a combination thereof.20. The acellular oxy gen-carrying perfusate of aspect 1 , wherein the hematopoietic growth factors comprise erythropoietin, thrombopoietin, insulin- like growth factor (IGF)-l, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), interleukin-3 (IL-3), or a combination thereof.21. The acellular oxy gen-carrying perfusate of any one of aspects 1-20, further comprising a crystalloid solution.22. The acellular oxygen-carrying perfusate of aspect 21, wherein the crystalloid solution is saline or Ringer's lactate solution.23. The acellular oxygen-carrying perfusate of any one of aspects 1-22, wherein the LtEc is oxygenated.24. The acellular oxygen-carrying perfusate of any one of aspects 1-23, further comprising a therapeutic agent.25. The acellular oxygcn-carrying perfusate of aspect 24, wherein the therapeutic agent is a drug, a gene therapy agent, a gene editing agent, a chemotherapeutic agent, a radiotherapeutic agent, or a cell.26. A method for perfusion of an organ comprising perfusing the organ with the acellular oxygen-carrying perfusate of any one of aspects 1-25.27. The method of aspect 26, wherein the perfusion is in situ or ex vivo normothermic organ perfusion, mid-thermic organ perfusion, sub-normothermic organ perfusion, or hypothermic organ perfusion.28. The method of aspect 26, wherein temperature of the acellular oxygen-carrying perfusate is maintained in a range from 20 °C to 40 °C, 1 °C to 10 °C, 13 °C to 24 °C, or 25 °C to 34 °C during perfusion.29. The method of aspect 26, wherein temperature of the organ or a container holding the organ is maintained in a range from 20 °C to 40 °C, 1 °C to 10 °C, 13 °C to 24 °C, or 25 °C to 34 °C during perfusion.30. The method of any one of aspects 26-29, wherein the organ is a kidney, a heart, a liver, a lung, a stomach, a small intestine, a large intestine, a pancreas, a gonad, a limb, an extremity, or a tissue graft or a portion thereof.31. The method of any one of aspects 26-30, wherein the organ is obtained from a live organ donor or an organ donor after circulatory death.32. The method of any one of aspects 26-31, wherein the acellular oxygen-carrying perfusate is oxygenated with a gas mixture having 1 % to 99% oxygen.33. The method of any one of aspects 26-31 , wherein the acellular oxygen-carrying perfusate is oxygenated with a gas mixture having 95% to 99% oxygen.34. The method of any one of aspects 26-31, wherein the acellular oxygen-carrying perfusate is deoxygenated for storage.35. The method of any one of aspects 26-32, further comprising adding a therapeutic agent or nutrient to the acellular oxygen-carrying perfusate.36. The method of aspect 35, wherein the therapeutic agent or nutrient is heparin, prostacycline, glucose, insulin, a bile salt, an amino acid, a fatty acid, a lipid, a vitamin, a mineral, a hormone, a cytokine, a steroid, a diuretic, a vasoactive molecule, an antibiotic, an antibody, or a cell.37. The method of aspect 35, wherein the therapeutic agent is a recombinant nucleic acid or gene editing system.38. The method of aspect 37, wherein the recombinant nucleic acid or gene editing system is encapsulated in a lipid nanoparti clc (LNP).39. The method of aspect 37 or 38, wherein the recombinant nucleic acid is a DNA or RNA encoding a therapeutic protein or regulatory RNA.40. The method of any one of aspects 37-39, wherein the recombinant nucleic acid comprises a viral vector or plasmid.41. The method of aspect 40, wherein the viral vector is an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.42. The method of aspect 40 or 41 , wherein expression of the therapeutic protein or regulatory RNA is inducible.43. The method of any one of aspects 39-42, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of a therapeutic protein.44. The method of aspect 37, wherein 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).45. The method of any one of aspects 37-44, wherein the recombinant nucleic acid or gene editing system is delivered in the perfusate to the organ.46. The method of any one of aspects 35-45, wherein the therapeutic agent is administered locally to a site on the organ during ex vivo perfusion of the organ.47. The method of any one of aspects 35-46, wherein the therapeutic agent is toxic when administered to a subject in vivo.48. The method of aspect 47, wherein the therapeutic agent is a gene therapy agent, a chemotherapeutic agent, or a radiotherapeutic agent.49. The method of any one of aspects 26-48, further comprising measuring a level of a biomarker in a sample of the perfusate to determine fitness of the organ for transplant.50. The method of any one of aspects 26-49, further comprising genetically modifying the organ during perfusion.51. The method of aspect 50, wherein said genetically modifying the organ comprises converting a disease-associated allele to a wild-type allele.52. The method of any one of aspects 26-51, further comprising resecting a tumor or surgically repairing the organ prior to transplantation into a recipient.53. The method of any one of aspects 26-52, further comprising measuring temperature of the acellular oxygen-carrying perfusate, flow-rate of the acellular oxygencarrying perfusate, pH of the acellular oxygen-carrying perfusate, concentration of oxygen in the acellular oxygen-carrying perfusate, concentration of glucose in the acellular oxygen-carrying perfusate, concentration of LtEc in the acellular oxygen-carrying perfusate, concentration ofsodium in the acellular oxygen-carrying perfusate, concentration of potassium in the acellular oxygen-carrying perfusate, concentration of calcium in the acellular oxygen-carrying perfusate, concentration of carbon dioxide in the acellular oxygen-carrying perfusate, percent saturation of oxygen in the acellular oxygcn-carrying perfusate, or concentration of lactate in the acellular oxygen-carrying perfusate, or any combination thereof.54. A method for transfusion of a subject comprising transfusing the subject with the acellular oxygen-carrying perfusate of any one of aspects 1-25.55. The method of aspect 54, wherein the subject has any blood type.56. The method of aspect 54 or 55, wherein the subject has acute blood loss from trauma, a surgical procedure, or a hemorrhage.57. The method of aspect 54 or 55, wherein the subject has chronic anemia from a nutrient deficiency, a malignancy, or a genetic disorder.58. The method of any one of aspects 54-57, wherein the subject is unable or unwilling to receive a transfusion with a human blood product.59. A method of performing liquid hyperbaric oxygen therapy, the method comprising infusing the acellular oxygen-carrying perfusate of any one of aspects 1-25 into a tissue, limb, organ, or organism in need of the liquid hyperbaric oxygen therapy, wherein the acellular oxygen-carrying perfusate is oxygenated in a hyperbaric oxygen chamber.60. The method of aspect 59, wherein the liquid hyperbaric oxygen therapy is used to treat a subject for necrotizing fasciitis, gas gangrene, hemorrhagic cystitis, poor wound healing, a skin graft, or a thermal or radiation burn.61. A method of treating ischemia in a subject, the method comprising infusing the acellular oxygen-carrying perfusate of any one of aspects 1-25 into an ischemic tissue in the subject.62. The method of aspect 61, wherein the acellular oxygen-carrying perfusate is oxygenated with a gas mixture having 1 % to 99% oxygen.63. The method of aspect 61, wherein the acellular oxygcn-carrying perfusate is oxygenated with a gas mixture having 95% to 99% oxygen.64. The method of any one of aspects 61-63, wherein the ischemia is caused by a stroke, a transient ischemic attack, a myocardial infarction, acute limb ischemia, or ischemic bowel syndrome.65. A method of supporting cellular respiration in a cell or tissue, the method comprising delivering the acellular oxygen-carrying perfusate of any one of aspects 1-25 to the cell or tissue.66. The method of aspect 65, wherein the acellular oxygen-carrying perfusate is delivered to the cell or tissue in vitro, ex vivo, or in vivo.67. A method of performing radiation therapy of a tumor, the method comprising: delivering the acellular oxygen-carrying perfusate of any one of aspects 1-25 to the tumor; and administering the radiation therapy to the tumor.68. The method of aspect 67, wherein the tumor is hypoxic.69. The method of aspect 67 or 68, wherein the tumor has previously been resistant to radiation therapy.70. The method of any one of aspects 67-69, wherein said delivering is performed prior to or during radiation therapy.71. The method of any one of aspects 67-70, wherein said delivering is performed by infusion, perfusion, or injection locally into the tumor.72. A system comprising:a perfusion machine comprising a reservoir, wherein the reservoir comprises the acellular oxygen-carrying perfusate of any one of aspects 1-25; and an oxygenator, wherein the oxygenator oxygenates the acellular oxygen-carrying perfusate.73. The system of aspect 72, further comprising a container for holding an organ or tissue.74. The system of aspect 72 or 73, wherein the perfusion machine performs normothermic organ perfusion, mid-thermic organ perfusion, sub-normothermic organ perfusion, or hypothermic organ perfusion.75. The system of any one of aspects 72-74, wherein the perfusion machine performs perfusion in situ or ex vivo.76. The system of any one of aspects 72-75, further comprising a temperature controller, wherein the temperature controller maintains the acellular oxygon-carrying perfusate at a desired temperature.77. The system of aspect 76, wherein the temperature controller maintains the temperature of the acellular oxygen-carrying perfusate or the container holding the organ or tissue in a range from 20 °C to 40 °C, 1 °C to 10 °C, 13 °C to 24 °C, or 25 °C to 34 °C during perfusion.78. The system of aspect 76 or 77, wherein the temperature controller is a water bath, an ice bath, a thermoelectric temperature controller, or a heat exchanger.EXAMPLES

[0226] 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 tonumbers 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: DEVELOPING AN ACELLULAR BLOOD SUBSTITUTE IN A NORMOTHERMIC EX VIVO KIDNEY PERFUSION MODENormothermic ex vivo kidney perfusion (NEVKP) technology:

[0227] NEVKP is an emerging area of innovation that promises to transform existing paradigms in kidney disease. NEVKP has many clinical and translational applications, ranging from preserving donor kidneys to testing novel kidney-directed therapeutics.1 6By sustaining the kidney’s complex biology in a controlled setting for an extended period of time, NEVKP can also be a valuable tool for basic investigation that transcends the limitations of in vivo and in vitro models.7 1(1However, while normothermic ex vivo organ perfusion devices have been developed for the heart, lung, and liver, NEVKP technology is not as mature and is typically limited to short durations (>24 hr).11

[0228] One critical barrier to extending the duration of NEVKP is the current reliance on red blood cells (RBC) for oxygen transport in the perfusate.11,12In addition to being costly to obtain and difficult to store, RBCs rapidly degrade in mechanical circuits.13-16The breakdown of hemoglobin (Hb) leads to oxidative stress, induction of tubular apoptosis, and other side effects associated with the release of free hemoglobin. These toxic degradation products are acutely damaging to the kidney.17-20We found that the upper limits of NEVKP duration were linked to RBC integrity, and additional challenges of using RBCs included heterogeneous quality, allogeneic compatibility issues, and risk of infectious contamination.21-24

[0229] There is a great need to develop an alternative to red blood cells as a life-sustaining oxygen carrier due to the scarcity of banked red blood cells (RBCs), their limited lifespan in storage, and contraindications that prohibit their use in various settings. Prior and ongoing attempts to use hemoglobin-based oxygen carriers (HBOCs) such as polymerized or lipid- encapsulated hemoglobin have had muted success to date as these products are unstable and costly to produce.

[0230] Early studies showed that pure hemoglobin tetramers rapidly dissociate after transfusion and extravasate into tissues to induce renal toxicity and other complications. Most modern HBOCs are thus cross-linked in some way to prevent their dissociation while preserving their ability to transport oxygen. For example, polymerized bovine hemoglobin (polybHb; alsoknown as HBOC-201) has been used for NEVKP with both human and porcine kidneys for up to 6 hours. These experiments showed that the performance of polybHbs was comparable to RBCs.25,26However, clinical trials using HBOC-201 in vivo have revealed significant disadvantages such as autoxidation and NO scavenging.27Specifically, the iron within the heme of mammalian Hbs is prone to autoxidation, which leads to the generation of harmful reactive oxygen species (e.g., O2- and H2O2) that induce oxidative stress and tissue damage.28Hbs also react with NO in the bloodstream to form nitrate (Figure I).28Since NO is a gaseous hormone that promotes vasodilation in vivo, this NO scavenging reaction induces severe vasoconstriction. These reactions also oxidize the heme iron and eliminate oxygen transport, thereby limiting the effectiveness and duration of HBOCs in NEVKP. To address this problem, newer products like Erythromer™ encapsulate the Hb inside a liposome to block interactions with NO in the bloodstream, but they have yet to be rigorously evaluated in vivo.29

[0231] To overcome this challenge, we have identified Lumbricus terrestris erythrocruorin (LtEc), the oxygen-carrying molecule of the common earthworm, as an alternative material given its molecular stability, favorable oxygen-binding profile, and natural abundance. L. terrestris erythrocruorin (LtEc) has many properties suggesting it would be an ideal oxygen carrier for NEVKP. Having evolved to transport oxygen in an extracellular environment, LtEc is molecularly stable in a wide range of temperatures, has a low oxidation rate, and does not scavenge nitrous oxide (NO), an important vasodilator. Its size (30 nm) is favorable for perfusing the kidney as it would likely not get lost to glomerular filtration (6-8 nm cutoff) but remain able to perfuse vasoconstricted capillaries that are inaccessible to RBCs (8 pm). Together with preliminary results from our lab demonstrating that LtEc can sustain respiration in NEVKP, these properties strongly justify investigating the utility of LtEc in NEVKP.

[0232] We use a normothermic ex vivo kidney perfusion (NEVKP) model to evaluate the organ-sustaining properties of LtEc and optimize a formulation for kidney perfusion. An LtEc- based blood substitute is being developed for organ perfusion with the eventual goal of using it for life-saving transfusions.Introduction to LtEc.

[0233] While mammals evolved red blood cells to protect their hemoglobin, most annelids secrete their naturally acellular hemoglobins (i.e., erythrocruorins, Ecs) directly into the bloodstream. Consequently, Ecs have evolved to avoid the oxidation, NO scavenging, and other issues that hinder other HBOCs.30For example, while mammalian hemoglobins assemble into tetramers via electrostatic interactions, the erythrocruorin of the common earthworm Lumbricusterrestris (LtEc) is an assembly of 144 globins held together by a network of intra- and intersubunit disulfide bonds, dozens of calcium binding sites, and 36 linker subunits that prevent the dissociation of LtEc (Figure 2).31The subunits form a hexagonal bilayer (HBL)32with a high molecular weight (3,600,000 g / mol) and diameter (D = 30 nm) similar to glutaraldehyde crosslinked HBOCs (e.g., Polyheme and HBOC-201).28,33The HBL structure of LtEc is extremely stable and resists dissociation in urea and human plasma.34Indeed, we have shown that LtEc can be stored for up to 6 months at temperatures up to 40°C.33,36

[0234] Another unique characteristic of LtEc is the relatively small volume of the heme pockets in its globin subunits, which are partially blocked by a bulky aromatic residue.37These adaptations do not hinder O2 binding; instead, they stabilize the bound O2 and decrease heme oxidation by blocking the escape of superoxide (O2 ).38Furthermore, the compact structure of the heme pocket also blocks NO from reacting with the oxygen bound to the heme group, allowing LtEc to be transfused into mice and hamsters without inducing oxidative stress, vasoconstriction, or hypertension.30,32,34,39-43In summary, the structure of LtEc prevents subunit dissociation, heme oxidation, and NO scavenging observed with other HBOCs. Earthworms are also readily available from the bait industry and LtEc purification via tangential flow filtration is easily scalable.Arenicola marina Ec (AmEc, a.k.a. HEMO21ife™):

[0235] The Ec of the marine lugworm Arenicola marina (AmEc) is another oxygen earner that does not induce vasoconstriction or oxidative stress in rodents. AmEc has also been used to perfuse the liver,44,45heart,46pancreas,47lung,44,48,49and kidney50-53ex vivo. However, it is important to note some significant differences between LtEc and AmEc:1) AmEc has only been used for hypothermic (not normothermic) organ perfusion56;2) AmEc dissociates into smaller dodecamers in plasma at 37°C, while LtEc retains in structure.3) Lugworms must be harvested or cultivated while earthworms are readily available from the bait industry4) AmEc has a higher O2 affinity (P50 = 7 mmHg)56than LtEc (P50 = 28 mmHg)39or RBCs (P50 = 26 mmHg).

[0236] We will determine whether the lower oxygen affinity and higher structural stability of LtEc at 37°C make it a better oxygen carrier for NEVKP than AmEc. Many of the proposed applications of NEVKP require recapitulating the biological processes of the kidney, most of which depend on normothermic respiration and metabolism. The practical advantages ofsustaining a kidney in normothermia therefore justify the development of LtEc as an oxygen carrier.Results

[0237] Overview. A primary goal was to determine the optimal dose of LtEc to sustain NEVKP and to extend the duration of NEVKP up to 7 days using LtEc. As shown in E1G. 3, NEVKP experiments directly compare LtEc to autologous RBCs, polybHb, and Arenicola Ec.Safety and efficacy of LtEc in exchange transfusion experiments with hamsters

[0238] Despite the drastic structural differences between LtEc and Hbs, the oxygen affinity of LtEc (e.g., P50 = 28 mm Hg) is almost identical to human RBCs (P50 ~ 30 mm Hg).30Indeed, we have shown that exchange transfusions of LtEc into hamsters effectively maintains tissue oxygenation (FIG. 4A).61,62Furthermore, while polymerized bovine hemoglobins significantly increased mean arterial pressure (MAP) in hamsters, transfusions of LtEc did not increase MAP (FIG. 4B).61,62Finally, pharmacokinetic analyses revealed a circulation half-life of 14 hours for LtEc and a complete lack of heme oxidation in vivo (FIG. 4C), indicating that LtEc can maintain O2 delivery in vivo. The lack of oxidation observed with LtEc can be attributed to its positive redox potential (+112 mV), which indicates that it can be reduced by ascorbic acid and other serum components.36,63NEVKP with 0.3 g / dL LtEc

[0239] We recently conducted an experiment investigating the suitability of LtEc for NEVKP. Using a pair of kidneys (~60 g each) from a juvenile female farm pig, we compared short-term perfusion outcomes between autologous serum alone (Kidney A) vs. autologous serum with 0.3 g / dL LtEc (Kidney B). This concentration was chosen to approximate the concentrations of LtEc and other IIBOCs used for transfusions and organ perfusion experiments.59,64,65While Kidney A stopped producing urine after 30 minutes, Kidney B made urine throughout the duration of NEVKP (60 mL total over 2.5 hours). Kidney B consumed more glucose, produced more CO2, and had lower intrarenal resistance compared to Kidney A (Table 1). Importantly, adding LtEc to our perfusate did not lead to precipitation at any point before or during NEVKP. Altogether, these results support the use of LtEc as an oxygen carrier in NEVKP.Table 1 - Physiologic comparisons in LtEc pilot experiment over 2.5 hours of NEVKP.Determining the optimal concentration of LtEc to maintain NEVKP for 24 hours

[0240] Overview: We are testing a range of LtEc concentrations (0.1, 0.5, and 2.5 g / dL) alongside porcine RBCs in NEVKP with matched kidneys over a period of 24 hours. These proposed LtEc concentrations are lower than the Hb concentration in RBC-based perfusates (7-8 g / dL), but they are similar to the concentrations that have been used for perfusion with other HBOCs.2,28Our goal is to identify a concentration of LtEc that can sustain kidney function (e.g., O2 consumption, urine production) in a manner comparable to RBCs while minimizing toxicity (e.g., oxidative stress, kidney injury).

[0241] After the optimal concentration of LtEc has been determined, we will then conductNEVKP experiments in matched kidneys to directly compare LtEc to a polybHb and samples of an Ec from the lugworm Arenicola cristata (AcEc). While it would be preferable to compare LtEc to AmEc and the polybEIb HBOC-201, those materials are not commercially available, so a similar polybHb and purified AcEc will be used as a highly similar substitute for AmEc. Nonetheless, we hypothesize that LtEc will be more structurally stable than AcEc and exhibit less heme oxidation than the polybHb while effectively maintaining kidney function.Determining the optimal dose of LtEc for 24 hours of NEVKP (0.1, 0.5, or 2.5 g / dL LtEc)LtEc purification and characterization:

[0242] Earthworms are purchased from Wholesale Bait Company (Fairfield, OH). LtEc are purified and sterilized via homogenization, centrifugation, and tangential flow filtration as previously described.59Batches of LtEc are only be used for NEVKP if they satisfy the following criteria: <10% Fe3+, >95% HBL, 100% purity, <0.5 U / mL endotoxin, >100 mg / mL LtEc, and P50 = 24-28 mm Hg. Aliquots of each batch are also be tested for contamination by diluting the LtEc in LB broth and incubating the sample at 37°C for 2 weeks to detect growth. Before and after NEVKP, LtEc concentrations and oxidation levels are measured via UV-Vis spectroscopy toestimate clearance and oxidation rates.36We do not expect LtEc to dissociate during NEVKP, since it maintains its HBL structure during 10-12 hours of diafiltration with a peristaltic pump during purification, but we are still analyzing LtEc samples via analytical size exclusion chromatography (SEC) on a Shimadzu UPLC system to ensure that it docs not dissociate like AmEc.Perfusate composition:

[0243] All perfusates are composed of crystalloid, bovine serum albumin (BSA), a nutrient mixture, and an oxygen carrier (RBC or HBOC) at the specified concentration (Table 2). This recipe is based on a previously reported perfusate (Solution 1) that we have reproduced and modified in our lab.66RBCs are be prepared via centrifugation of autologous whole blood, which are collected during the nephrectomy procedure. LtEc is sterilized via 0.22 mm filtration for these experiments. The final perfusate is balanced with electrolytes and osmotically active agents (Dextran 40, mannitol) and fortified with prophylactic antibiotics (e.g. ceftriaxone 100 mg and / or gentamicin 5 mg).Table 2 - Experimental conditions to be tested.NEVKP:

[0244] All kidney perfusion experiments arc conducted in triplicate using pairs of kidneys obtained from female juvenile (20-40 kg) farm pigs (N=9 animals; 3 animals for each LtEc concentration). Kidneys are surgically removed from each animal prior to euthanasia. Once removed, each kidney is flushed using UW solution (Viaspan) and kept on ice during transport to the lab and surgical preparation. Cold storage time is limited to 2 hours to minimize the impact of cold storage on kidney function across all experiments. Kidneys are cannulated and connected to identical NEVKP circuits (FIG. 5) that are primed with perfusate according to the experimental conditions shown in Table 2 and warmed to 37°C. Perfusates are oxygenated with a mixture of 95-97% 02 / 3-5% CO2 (target pCO2 = 40 mm Hg) and pumped through the kidneys at 100-500 mL / min to achieve a target MAP of 70-90 mm Ilg. Perfusate blood gases pre- and post-kidney are measured continuously using Terumo CDI-500 devices to measure O2 consumption and CO2production. Perfusate electrolytes (Na, K, Ca, and Glu) are measured every 3-4 hours using an iSTAT (Abbott). Urine output is collected and quantified via an indwelling ureteral catheter. Urine samples are saved for post-hoc detection of cleared LtEc and analysis of other solutes. Fluids are administered to keep perfusate volume constant in compensation for urine excretion and nutritional supplementation is given to account for kidney metabolism. LtEc concentrations in the perfusate are also be measured via UV-Vis spectrophotometry and supplemented as necessary. The administered volume of fluid, LtEc, and nutritional supplementation are used to evaluate ex vivo kidney performance.

[0245] After 24 hours of perfusion, the kidneys are removed from the circuit and flushed with IL of heparinized saline to collect the remaining LtEc, which are characterized for oxidation and dissociation. Kidney tissue is biopsied and stored in formalin for histologic assessments of necrosis, apoptosis, and osmotic tubular changes. Perfusate samples collected during / after NEVKP are used for serologic assessments (electrolytes, lactate) and ELISAs to detect markers of oxidative stress (e.g., 4-HNE), inflammation (IL-6), heme release / toxicity (e.g., HOI), complement activation (C3a, C5a), hypoxia (HIFla), and kidney injury (NGAL, KIM-1).Comparison of LtEc vs RBCs during NEVKP:

[0246] All kidney perfusion experiments are conducted in triplicate using pairs of kidneys obtained from female juvenile (20-40 kg) Yorkshire pigs (N=9 animals; 3 animals for each LtEc concentration). Kidneys are surgically removed from each animal prior to euthanasia. Kidneys are cannulated and connected to identical NEVKP circuits (FIG. 5) that are primed with perfusate according to the experimental conditions shown in Table 2 and warmed to 37°C. Perfusates are oxygenated using a 95% 02 / 5% CO2 mixture and delivered the kidneys at 100-500 mL / min to achieve a target MAP of 70-90 mm Hg. Perfusate blood gases pre- and post-kidney are measured continuously using Terumo CDI-500 devices to measure O2 consumption and CO2 production. Perfusate electrolytes (Na, K, Ca, and Glu) are measured every 3-4 hours using an iSTAT (Abbott). Urine output is collected and quantified via an indwelling ureteral catheter. Urine samples are saved for post-hoc detection of cleared LtEc and analysis of other solutes. Fluids are administered to keep perfusate volume constant in compensation for urine excretion and nutritional supplementation are given to account for kidney metabolism. LtEc concentrations in the perfusate will also be measured via UV-Vis spectrophotometry and supplemented as necessary. The required volume of fluid, LtEc, and nutritional supplementation will also be used to evaluate ex vivo kidney performance. Following perfusion, kidney sections are prepared forhistological examination to evaluate for signs of ischemic necrosis, tissue edema, and glomerular pathology.Direct comparison of LtEc to other HBOCs (polybHb and Arenicola cristata erythrocruorin).Preparation of polybHb and AcEc:

[0247] Lugworms Arenicola cristata) are purchased from Gulf of Maine, Inc. and Arenicola cristata Ec (AcEc) are purified using tangential flow filtration (TFF) as described for LtEc. Bovine RBCs are purchased from Fisher Scientific, lysed in ice cold water, and then TFF are used to purify bHb. The bHb will then be polymerized by mixing it with glutaraldehyde at a 40:1 molar ratio for 1 hour at 25°C.29The resulting polybHb is stabilized by reducing its Schiff bases with sodium borohydride and purified via subsequent rounds of diafiltration with 500 kDa MWCO TFF filters to remove unreacted glutaraldehyde and bHb tetramers. The polybHb and AcEc are only used for NEVKP if they pass the quality checks described for LtEc.NEVKP of LtEc, AcEc, and polybHb:

[0248] NEVKP experiments comparing LtEc vs AcEc and LtEc versus polybHb are conducted in triplicate (N = 6 pigs). The same metrics described for kidney respiration, function, and toxicity are used to compare the performance of each HBOC. Each HBOC is used at the optimal concentration described in the literature (0.1 g / dL AmEc and 3 g / dL polybHb). To ensure an unbiased assessment of HBOC performance. All HBOCs (LtEc, AcEc, and polybHb) are delivered in a blinded fashion.Evaluation of HBOC performance:

[0249] Primary outcomes include 1) duration, volume, and quality of urine output; 2) production of CO2 and lactate; and 3) markers of stress and toxicity as described above. Additional measures include each HBOC’s molecular stability over time as determined by the concentration of dissociated globin subunits in the perfusate and urine. The ideal HBOC for NEVKP will maintain kidney function (e.g., urine and CO2 production) and minimize dissociation, clearance, oxidation, and toxicity.Evaluate the durability and efficacy of LtEc and gLtEc in NEVKP for up to 7 days

[0250] Overview: We hypothesize that the high stability of LtEc compared to RBCs will allow us to extend NEVKP for up to 7 days in porcine kidneys. Prolonging NEVKP by this order of magnitude will unlock myriad possibilities, both clinically and translationally. Wc will begin by attempting to prolong the half-life of LtEc by cross-linking it with glutaraldehyde (gLtEc) and comparing it to native LtEc in 24-hour NEVKP experiments (Aim 2a).67We then use a phased approach to develop methods of extending NEVKP with an LtEc-based perfusate up to 3 days. We then extend NEVKP to 7 days. While our primary goal is to maintain kidney function ex vivo for 7 days, we also attempt autotransplantation experiments to demonstrate that the perfused kidneys also maintain their function in vivo.Evaluate the molecular durability of LtEc and gLtEc in NEVKP

[0251] Preparation of gLtEc: Native LtEc is cross-linked with glutaraldehyde at a molar ratio of 32: 1 (glutaraldehyde to LtEc). We have shown that this ratio maximizes the thermal stability of LtEc and completely prevents it from dissociating into smaller oligomers.67Schiff bases are reduced with sodium borohydride. TFF is used to purify the gLtEc, and the same quality checks used for LtEc arc performed.Determining the half-life and oxidation rate of LtEc vs gLtEc in NEVKP:NEVKP is conducted in triplicate (N = 3 pigs) for 24 hours using pairs of kidneys that are perfused with LtEc or gLtEc at the optimal concentration determined, as described above. The materials are blinded to the UCSF NEVKP group to ensure an unbiased comparison and the same metrics described above are assessed during NEVKP. The perfusate and urinary concentrations of LtEc and gLtEc are measured over time to compare the half-lives, dissociation kinetics, and oxidation rates of LtEc and gLtEc during NEVKP.Long-term NEVKP perfusate composition:

[0252] A perfusate composed of crystalloid, bovine serum albumin, nutrient media, and LtEc in its optimal form and concentration is prepared, with additional electrolytes and antibiotics. To allow for daily scheduled perfusate changes with a circuit priming volume of 0.5L, we prepare and store 1.5 L perfusate for experiments targeting 3-day NEVKP and 3.5L of perfusate for experiments targeting 7-day NEVKP.Extending the duration of NEVKP to 3 days:

[0253] A minimum of 3 female juvenile farm pigs are used for the first phase of prolonged NEVKP experiments (all experiments have been approved 06 / 07 / 2024 under UCSF IACUC protocol AN203191). Each animal undergoes a survival procedure to remove the left kidney. NEVKP is then be conducted as described above, with special care to maintain sterility during all steps. Scheduled perfusate changes are conducted every 24 hours (or as necessary) to maintain perfusate volume and LtEc concentration, while physiologic parameters are monitored to track kidney viability. Nutritional assessments via glucose tests (iSTAT), amino acid analysis (Quest labs), and lipid profiling (Quest labs) are performed daily and reviewed after each experiment to guide nutritional supplementation. The UCSF NEVKP team provides 24-hour monitoring of the NEVKP experiment for this duration.

[0254] At the conclusion of 72-hour perfusion, the perfused kidney is assessed to determine its suitability for autotransplantation. Kidneys are qualitatively stratified (good, fair, and poor) according to viability (CO2 and lactate production), function (urine output), and sterility (gram stain of perfusate and urine). For kidneys deemed “good” or “fair,” we will proceed with a second survival surgery that includes autotransplantation of the ex vivo perfused kidney with concurrent removal of the contralateral kidney. The animals arc then be observed for 48 hours postoperatively to determine whether kidney function (urine production, electrolyte homeostasis) can be supported by the autotransplanted organ. The kidney tissue will be examined on necropsy for histologic integrity and evidence of tubular necrosis or glomerular changes. Autotransplantation experiments in pigs following ex vivo perfusion have also been demonstrated by other groups.71 72

[0255] Extending the duration of NEVKP to 7 days: If 72-hour NEVKP successfully attained, we will attempt to extend NEVKP to 7 days with subsequent auto-transplantation using the same methods described above in at least 3 animals. Like the 3-day NEVKP experiments, our primary measure of success will be preservation of renal function in the animal after the ex vivo kidney is auto-transplanted and the contralateral kidney is removed. Given the potential impact to the field and the challenging logistics of executing this experiment for the first time, it should be emphasized that achieving 72-hour NEVKP would represent a significant advancement in NEVKP, and demonstrating 7-day NEVKP with preserved renal function in just one animal would be a monumental and unprecedented breakthrough.References

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[0328] 73. Spivack, K. et al. Increasing the stability of Lumbricus terrestris erythrocruorin via poly(acrylic acid) conjugation. Artif Cells Nanomed Biotechnol 46, 1137-1144 (2018).

[0329] 74. lani, V. P. et al. Polyethylene Glycol Camouflaged EarthwormHemoglobin. PLoS One 12, e0170041 (2017).

[0330] 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.

[0331] 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 arc 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

CLAIMSWHAT is CLAIMED IS:

1. An acellular oxygcn-carrying perfusate comprising Lumbricus terrestris erythrocruorin (LtEc).

2. The acellular oxygen-carrying perfusate of claim 1 , wherein the LtEc is at a concentration in a range from 0.1 g / dL to 2.5 g / dL.

3. The acellular oxygen-carrying perfusate of claim 2, wherein the LtEc is at a concentration of about 0.1 g / dL, about 0.5 g / dL, or about 2.5 g / dL.

4. The acellular oxygen-carrying perfusate of any one of claims 1-3, wherein the LtEc comprises: a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:1; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:3; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:5; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:7; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:9; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:11; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 13; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:L5; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 17; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 19;a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:21; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:23; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:25; a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:27; and a polypeptide comprising or consisting of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:29.

5. The acellular oxy gen-carrying perfusate of any one of claims 1-4, wherein the LtEc is cross-linked with a cross-linking agent such that subunits of LtEc do not dissociate.

6. The acellular oxygen-carrying perfusate of any one of claims 1-5, further comprising one or more of nutrients, electrolytes, reducing agents, antibiotics, anticoagulants, osmotic agents, colloids, diuretics, vasodilators, insulin, growth factors, immunosuppressive agents, or a combination thereof.

7. The acellular oxygen-carrying perfusate of claim 6, wherein the nutrients comprise carbohydrates, amino acids, nucleotides, fatty acids, vitamins, or a combination thereof.

8. The acellular oxy gen-carrying perfusate of claim 7, wherein the amino acids comprise alanine, aspartate, asparagine, glycine, glutamate, glutamine, lysine, arginine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine, isoleucine, leucine, valine, histidine, cysteine, proline, or a combination thereof.

9. The acellular oxygen-carrying perfusate of claim 7, wherein the fatty acids comprise oleic acid, linoleic acid, palmitic acid, stearic acid, or a combination thereof.

10. The acellular oxygen-carrying perfusate of claim 7, wherein the carbohydrates comprise glucose.

11. The acellular oxygen-carrying perfusate of claim 7, wherein the nucleotides comprise adenosine and its metabolites like adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP).

12. The acellular oxygen-carrying perfusate of claim 6, wherein the electrolytes comprise sodium (Na+), chloride (Cl ), potassium (K+), calcium (Ca2+) magnesium (Mg2+), bicarbonate (HCO3 ), acetate, citrate, or a combination thereof.

13. The acellular oxygen-carrying perfusate of claim 12, wherein the acellular oxygen-carrying perfusate comprises Ca2+.

14. The acellular oxy gen-carrying perfusate of claim 6, wherein the reducing agents comprise ascorbic acid, glutathione, dithiothreitol (DTT), tris(2-carboxyethyl)phosphine hydrochloride (TCEP), N-acetyl-L-cysteine, or a combination thereof.

15. The acellular oxygen-carrying perfusate of claim 6, wherein the osmotic agents comprise mannitol, glycerol, sorbitol, dextrose, or ribose.

16. The acellular oxygen-carrying perfusate of claim 6, wherein the colloids comprise albumin, dextran, hydroxyethyl starch (HES), or a combination thereof.

17. The acellular oxy gen-carrying perfusate of claim 6, wherein the vasodilators comprise nitroglycerin, hydralazine, or sodium nitroprusside.

18. The acellular oxy gen-carrying perfusate of claim 6, wherein the antibiotics comprise ceftriaxone, gentamicin, penicillin, streptomycin, or a combination thereof.

19. The acellular oxy gen-carrying perfusate of claim 6, wherein the growth factors comprise one or more hematopoietic growth factors or interleukins, or a combination thereof.

20. The acellular oxy gen-carrying perfusate of claim 19, wherein the hematopoietic growth factors comprise erythropoietin, thrombopoietin, insulin- like growth factor (IGF)-l, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulatingfactor (GM-CSF), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), interleukin-3 (IL-3), or a combination thereof.

21. The acellular oxygcn-carrying perfusate of any one of claims 1-20, further comprising a crystalloid solution.

22. The acellular oxygen-carrying perfusate of claim 21, wherein the crystalloid solution is saline or Ringer's lactate solution.

23. The acellular oxygen-carrying perfusate of any one of claims 1-22, wherein the LtEc is oxygenated.

24. The acellular oxygen-carrying perfusate of any one of claims 1-23, further comprising a therapeutic agent.

25. The acellular oxygen-carrying perfusate of claim 24, wherein the therapeutic agent is a drug, a gene therapy agent, a gene editing agent, a chemotherapeutic agent, a radiotherapeutic agent, or a cell.

26. A method for perfusion of an organ comprising perfusing the organ with the acellular oxygen-carrying perfusate of any one of claims 1-25.

27. The method of claim 26, wherein the perfusion is in situ or ex vivo normothermic organ perfusion, mid-thermic organ perfusion, sub-normothermic organ perfusion, or hypothermic organ perfusion.

28. The method of claim 26, wherein temperature of the acellular oxygen-carrying perfusate is maintained in a range from 20 °C to 40 °C, 1 °C to 10 °C, 13 °C to 24 °C, or 25 °C to 34 °C during perfusion.

29. The method of claim 26, wherein temperature of the organ or a container holding the organ is maintained in a range from 20 °C to 40 °C, 1 °C to 10 °C, 13 °C to 24 °C, or 25 °C to 34 °C during perfusion.

30. The method of any one of claims 26-29, wherein the organ is a kidney, a heart, a liver, a lung, a stomach, a small intestine, a large intestine, a pancreas, a gonad, a limb, an extremity, or a tissue graft or a portion thereof.

31. The method of any one of claims 26-30, wherein the organ is obtained from a live organ donor or an organ donor after circulatory death.

32. The method of any one of claims 26-31, wherein the acellular oxygen-carrying perfusate is oxygenated with a gas mixture having 1 % to 99% oxygen.

33. The method of any one of claims 26-31, wherein the acellular oxygen-carrying perfusate is oxygenated with a gas mixture having 95% to 99% oxygen.

34. The method of any one of claims 26-31, wherein the acellular oxygen-carrying perfusate is deoxygenated for storage.

35. The method of any one of claims 26-32, further comprising adding a therapeutic agent or nutrient to the acellular oxygen-carrying perfusate.

36. The method of claim 35, wherein the therapeutic agent or nutrient is heparin, prostacycline, glucose, insulin, a bile salt, an amino acid, a fatty acid, a lipid, a vitamin, a mineral, a hormone, a cytokine, a steroid, a diuretic, a vasoactive molecule, an antibiotic, an antibody, or a cell.

37. The method of claim 35, wherein the therapeutic agent is a recombinant nucleic acid or gene editing system.

38. The method of claim 37, wherein the recombinant nucleic acid or gene editing system is encapsulated in a lipid nanoparticle (LNP).

39. The method of claim 37 or 38, wherein the recombinant nucleic acid is a DNA or RNA encoding a therapeutic protein or regulatory RNA.

40. The method of any one of claims 37-39, wherein the recombinant nucleic acid comprises a viral vector or plasmid.

41. The method of claim 40, wherein the viral vector is an adcno-associatcd viral vector, an adenoviral vector, a lentiviral vector, or a retroviral vector.

42. The method of claim 40 or 41, wherein expression of the therapeutic protein or regulatory RNA is inducible.

43. The method of any one of claims 39-42, wherein the RNA is a messenger RNA (mRNA), wherein translation of the mRNA results in production of a therapeutic protein.

44. The method of claim 37, wherein 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).

45. The method of any one of claims 37-44, wherein the recombinant nucleic acid or gene editing system is delivered in the perfusate to the organ.

46. The method of any one of claims 35-45, wherein the therapeutic agent is administered locally to a site on the organ during ex vivo perfusion of the organ.

47. The method of any one of claims 35-46, wherein the therapeutic agent is toxic when administered to a subject in vivo.

48. The method of claim 47, wherein the therapeutic agent is a gene therapy agent, a chemotherapeutic agent, or a radiotherapeutic agent.

49. The method of any one of claims 26-48, further comprising measuring a level of a biomarker in a sample of the perfusate to determine fitness of the organ for transplant.

50. The method of any one of claims 26-49, further comprising genetically modifying the organ during perfusion.

51. The method of claim 50, wherein said genetically modifying the organ comprises converting a disease-associated allele to a wild-type allele.

52. The method of any one of claims 26-51, further comprising resecting a tumor or surgically repairing the organ prior to transplantation into a recipient.

53. The method of any one of claims 26-52, further comprising measuring temperature of the acellular oxygen-carrying perfusate, flow-rate of the acellular oxygencarrying perfusate, pH of the acellular oxygen-carrying perfusate, concentration of oxygen in the acellular oxygen-carrying perfusate, concentration of glucose in the acellular oxygen-carrying perfusate, concentration of LtEc in the acellular oxygen-carrying perfusate, concentration of sodium in the acellular oxygen-carrying perfusate, concentration of potassium in the acellular oxygen-carrying perfusate, concentration of calcium in the acellular oxygen-carrying perfusate, concentration of carbon dioxide in the acellular oxygen-carrying perfusate, percent saturation of oxygen in the acellular oxygen-carrying perfusate, or concentration of lactate in the acellular oxygcn-carrying perfusate, or any combination thereof.

54. A method for transfusion of a subject comprising transfusing the subject with the acellular oxygen-carrying perfusate of any one of claims 1-25.

55. The method of claim 54, wherein the subject has any blood type.

56. The method of claim 54 or 55, wherein the subject has acute blood loss from trauma, a surgical procedure, or a hemorrhage.

57. The method of claim 54 or 55, wherein the subject has chronic anemia from a nutrient deficiency, a malignancy, or a genetic disorder.

58. The method of any one of claims 54-57, wherein the subject is unable or unwilling to receive a transfusion with a human blood product.

59. A method of performing liquid hyperbaric oxygen therapy, the method comprising infusing the acellular oxygen-carrying perfusate of any one of claims 1-25 into atissue, limb, organ, or organism in need of the liquid hyperbaric oxygen therapy, wherein the acellular oxygen-carrying perfusate is oxygenated in a hyperbaric oxygen chamber.

60. The method of claim 59, wherein the liquid hyperbaric oxygen therapy is used to treat a subject for necrotizing fasciitis, gas gangrene, hemorrhagic cystitis, poor wound healing, a skin graft, or a thermal or radiation burn.

61. A method of treating ischemia in a subject, the method comprising infusing the acellular oxygen-carrying perfusate of any one of claims 1-25 into an ischemic tissue in the subject.

62. The method of claim 61, wherein the acellular oxygen-carrying perfusate is oxygenated with a gas mixture having 1 % to 99% oxygen.

63. The method of claim 61, wherein the acellular oxygen-carrying perfusate is oxygenated with a gas mixture having 95% to 99% oxygen.

64. The method of any one of claims 61-63, wherein the ischemia is caused by a stroke, a transient ischemic attack, a myocardial infarction, acute limb ischemia, or ischemic bowel syndrome.

65. A method of supporting cellular respiration in a cell or tissue, the method comprising delivering the acellular oxy gen-carrying perfusate of any one of claims 1-25 to the cell or tissue.

66. The method of claim 65, wherein the acellular oxygen-carrying perfusate is delivered to the cell or tissue in vitro, ex vivo, or in vivo.

67. A method of performing radiation therapy of a tumor, the method comprising: delivering the acellular oxygen-carrying perfusate of any one of claims 1-25 to the tumor; and administering the radiation therapy to the tumor.

68. The method of claim 67, wherein the tumor is hypoxic.

69. The method of claim 67 or 68, wherein the tumor has previously been resistant to radiation therapy.

70. The method of any one of claims 67-69, wherein said delivering is performed prior to or during radiation therapy.

71. The method of any one of claims 67-70, wherein said delivering is perfo lined by infusion, perfusion, or injection locally into the tumor.

72. A system comprising: a perfusion machine comprising a reservoir, wherein the reservoir comprises the acellular oxygen-carrying perfusate of any one of claims 1-25; and an oxygenator, wherein the oxygenator oxygenates the acellular oxygen-carrying perfusate.

73. The system of claim 72, further comprising a container for holding an organ or tissue.

74. The system of claim 72 or 73, wherein the perfusion machine performs normothermic organ perfusion, mid-thermic organ perfusion, sub-normothermic organ perfusion, or hypothermic organ perfusion.

75. The system of any one of claims 72-74, wherein the perfusion machine performs perfusion in situ or ex vivo.

76. The system of any one of claims 72-75, further comprising a temperature controller, wherein the temperature controller maintains the acellular oxygen-carrying perfusate at a desired temperature.

77. The system of claim 76, wherein the temperature controller maintains the temperature of the acellular oxygen-carrying perfusate or the container holding the organ or tissue in a range from 20 °C to 40 °C, 1 °C to 10 °C, 13 °C to 24 °C, or 25 °C to 34 °C during perfusion.

78. The system of claim 76 or 77, wherein the temperature controller is a water bath, an ice bath, a thermoelectric temperature controller, or a heat exchanger.

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