H-NOX PROTEINS FOR ORGAN PRESERVATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/234,454, filed August 18, 2021, which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] This application pertains to the use of polymeric H-NOX proteins in methods of preserving at least one organ in situ or ex situ in a donation after brain death of the donor or a donation after cardiac death of the donor.

BACKGROUND

[0003] H-NOX proteins (named for Heme-Nitric oxide and OXygen binding domain) are members of a highly-conserved, well-characterized family of hemoproteins (Iyer, LM et al. (2003) BMC Genomics 4( 1 ): 5; Karow, DS et al. (2004) Biochemistry 43(31): 10203-10211; Boon, EM et al. (2005) Nature Chem. Biol. 1 :53-59; Boon, EM et al. (2005) Curr. Opin. Chem. Biol. 9(5):441-446; Boon, EM et al. (2005) Inorg. Biochem. 99(4):892-902; Cary, SP et al. (2005) Proc Natl Acad Sci USA 102(37): 13064-9; Karow DS et al. (2005) Biochemistry 44(49): 16266-74; Cary, SP et al. (2006) Trends Biochem Sci 31(4):231-9; Boon, EM et al. (2006) J Biol Chem 281(31):21892-902; Winger, JA et al. (2007) J Biol Chem. 282(2): 897-907). H-NOX proteins are nitric-oxide-neutral, unlike previous hemoglobin-based oxygen carriers, H- NOX do not scavenge circulating nitric oxide, and thus are not associated with hypertensive or renal side effects. The intrinsic low NO reactivity (and high NO stability) makes wild-type and mutant H-NOX proteins desirable blood substitutes because of the lower probability of inactivation of H-NOX proteins by endogenous NO and the lower probability of scavenging of endogenous NO by H-NOX proteins. Importantly, the presence of a distal pocket tyrosine in some H-NOX proteins (Pellicena, P. et al. (2004) Proc Natl. Acad Sci USA 101(35): 12854- 12859) is suggestive of undesirable, high NO reactivity, contraindicating use as a blood substitute. For example, by analogy, a Mycobacterium tuberculosis hemoglobin protein, with a structurally analogous distal pocket tyrosine, reacts extremely rapidly with NO, and is used by the Mycobacterium to effectively scavenge and avoid defensive NO produced by an infected host (Ouellet, H. et al. (2002) roc. Natl. Acad. Sci. U SA 99(9):5902-5907). However, it was surprisingly discovered that H-NOX proteins actually have a much lower NO reactivity than that of hemoglobin making their use as blood substitutes possible.

[0004] It was discovered that H-NOX proteins that bind NO but not O2 can be converted to H- NOX proteins that bind both NO and O2 by the introduction of a single amino acid mutation (see WO 2007/139791 and WO 2007/139767). Thus, the affinity of H-NOX proteins for O ₂ and NO and the ability of H-NOX proteins to discriminate between O2 and NO ligands can be altered by the introduction of one or more amino acid mutations, allowing H-NOX proteins to be tailored to bind O2 or NO with desired affinities. Additional mutations can be introduced to further alter the affinity for O2 and/or NO. The H-NOX protein family can therefore be manipulated to exhibit improved or optimal kinetic and thermodynamic properties for O2 delivery. For example, mutant H-NOX proteins have been generated with altered dissociation constants and/or off rates for O2 binding that improve the usefulness of H-NOX proteins for a variety of clinical and industrial applications. The ability to tune H-NOX proteins to bind and deliver O2 is a therapeutic avenue that addresses and overcomes the central shortcomings of current O2 carriers. [0005] Organ donation is the harvesting of organs from a human body, called donor, for the purpose of treating a patient, called recipient, whose organs are seriously damaged.

[0006] One of the difficulties of this donation remains the organ preservation time. Indeed, in normothermia (37° C), before and/or after harvesting from the donor, an organ undergoes a period of warm ischemia, i.e. a period where the organ is no longer perfused by the donor's blood, and is not yet refrigerated. It deteriorates rapidly and is no longer supplied with oxygen. The acceptable time for ensuring the subsequent resumption of function of the transplant organ varies from one organ to another, when said organ is preserved in hypothermia (re. at around 4° C). For example, it is approximately 4 to 6 hours for a heart or a lung, 8 to 12 hours for a liver, 24 to 48 hours for a kidney and 8 to 10 hours for a pancreas or an intestine. The transplant must therefore be carried out within a well-defined period, in order to ensure that the organ functionality is maintained.

[0007] Moreover, hypothermia is the essential component of storage. As soon as it is removed, the transplant organ is cooled, in order to rapidly bring its temperature down from 37° C to 4° C; for this, the organ is rinsed with a preserving solution via the vessels and then simply immersed in this preserving solution kept at low temperature by crushed ice according to guaranteed aseptic conditions. The decrease in the temperature of the tissues leads to a decrease in cell metabolism, i.e. a slowing down of the catalytic enzymatic activity required for cell viability, without however stopping it (Belzer F. O., Southard J. H. Principles of solid-organ preservation by cold storage. Transplantation 1988; 45(4): 673-676). The transplant organ placed at 4° C experiences a decrease in its metabolism of approximately 85%. Hypothermia thus makes it possible to combat the harmful effects of oxygen starvation and nutrient starvation induced by the arrest of blood circulation and defers cell death, responsible for tissue necrosis.

[0008] In the face of shortages of donations, preservation of the transplant organ and oxygenation thereof, over a longer period of time, are essential preoccupations; this allows the quality of the organ to be maintained, prolonged survival of the organ, and thus a successful transplant. Indeed, even though the metabolism of a transplant organ preserved at 4° C is reduced, it still needs oxygen, like all aerobic tissues.

[0009] In addition, the majority of blood substitutes available today, such as perfluorocarbons (PFCs), HBOCs or human blood, are capable of oxygenating organs, but cannot be used at just any temperature. In particular, they are not functional or stable at 4° C. Moreover, PFCs are not oxygen transporters, but solutes capable of dissolving a large amount of oxygen according to the partial oxygen pressure. They cannot therefore be used simply, and can create oxidative stress problems.

[0010] All references cited herein, including patent applications and publications, are incorporated herein by reference in their entirety.

SUMMARY

[0011] The present disclosure is based in part on the surprising discovery that polymeric H- NOX proteins preferentially oxygenate pathologically hypoxic tissues such as brain during ischemic stroke or heart during global hypoxemia, and provide a longer oxygenation window due to a longer circulation half-life compared to monomeric H-NOX proteins. Accordingly, the present disclosure provides proteins, compositions, kits and methods for the delivery of oxygen; for example, to preserve and oxygenate a donor's organs under optimum conditions, in order to maintain their functions before they are harvested from the donor.

[0012] In at least one embodiment of the present disclosure is directed to a method for preserving an organ for donation after brain or cardiac death in a donor, the method comprising administering to the donor a composition comprising at least one H-NOX protein, a stabilizing solution and/or an organ preservation solution, wherein the composition is at a temperature of between 0°C and 37° C.

[0013] In at least one embodiment, the stabilizing solution is an aqueous solution comprising salts, and comprises a pH of between 6.5 and 7.6. In at least one embodiment, the solution is an aqueous solution comprising sodium ions.

[0014] In at least one embodiment, the stabilizing solution is an aqueous solution comprising 20 mM sodium citrate, 250 mM glucose, 10 mM glutathione, and 0.1% pol oxamer 188 at pH 6.8 ± 0.2.

[0015] In at least one embodiment, the organ preservation solution is an aqueous solution having a pH of between 6.5 and 7.5 and comprising salts; sugars; antioxidants; active agents. In at least one embodiment, the solution is an aqueous solution comprising chloride, sulfate, sodium, calcium, magnesium or potassium ions; sugars selected from mannitol, ramose, sucrase, glucose, fructose, lactobionate and gluconate; glutathione; active agents selected from xanthine oxidase inhibitors, lactates, and amino acids, and optionally colloids selected from hydroxyethyl starch, poly-ethylene glycol and dextran.

[0016] In at least one embodiment, the H-NOX protein is present at a concentration, relative to the final volume of composition, of between 0.001 mg/ml and 100 mg/ml, and in that the composition has an osmolarity of between 250 and 350 mOsm/1. In at least one embodiment, the H-NOX protein is present at a concentration, relative to the final volume of composition, of between 0.5 mg/ml and 5 mg/ml, and the composition has an osmolarity of between 275 and 310 mOsm/1.

[0017] In at least one embodiment, the present disclosure is directed to a method for preserving an organ ex situ in a donation after brain death donor or a donation after cardiac death donor, comprising the following steps: a) perfusion of said deceased donor with a composition of the present disclosure; then b) harvesting of the organ to be transplanted; then c) static or dynamic-perfusion preservation of said organ obtained in b), at a temperature of between 0° C and 37° C, for a time predetermined according to said organ, in the composition or the aqueous solution defined in step a).

[0018] In at least one embodiment, the present disclosure is directed to the use of a composition comprising at least one H-NOX protein, a stabilizing solution and / or an organ preservation solution, said composition having a temperature of between 0°C and 37°C, to preserve an organ in a dead donor who died in brain death or died of cardiac arrest.

[0019] In at least one embodiment, the use is characterized in that the H-NOX protein is T. tengcongensis H-NOX, a L. pneumophilia 2 H-NOX, a H. sapiens pi, a R. norvegicus i, a C. lupus H-NOX domain, a Z>. melangaster pi, a Z>. melangaster CG14885-PA, a C. elegans GCY- 35, a TV. punctiforme H-NOX, C. crescentus H-NOX, a S. oneidensis H-NOX, or C. acetobutylicum H-NOX. In at least one embodiment, the use is characterized in that the stabilizing solution is an aqueous solution comprising salts, preferably sodium ions, and gives the composition according to the invention a pH inclusive between 6.5 and 7.6. In at least one embodiment, the use is characterized in that the stabilizing solution is an aqueous solution comprising 20 mM sodium citrate, 250 mM glucose, 10 mM glutathione, and 0.1% poloxamer 188 at pH 6.8 ± 0.2. In at least one embodiment, the use is characterized in that the organ preservation solution is an aqueous solution having a pH of between 6.5 and 7.5 and comprising salts, preferably chloride, sulphate, sodium and calcium ions, magnesium and potassium; sugars, preferably mannitol, raffinose, sucrose, glucose, fructose, lactobionate, or gluconate; antioxidants, preferably glutathione; active agents, preferably xanthine oxidase inhibitors such as allopurinol, lactates or amino acids such as histidine, glutamic acid (or glutamate), tryptophan; and optionally colloids such as hydroxyethyl starch, polyethylene glycol or dextran. In at least one embodiment, the use is characterized in that the H-NOX protein is present at a concentration, relative to the final volume of composition, of between 0.001 mg / ml and 100 mg / ml, preferentially between 0.005 mg / ml and 20 mg / ml, more preferably between 0.5 mg / ml and 5 mg / ml, and in that the composition has an osmolarity of between 250 and 350 mOsm / L, preferably between 275 and 310 mOsm / L, preferably about 302 mOsm / L.

[0020] In at least one embodiment, the present disclosure is directed to a method of preserving an ex situ organ in a deceased donor in a brain dead or died state of cardiac arrest, comprising the steps of: a) infusing said deceased donor with a composition according to the present disclosure, or with an aqueous solution according to the present disclosure; then removal of the organ to be transplanted; then c) maintaining in static or dynamic perfusion said organ obtained in b), at a temperature between 0°C and 37°C, preferably between 2 °C and 25 ° C, more preferably about 4 °C or 37°C, during a time determined according to said organ, in the composition or the aqueous solution defined in step a). [0021] In at least one embodiment, the present disclosure is directed to a method of preserving an ex situ organ in a deceased donor, comprising the steps of: a) removal of the organ to be transplanted and b) maintaining in static or dynamic perfusion said organ obtained in a), at a temperature between 0°C and 37°C, preferably between 2 °C and 25 ° C, more preferably about 4 °C or 37°C, during a time determined according to said organ, in a composition according to according to the present disclosure, or with an aqueous solution according to the present disclosure.

[0022] In at least one embodiment, the present disclosure is directed to a method for preserving an organ ex situ, comprising the steps of: a) harvesting of the organ to be transplanted; then b) maintaining in static or dynamic perfusion said organ obtained in a), at a temperature of between 0° C and 37° C, for a time predetermined according to said organ, in the composition or the aqueous solution of the present disclosure.

[0023] In at least one embodiment, the present disclosure is directed to a composition having a pH of 6.5 to 7.6, which comprises: at least one H-NOX protein; calcium ions, preferably in an amount of between 0 and 0.5 mM; KOH, preferably in an amount of between 20 and 100 mM; NaOH, preferably in an amount of between 20 and 125 mM; KH2PO4, preferably in an amount of between 20 and 25 mM; MgCh, preferably in an amount of between 3 and 5 mM; at least one sugar chosen from raffinose and glucose, preferably in an amount of between 5 and 200 mM; adenosine, preferably in an amount of between 3 and 5 mM; glutathione, preferably in an amount of between 2 and 4 mM; allopurinol, preferably in an amount of between 0 and 1 mM; and at least one compound chosen from hydroxyethyl starch, polyethylene glycols of different molecular weights and human serum albumin, preferably in an amount of between 1 and 50 g / 1. [0024] In at least one embodiment, the present disclosure is directed to a composition having a pH of 6.5 to 7.6, which comprises at least one H-NOX protein; calcium ions, preferably in an amount of between 0 and 0.5 mM; NaOH, preferably in an amount of between 15 and 30 mM; HEPES, preferably in an amount of between 2 and 10 mM; KH2PO4, preferably in an amount of between 20 and 25 mM; mannitol, preferably in an amount of between 20 and 35 mM; glucose, preferably in an amount of between 3 and 10 mM; sodium gluconate, preferably in an amount of between 50 and 100 mM; magnesium gluconate, preferably in an amount of between 1 and 5 mM; ribose, preferably in an amount of between 2 and 5 mM; at least one compound chosen from hydroxyethyl starch, polyethylene glycols of different molecular weights and human serum albumin, preferably in an amount of between 1 and 50 g / 1; glutathione, preferably in an amount of between 2 and 4 mM; and adenine, preferably in an amount of between 3 and 5 mM.

DESCRIPTION OF THE DRAWINGS

[0025] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0026] FIGs. 1 A-1D show functional assessment at the pump. Resistance and flow were measured at 0, 1, 2, 3, 4, 6, 8, and 10 hours for both genetically match-controlled kidneys from donor #1 (FIGs. 1 A and 1C) and #2 (FIGs. IB and ID).

[0027] FIGs. 2A-2F show histopathological assessment. Tubular degeneration, necrosis, and apoptosis were evaluated at 0, 1, 2, 3, 4, 6, 8, and 10 hours for both genetically match-controlled kidneys from donor #1 (FIGs. 2 A, 2C, and 2E) and #2 (FIGs. 2B, 2D, and 2F).

[0028] FIG. 3 shows histopathological assessment. Cytoskeletal derangement was evaluated at 0, 1, 2, 3, 4, 6, 8, and 10 hours for both genetically match-controlled kidneys from donor #1 and #2.

DETAILED DESCRIPTION

[0029] The following description and examples illustrate embodiments of the present disclosure in detail.

[0030] It is to be understood that the present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are variations and modifications of the present disclosure, which are encompassed within its scope.

[0031] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0032] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. [0033] The present disclosure is based in part on the surprising discovery that polymeric H- NOX proteins preferentially oxygenate pathologically hypoxic tissues such as the brain during ischemic stroke or heart during global hypoxemia, and provide a longer oxygenation window due to a longer circulation half-life compared to monomeric H-NOX proteins. Accordingly, the present disclosure provides proteins, compositions, kits and methods for the delivery of oxygen; for example, to preserve and oxygenate a donor's organs under optimum conditions, in order to maintain their functions before they are harvested from the donor.

Definitions

[0034] Unless defined otherwise, the meanings of all technical and scientific terms used herein are those commonly understood by one of skill in the art to which this disclosure belongs. One of skill in the art will also appreciate that any methods and materials similar or equivalent to those described herein can also be used to practice or test the disclosure.

[0035] For use herein, unless clearly indicated otherwise, use of the terms “a”, “an,” and the like refers to one or more.

[0036] In this application, the use of “or” means “and/or” unless expressly stated or understood by one skilled in the art. In the context of a multiple dependent claim, the use of “or” refers back to more than one preceding independent or dependent claim.

[0037] Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

[0038] It is understood that aspect and embodiments of the disclosure described herein include “comprising,” “consisting,” and “consisting essentially of’ aspects and embodiments.

[0039] The terms “polypeptide” and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Such polymers of amino acid residues may contain natural or non-natural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. Both full- length proteins and fragments thereof are encompassed by the definition. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. Furthermore, for purposes of the present disclosure, a “polypeptide” refers to a protein which includes modifications, such as deletions, additions, and substitutions (generally conservative in nature), to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification. As used herein, a protein may include two or more subunits, covalently or non-covalently associated; for example, a protein may include two or more associated monomers.

[0040] The terms “nucleic acid molecule”, “nucleic acid” and “polynucleotide” may be used interchangeably, and refer to a polymer of nucleotides. Such polymers of nucleotides may contain natural and/or non-natural nucleotides, and include, but are not limited to, DNA, RNA, and PNA. “Nucleic acid sequence” refers to the linear sequence of nucleotides that comprise the nucleic acid molecule or polynucleotide.

[0041] As used herein, an “H-NOX protein” means a protein that has an H-NOX domain (named for Heme-Nitric oxide and OXygen binding domain). An H-NOX protein may or may not contain one or more other domains in addition to the H-NOX domain. In some examples, an H-NOX protein does not comprise a guanylyl cyclase domain. An H-NOX protein may or may not comprise a polymerization domain.

[0042] As used herein, a “polymeric H-NOX protein” is an H-NOX protein comprising two or more H-NOX domains. The H-NOX domains may be covalently or non-covalently associated. [0043] As used herein, an “H-NOX domain” is all or a portion of a protein that binds nitric oxide and/or oxygen by way of heme. The H-NOX domain may comprise heme or may be found as an apoproprotein that is capable of binding heme. In some examples, an H-NOX domain includes six alpha-helices, followed by two beta-strands, followed by one alpha-helix, followed by two beta strands. In some examples, an H-NOX domain corresponds to the H-NOX domain of Thermoanaerobacter tengcongensis H-NOX set forth in SEQ ID NO:2. For example, the H-NOX domain may be at least about 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% identical to the H-NOX domain of Thermoanaerobacter tengcongensis H-NOX set forth in SEQ ID NO:2. In some embodiments, the H-NOX domain may be 10%- 20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-99% or 100% identical to the H-NOX domain of Thermoanaerobacter tengcongensis H- NOX set forth in SEQ ID NO:2.

[0044] As used herein, a “polymerization domain” is a domain (e.g. a polypeptide domain) that promotes the association of monomeric moi eties to form a polymeric structure. For example, a polymerization domain may promote the association of monomeric H-NOX domains to generate a polymeric H-NOX protein. An exemplary polymerization domain is the foldon domain of T4 bacteriophage, which promotes the formation of trimeric polypeptides. Other examples of polymerization domains include, but are not limited to, Arc, POZ, coiled coil domains (including GCN4, leucine zippers, Velcro), uteroglobin, collagen, 3-stranded coiled coils (matrilin-1), thrombospondins TRPV1-C, P53, Mnt, avidin, streptavidin, Bcr-Abl, COMP, verotoxin subunit B, CamKII, RCK, and domains from N ethylmaleimide-sensitive fusion protein, STM3548, KaiC, TyrR, Hcpl, CcmK4, GP41, anthrax protective antigen, aerolysin, a- hemolysin, C4b-binding protein, Mi-CK, arylsulfatase A, and viral capsid proteins.

[0045] As used herein, an “amino acid linker sequence” or an “amino acid spacer sequence” is a short polypeptide sequence that may be used to link two domains of a protein. In some embodiments, the amino acid linker sequence is one, two, three, four, five, six, seven, eight, nine, ten or more than ten amino acids in length. Exemplary amino acid linker sequences include but are not limited to a Gly-Ser-Gly sequence and an Arg-Gly-Ser sequence.

[0046] As used herein, a “Hise tag” refers to a peptide comprising six His residues attached to a polypeptide. A Hise tag may be used to facilitate protein purification; for example, using chromatography specific for the Hise tag. Following purification, the Hise tag may be cleaved using an exopeptidase.

[0047] The term “substantially similar” or “substantially the same,” as used herein, denotes a sufficiently high degree of similarity between two or more numeric values such that one of skill in the art would consider the difference between the two or more values to be of little or no biological and/or statistical significance within the context of the biological characteristic measured by said value. In some embodiments the two or more substantially similar values differ by no more than about any one of 5%, 10%, 15%, 20%, 25%, or 50%.

[0048] The phrase “substantially reduced,” or “substantially different,” as used herein, denotes a sufficiently high degree of difference between two numeric values such that one of skill in the art would consider the difference between the two values to be of statistical significance within the context of the biological characteristic measured by said values. In some embodiments, the two substantially different numeric values differ by greater than about any one of 10%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%. In some embodiment, the two substantially different numeric values differ by about any one of 10%-20%, 20%-30%, 30%- 40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-99% or 100%. [0049] A “native sequence” polypeptide comprises a polypeptide having the same amino acid sequence as a polypeptide found in nature. Thus, a native sequence polypeptide can have the amino acid sequence of naturally occurring polypeptide from any organism. Such native sequence polypeptide can be isolated from nature or can be produced by recombinant or synthetic means. The term “native sequence” polypeptide specifically encompasses naturally occurring truncated or secreted forms of the polypeptide e.g., an extracellular domain sequence), naturally occurring variant forms (e.g., alternatively spliced forms) and naturally occurring allelic variants of the polypeptide.

[0050] A polypeptide “variant” means a biologically active polypeptide having at least about 80% amino acid sequence identity with the native sequence polypeptide after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Such variants include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of the polypeptide. In some embodiments, a variant will have at least about any one of 80%, 90% or 95% amino acid sequence identity with the native sequence polypeptide. In some embodiments, a variant will have about any one of 80%- 90%, 90%-95% or 95% -99% amino acid sequence identity with the native sequence polypeptide.

[0051] As used herein, a “mutant protein” means a protein with one or more mutations compared to a protein occurring in nature. In one embodiment, the mutant protein has a sequence that differs from that of all proteins occurring in nature. In various embodiments, the amino acid sequence of the mutant protein is at least about any of 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 97, 98, 99, or 99.5% identical to that of the corresponding region of a protein occurring in nature. In some embodiments, the amino acid sequence of the mutant protein is at least about any of 10%-20%, 20%-30%, 30%-40%, 40%-50%, 50%-60%, 60%-70%, 70%-80%, 80%-90%, 90%-95%, 95%-99% or 100% identical to that of the corresponding region of a protein occurring in nature. In some embodiments, the mutant protein is a protein fragment that contains at least about any of 25, 50, 75, 100, 150, 200, 300, or 400 contiguous amino acids from a full-length protein. In some embodiments, the mutant protein is a protein fragment that contains about any of 25-50, 50-75, 75-100, 100-150, 150-200, 200-300, or 300-400 contiguous amino acids from a full-length protein. Sequence identity can be measured, for example, using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). This software program matches similar sequences by assigning degrees of homology to various amino acids replacements, deletions, and other modifications.

[0052] As used herein, a “mutation” means an alteration in a reference nucleic acid or amino acid sequence occurring in nature. Exemplary nucleic acid mutations include an insertion, deletion, frameshift mutation, silent mutation, nonsense mutation, or missense mutation. In some embodiments, the nucleic acid mutation is not a silent mutation. Exemplary protein mutations include the insertion of one or more amino acids (e.g., the insertion of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), the deletion of one or more amino acids (e.g., a deletion of N-terminal, C-terminal, and/or internal residues, such as the deletion of at least about any of 5, 10, 15, 25, 50, 75, 100, 150, 200, 300, or more amino acids or a deletion of about any of 5-10, 10-15, 15-25, 25-50, 50-75, 75-100, 100-150, 150-200, 200-300, or 300-400 amino acids), the replacement of one or more amino acids (e.g., the replacement of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), or combinations of two or more of the foregoing. The nomenclature used in referring to a particular amino acid mutation first identifies the wild-type amino acid, followed by the residue number and finally the substitute amino acid. For example, Y140L means that tyrosine has been replaced by a leucine at residue number 140. Likewise, a variant H-NOX protein may be referred to by the amino acid variations of the H-NOX protein. For example, a T. tengcongensis Y140L H-NOX protein refers to a T. tengcongensis H-NOX protein in which the tyrosine residue at position number 140 has been replaced by a leucine residue and a T. tengcongensis W9F/Y140L H-NOX protein refers to a T. tengcongensis H-NOX protein in which the tryptophan residue at position 9 has been replaced by a phenylalanine residue and the tyrosine residue at position number 140 has been replaced by a leucine residue. [0053] An “evolutionary conserved mutation” is the replacement of an amino acid in one protein by an amino acid in the corresponding position of another protein in the same protein family.

[0054] As used herein, “derived from” refers to the source of the protein into which one or more mutations is introduced. For example, a protein that is “derived from a mammalian protein” refers to protein of interest that results from introducing one or more mutations into the sequence of a wild-type (/.< ., a sequence occurring in nature) mammalian protein.

[0055] As used herein, “Percent (%) amino acid sequence identity” and “homology” with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGNTM (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0056] As used herein, a “k _O ff” refers to a dissociation rate, such as the rate of release of O2 or NO from a protein. A lower numerical lower k _O ff indicates a slower rate of dissociation.

[0057] As used herein, “kon” refers to an association rate, such as the rate of binding of O2 or NO to a protein. A lower numerical lower k _on indicates a slower rate of association.

[0058] As used herein, “dissociation constant” refers to a “kinetic dissociation constant” or a “calculated dissociation constant.” A “kinetic dissociation constant” or “KD” is a ratio of kinetic off-rate (k _O ff) to kinetic on-rate (kon), such as a KD value determined as an absolute value using standard methods (e.g., standard spectroscopic, stopped-flow, or flash-photolysis methods) including methods known to the skilled artisan and/or described herein. “Calculated dissociation constant” or “calculated KD” refers to an approximation of the kinetic dissociation constant based on a measured k _O ff. A value for the k _on is derived via the correlation between kinetic KD and k _O ff as described herein. [0059] As used herein, “oxygen affinity” is a qualitative term that refers to the strength of oxygen binding to the heme moiety of a protein. This affinity is affected by both the k _O ff and k _on for oxygen. A numerically lower oxygen KD value means a higher affinity.

[0060] As used herein, “NO affinity” is a qualitative term that refers to the strength of NO binding to a protein (such as binding to a heme group or to an oxygen bound to a heme group associated with a protein). This affinity is affected by both the k _O ff and k _on for NO. A numerically lower NO KD value means a higher affinity.

[0061] As used herein, “NO stability” refers to the stability or resistance of a protein to oxidation by NO in the presence of oxygen. For example, the ability of the protein to not be oxidized when bound to NO in the presence of oxygen is indicative of the protein’s NO stability. In some embodiments, less than about any of 50, 40, 30, 10, or 5% of an H-NOX protein is oxidized after incubation for about any of 1, 2, 4, 6, 8, 10, 15, or 20 hours at 20 °C.

[0062] As used herein, “NO reactivity” refers to the rate at which iron in the heme of a hemebinding protein is oxidized by NO in the presence of oxygen. A lower numerical value for NO reactivity in units of s' ¹ indicates a lower NO reactivity

[0063] As used herein, an “autoxidation rate” refers to the rate at which iron in the heme of a heme-binding protein is autoxidized. A lower numerical autoxidation rate in units of s' ¹ indicates a lower autoxidation rate.

[0064] The term “vector” is used to describe a polynucleotide that may be engineered to contain a cloned polynucleotide or polynucleotides that may be propagated in a host cell. A vector may include one or more of the following elements: an origin of replication, one or more regulatory sequences (such as, for example, promoters and/or enhancers) that regulate the expression of the polypeptide of interest, and/or one or more selectable marker genes (such as, for example, antibiotic resistance genes and genes that may be used in colorimetric assays, e.g., P-galactosidase). The term “expression vector” refers to a vector that is used to express a polypeptide of interest in a host cell.

[0065] A “host cell” refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Exemplary prokaryotic cells include bacterial cells; for example, E. coli cells. [0066] The term “isolated” as used herein refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or produced. For example, a polypeptide is referred to as “isolated” when it is separated from at least some of the components of the cell in which it was produced. Where a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be “isolating” the polypeptide. Similarly, a polynucleotide is referred to as “isolated” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, e.g., in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as “isolated”.

[0067] The terms “individual” or “subject” are used interchangeably herein to refer to an animal; for example a mammal. In some embodiments, methods of treating mammals, including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are provided. In some examples, an “individual” or “subject” refers to an individual or subject in need of treatment for a disease or disorder.

[0068] A “disease” or “disorder” as used herein refers to a condition where treatment is needed.

[0069] The terms “inhibition” or “inhibit” refer to a decrease or cessation of any phenotypic characteristic or to the decrease or cessation in the incidence, degree, or likelihood of that characteristic. To “reduce” or “inhibit” is to decrease, reduce or arrest an activity, function, and/or amount as compared to a reference. In certain embodiments, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 20% or greater. In another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 50% or greater. In yet another embodiment, by “reduce” or “inhibit” is meant the ability to cause an overall decrease of 75%, 85%, 90%, 95%, or 99%.

[0070] A “reference” as used herein, refers to any sample, standard, or level that is used for comparison purposes. A reference may be obtained from a healthy and/or non-diseased sample. In some examples, a reference may be obtained from an untreated sample. In some examples, a reference is obtained from a non-diseased on non-treated sample of a subject individual. In some examples, a reference is obtained from one or more healthy individuals who are not the subject or patient.

[0071] “Preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease.

[0072] An “effective amount” of an agent refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

[0073] The terms “pharmaceutical formulation” and “pharmaceutical composition” refer to a preparation which is in such form as to permit the biological activity of the active ingredient(s) to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. Such formulations may be sterile and essentially free of endotoxins.

[0074] A “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a “pharmaceutical composition” for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed.

[0075] A “sterile” formulation is aseptic or essentially free from living microorganisms and their spores.

[0076] Administration “in combination with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive or sequential administration in any order.

[0077] The term “concurrently” is used herein to refer to administration of two or more therapeutic agents, where at least part of the administration overlaps in time or where the administration of one therapeutic agent falls within a short period of time relative to administration of the other therapeutic agent. For example, the two or more therapeutic agents are administered with a time separation of no more than about 60 minutes, such as no more than about any of 30, 15, 10, 5, or 1 minutes.

[0078] The term “sequentially” is used herein to refer to administration of two or more therapeutic agents where the administration of one or more agent(s) continues after discontinuing the administration of one or more other agent(s). For example, administration of the two or more therapeutic agents are administered with a time separation of more than about 15 minutes, such as about any of 20, 30, 40, 50, or 60 minutes, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 1 month.

[0079] As used herein, “in conjunction with” refers to administration of one treatment modality in addition to another treatment modality. As such, “in conjunction with” refers to administration of one treatment modality before, during or after administration of the other treatment modality to the individual.

[0080] The term “package insert” is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

[0081] An “article of manufacture” is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder (e.g., cancer), or a probe for specifically detecting a biomarker described herein. In certain embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.

H-NOX Proteins

Overview of H-NOX Protein Family

[0082] Unless otherwise indicated, any wild-type or mutant H-NOX protein can be used in the compositions, kits, and methods as described herein. As used herein, an “H-NOX protein” means a protein that has an H-NOX domain (named for Heme-Nitric oxide and OXygen binding domain). An H-NOX protein may or may not contain one or more other domains in addition to the H-NOX domain. H-NOX proteins are members of a highly-conserved, well-characterized family of hemoproteins (Iyer, L. M. et al. (February 3, 2003). BMC Genomics 4(1):5; Karow, D. S. et al. (August 10, 2004). Biochemistry 43(31): 10203-10211; Boon, E. M. et al. (2005). Nature Chem. Biol. 1 :53-59; Boon, E. M. et al. (October 2005). Curr. Opin. Chem. Biol. 9(5):441-446; Boon, E. M. et al. (2005). J. Inorg. Biochem. 99(4): 892-902). H-NOX proteins are also referred to as Pfam 07700 proteins or HNOB proteins (Pfam - A database of protein domain family alignments and Hidden Markov Models, Copyright (C) 1996-2006 The Pfam Consortium; GNU LGPL Free Software Foundation, Inc., 59 Temple Place - Suite 330, Boston, MA 02111-1307, USA). In some embodiments, an H-NOX protein has, or is predicted to have, a secondary structure that includes six alpha-helices, followed by two beta-strands, followed by one alpha-helix, followed by two beta-strands. An H-NOX protein can be an apoprotein that is capable of binding heme or a holoprotein with heme bound. An H-NOX protein can covalently or non-covalently bind a heme group. Some H-NOX proteins bind NO but not O2, and others bind both NO and O2. H-NOX domains from facultative aerobes that have been isolated bind NO but not O2. H-NOX proteins from obligate aerobic prokaryotes, C. elegans, and D. melanogaster bind NO and O2. Mammals have two H-NOX proteins: pi and P2. An alignment of mouse, rat, cow, and human H-NOX sequences shows that these species share >99% identity. In some embodiments, the H-NOX domain of an H-NOX protein or the entire H-NOX protein is at least about any of 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 97, 98, 99, or 99.5% identical to that of the corresponding region of a naturally-occurring Thermoanaerobacter tengcongensis H-NOX protein (e.g. SEQ ID NO:2) or a naturally-occurring sGC protein (e.g., a naturally- occurring sGC pi protein). In some embodiments, the H-NOX domain of an H-NOX protein or the entire H-NOX protein is at least about any of 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99, or 99-99.9% identical to that of the corresponding region of a naturally-occurring Thermoanaerobacter tengcongensis H-NOX protein (e.g. SEQ ID NO:2) or a naturally-occurring sGC protein (e.g., a naturally-occurring sGC pi protein). As discussed further herein, an H-NOX protein may optionally contain one or more mutations relative to the corresponding naturally-occurring H-NOX protein. In some embodiments, the H- NOX protein includes one or more domains in addition to the H-NOX domain. In particular embodiments, the H-NOX protein includes one or more domains or the entire sequence from another protein. For example, the H-NOX protein may be a fusion protein that includes an H- NOX domain and part or all of another protein, such as albumin (e.g., human serum albumin). In some embodiments, only the H-NOX domain is present. In some embodiments, the H-NOX protein does not comprise a guanylyl cyclase domain. In some embodiments, the H-NOX protein comprises a tag; for example, a Hise tag.

Polymeric H-NOX proteins

[0083] In some aspects, the disclosure provides polymeric H-NOX proteins comprising two or more H-NOX domains. The two or more H-NOX domains may be covalently linked or noncovalently linked. In some embodiments, the polymeric H-NOX protein is in the form of a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octomer, a nanomer, or a decamer. In some embodiments, the polymeric H-NOX protein comprises homologous H-NOX domains. In some embodiments, the polymeric H-NOX protein comprises heterologous H-NOX domains; for example, the H-NOX domains may comprise amino acid variants of a particular species of H-NOX domain or may comprise H-NOX domains from different species. In some embodiments, at least one of the H-NOX domains of a polymeric H-NOX protein comprises a mutation corresponding to an L144F mutation of T. tengcongensis H-NOX. In some embodiments, at least one of the H-NOX domains of a polymeric H-NOX protein comprises a mutation corresponding to a W9F/L144F mutation of T. tengcongensis H-NOX. In some embodiments, the polymeric H-NOX proteins comprise one or more polymerization domains. In some embodiments, the polymeric H-NOX protein is a trimeric H-NOX protein. In some embodiments, the polymeric H-NOX protein comprises at least one trimerization domain. In some embodiments, the trimeric H-NOX protein comprises three T. tengcongensis H-NOX domains. In some embodiments the trimeric H-NOX domain comprises three T. tengcongensis L144F H-NOX domains. In some embodiments the trimeric H-NOX domain comprises three T. tengcongensis W9F/L144F H-NOX domains

[0084] In some aspects of the disclosure, the polymeric H-NOX protein comprises two or more associated monomers. The monomers may be covalently linked or noncovalently linked. In some embodiments, monomeric subunits of a polymeric H-NOX protein are produced where the monomeric subunits associate in vitro or in vivo to form the polymeric H-NOX protein. In some embodiments, the monomers comprise an H-NOX domain and a polymerization domain. In some embodiments, the polymerization domain is covalently linked to the H-NOX domain; for example, the C-terminus of the H-NOX domain is covalently linked to the N-terminus or the C-terminus of the polymerization domain. In other embodiments, the N-terminus of the H-NOX domain is covalently linked to the N-terminus or the C-terminus of the polymerization domain. In some embodiments, an amino acid spacer is covalently linked between the H-NOX domain and the polymerization domain. An “amino acid spacer” and an “amino acid linker” are used interchangeably herein. In some embodiments, at least one of the monomeric subunits of a polymeric H-NOX protein comprises a mutation corresponding to an L144F mutation of T. tengcongensis H-NOX. In some embodiments, at least one of the monomeric subunits of a polymeric H-NOX protein comprises a mutation corresponding to a W9F/L144F mutation of T. tengcongensis H-NOX. In some embodiments the polymeric H-NOX protein is a trimeric H- NOX protein. In some embodiments, the monomer of a trimeric H-NOX protein comprises an H-NOX domain and a fol don domain of T4 bacteriophage. In some embodiments, the monomer of a trimeric H-NOX protein comprises a T. tengcongensis H-NOX domain and a foldon domain. In some embodiments, the monomer of a trimeric H-NOX protein comprises a T. tengcongensis L144F H-NOX domain and a foldon domain. In some embodiments, the monomer of a trimeric H-NOX protein comprises a T. tengcongensis W9F/L144F H-NOX domain and a foldon domain. In some embodiments, the trimer H-NOX protein comprises three monomers, each monomer comprising a T. tengcongensis L144F H-NOX domain and a foldon domain. In some embodiments, the H-NOX domain is linked to the foldon domain with an amino acid linker; for example a Gly-Ser-Gly linker. In some embodiments, at least one H- NOX domain comprises a tag. In some embodiments, at least one H-NOX domain comprises a Hise tag. In some embodiments, the Hise tag is linked to the foldon domain with an amino acid linker; for example an Arg-Gly-Ser linker. In some embodiments, all of the H-NOX domains comprise a Hise tag. In some embodiments, the trimeric H-NOX protein comprises the amino acid sequence set forth in SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:26 or SEQ ID NO:29.

[0085] The exemplary H-NOX domain from T. tengcongensis is approximately 26.7 kDal. In some embodiments, the polymeric H-NOX protein has an atomic mass greater than any of about 50 kDal, 75 kDal, 100 kDal, 125kDal, to about 150 kDal.

Sources of H-NOX Proteins and H-NOX domains

[0086] H-NOX proteins and H-NOX domains from any genus or species can be used in the compositions, kits, and methods described herein. In various embodiments, the H-NOX protein or the H-NOX domains of a polymeric H-NOX protein is a protein or domain from a mammal (e.g., a primate (e.g., human, monkey, gorilla, ape, lemur, etc), a bovine, an equine, a porcine, a canine, or a feline), an insect, a yeast, or a bacteria or is derived from such a protein. Exemplary mammalian H-NOX proteins include wild-type human and rat soluble guanylate cyclase (such as the pi subunit). Examples of H-NOX proteins include wild-type mammalian H-NOX proteins, e.g. H. sapiens, M. musculus, C. familiaris, B. Taurus, C. lupus and R. norvegicus; and wild-type non-mammalian vertebrate H-NOX proteins, e.g,. X. laevis, O. latipes, O. curivatus, and . rubripes. Examples of non-mammalian wild-type NO-binding H-NOX proteins include wildtype H-NOX proteins of D. melanogaster , A. gam bias, and M. sex la examples of nonmammalian wild-type Ch-binding H-NOX proteins include wild-type H-NOX proteins of C. elegans gcy-31, gcy-32, gcy-33, gcy-34, gcy-35, gcy-36, and gcy-37; D. melanogaster CG14885, CG14886, and CG4154; and M. sexta beta-3; examples of prokaryotic wild-type H- NOX proteins include T. lengcongensis. V. cholera, V. fischerii, N. punctiforme, D. de sulfur leans, L. pneumophila 1, L. pneumophila 2, and C. acetobutylicum.

[0087] NCBI Accession numbers for exemplary H-NOX proteins include the following: Homo sapiens 01 [gi :2746083], Rattus norvegicus 01 [gi :27127318], Drosophila melangaster 01 [gi:861203], Drosophila melangaster CG14885-PA [gi:23171476], Caenorhabditis elegans GCY-35 [fv.5‘ 2T&2'&Q6\, Nostocpunctiforme [gi:23129606], Caulobacter crescentus [gi: 16127222], Shewanella oneidensis [gi:24373702], Legionella pneumophila (ORF 2) [CUCGC 272624], Clostridium acetobutylicum [gi: 15896488], and Thermoanaerobacter tengcongensis [gi:20807169], Canis lupus H-NOX is provided by GenBank accession DQ008576. Nucleic acid and amino acid sequences of exemplary H-NOX proteins and domains are provided in Figure 37.

[0088] Exemplary H-NOX protein also include the following H-NOX proteins that are listed by their gene name, followed by their species abbreviation and Genbank Identifiers (such as the following protein sequences available as of May 21, 2006; May 22, 2006; May 21, 2007; or May 22, 2007, which are each hereby incorporated by reference in their entireties): Npun5905_Npu_23129606, alr2278_Ana_l 7229770, SO2144_Sone_24373702, Mdegl 343_Mde_23027521 , VCA0720_Vch_l 5601476,CC2992_Ccr_l 6127222, Rsph2043_Rhsp_22958463 (gi:46192757), Mmcl0739_Mcsp_22999020, Tar4_Tte_20807169,Ddes2822_Dde_23475919,CAC3243_Cac_15896488 ,gcy- 31_Ce_17568389, CG14885_Dm_24647455,GUCYlB3_Hs_4504215, HpGCS- betal_Hpul_14245738, Gycbetal00B_Dm_24651577, CG4154_Dm_24646993 (gi:NP_650424.2, gi:62484298), gcy-32_Ce_13539160,gcy-36_Ce_17568391 (gi:32566352, gi:86564713), gcy-35_Ce- 17507861 (gi:71990146), gcy-37_Ce_l 7540904 (gi:71985505), GCYla3_Hs_20535603, GCYla2-Hs_899477, or GYCa-99B_Dm_729270 (gi:68067738) (Lakshminarayan et al. (2003). BMG Genomics 4:5-13). The species abbreviations used in these names include Ana - Anabaena Sp: Ccr - Caulobacter crescentus,' Cac - Clostridium acetobutylicum; Dde - Desulfovibrio de sulfur icons Mcsp - Magnetococcus sp. Mde - Microbulbifer degradans Npu - Nostoc punctiforme; Rhsp - Rhodobacter sphaeroides; Sone - Shewanella oneidensis: Tte - Thermoanaerobacter tengcongensis; Veh - Vibrio cholerae Ce - Caenorhabditis elegans; Dm - Drosophila melanogaster Hpul - Hemicentrotus pulcherrimus Hs - Homo sapiens.

[0089] Other exemplary H-NOX proteins include the following H-NOX proteins that are listed by their organism name and Pfam database accession number (such as the following protein sequences available as of May 21, 2006; May 22, 2006; May 17, 2007; May 21, 2007; or May 22, 2007, which are each hereby incorporated by reference in their entireties): Caenorhabditis briggsae Q622M5 CAEBR, Caenorhabditis briggsae Q61P44 CAEBR, Caenorhabditis briggsae Q61R54 CAEBR, Caenorhabditis briggsae Q61V90 CAEBR, Caenorhabditis briggsae Q61A94 CAEBR, Caenorhabditis briggsae Q60TP4 CAEBR, Caenorhabditis briggsae Q60M10 CAEBR, Caenorhabditis elegans GCY37 CAEEL, Caenorhabditis elegans GCY31 CAEEL, Caenorhabditis elegans GCY36 CAEEL, Caenorhabditis elegans GCY32 CAEEL, Caenorhabditis elegans GCY35 CAEEL, Caenorhabditis elegans

GCY34 CAEEL, Caenorhabditis elegans GCY33 CAEEL, Oryzias curvinotus Q7T040 0RYCU, Oryzias curvinotus Q75WFO ORYCU, Oryzias latipes P79998 ORYLA, Oryzias latipes Q7ZSZ5 ORYLA, Tetraodon nigroviridis Q4SW38 TETNG, Tetraodon nigroviridis Q4RZ94 TETNG, Tetraodon nigroviridis Q4S6K5 TETNG, Fugu rubripes Q90VY5 FUGRU, Xenopus laevis Q6INK9 XENLA, Homo sapiens Q5T8J7 HUMAN, Homo sapiens GCYA2 HUMAN, Homo sapiens GCYB2 HUMAN, Homo sapiens GCYB1 HUMAN, Gorilla gorilla Q9N193 9PRIM, Pongo pygmaeus Q5RAN8 PONPY, Pan troglodytes Q9N192_PANTR, Macaca mulatto Q9N194_MACMU, Hylobates lar Q9N191 HYLLA, Mus musculus Q8BXH3 MOUSE, Mus musculus GCYB1 MOUSE, Mus musculus Q3UTI4 MOUSE, Mus musculus Q3UH83 MOUSE, Mus musculus Q6XE41 MOUSE, Mus musculus Q80YP4 MOUSE, Rattus norvegicus Q80WX7 RAT, Rattus norvegicus Q80WX8 RAT, Rattus norvegicus Q920Q1 RAT, Rattus norvegicus Q54A43 RAT, Rattus norvegicus Q80WY0 RAT, Rattus norvegicus Q80WY4 RAT, Rattus norvegicus Q8CH85 RAT, Rattus norvegicus Q80WY5 RAT, Rattus norvegicus GCYB1 RAT, Rattus norvegicus Q8CH90 RAT, Rattus norvegicus Q91XJ7 RAT, Rattus norvegicus Q80WX9 RAT, Rattus norvegicus GCYB2 RAT, Rattus norvegicus GCYA2 RAT, Canis familiaris Q4ZHR9 CANFA, Bos taurus GCYB I BOVIN, Sus scrofa Q4ZHR7 PIG, Gryllus bimaculatus Q59HN5 GRYBI, Manduca sexta O77106 MANSE, Manduca sexta O76340_MANSE, Apis mellifera Q5UAF0 APIME, Apis mellifera Q5FAN0_APIME, Apis mellifera Q6L5L6 APIME, Anopheles gambiae str PEST Q7PYK9 ANOGA, Anopheles gambiae str PEST Q7Q9W6 ANOGA, Anopheles gambiae str PEST Q7QF31 ANOGA, Anopheles gambiae str PEST Q7PS01 ANOGA, Anopheles gambiae str PEST

Q7PFY2 ANOGA, Anopheles gambiae Q7KQ93 ANOGA, Drosophila melanogaster Q24086 DROME, Drosophila melanogaster GCYH DROME, Drosophila melanogaster GCY8E DROME, Drosophila melanogaster GCYDA DROME, Drosophila melanogaster GCYDB DROME, Drosophila melanogaster Q9VA09 DROME, Drosophila pseudoobscura Q29CE1 DROPS, Drosophila pseudoobscura Q296C7 DROPS, Drosophila pseudoobscura Q296C8 DROPS, Drosophila pseudoobscura Q29BU7 DROPS, Aplysia californica Q7YWK7 APLCA, Hemicentrotus pulcherrimus Q95NK5 HEMPU, Chlamydomonas reinhardtii, Q5YLC2 CHLRE, Anabaena sp Q8 YUQ7 ANASP, Flavobacteria bacterium BBFL7 Q26GR8 9BACT, Psychroflexus torquis ATCC 700755 Q1VQE5 9FLAO, marine gamma proteobacterium HTCC2207 Q1YPJ5 9GAMM, marine gamma proteobacterium HTCC2207 Q1YTK4 9GAMM, Caulobacter crescentus Q9A451 CAUCR, Acidiphilium cryptum JF-5 Q2DG60 ACICY, Rhodobacter sphaeroides Q3JOU9 RHOS4, Silicibacter pomeroyi Q5LPV1 SILPO, Paracoccus denitrificans PD1222, Q3PC67 PARDE, Silicibacter sp TM1040 Q3QNY2 9RHOB, Jannaschia sp Q28ML8 JANSC, Magnetococcus sp MC-1 Q3XT27 9PROT, Legionella pneumophila Q5WXP0 LEGPL, Legionella pneumophila Q5WTZ5 LEGPL, Legionella pneumophila Q5X268 LEGPA, Legionella pneumophila Q5X2R2 LEGPA, Legionella pneumophila subsp pneumophila Q5ZWM9 LEGPH, Legionella pneumophila subsp pneumophila Q5ZSQ8 LEGPH, Colwellia psychrerythraea

Q47Y43 COLP3, Pseudoalter omonas atlantica T6c Q3CSZ5 ALTAT, Shewanella oneidensis Q8EF49 SHEON, Saccharophagus degradans Q21E20 SACD2, Saccharophagus degradans Q21ER7 SACD2, Vibrio angustum S14 Q1ZWE5 9VIBR, Vibrio vulnificus Q8DAE2 VIBVU, Vibrio alginolyticus 12G01 Q1VCP6 VIBAL, Vibrio sp DAT722 Q2FA22 9VIBR, Vibrio parahaemolyticus Q87NJ1 VIBPA, Vibrio fischeri Q5E1F5 VIBF1, Vibrio vulnificus Q7MJS8 VIBVY, Photobacterium sp SKA34 Q2C6Z5 9GAMM, Hahella chejuensis

Q2SFY7 HAHCH, Oceanospirillum sp MED92 Q2BKV0 9GAMM, Oceanobacter sp RED65 Q1N035 9GAMM, Desulfovibrio desulfuricans Q310U7 DESDG, Halothermothrix orenii H 168 Q2AIW5 9FIRM, Thermoanaerobacter tengcongensis Q8RBX6 THETN, Caldicellulosiruptor saccharolyticus DSM 8903 Q2ZH17 CALSA, Clostridium acetobutylicum Q97E73 CLOAB, Alkaliphilus metalliredigenes QYMF Q3C763 9CLOT, Clostridium tetani Q899J9 CLOTE, and Clostridium beijerincki NCIMB 8052 Q2WVNO CLOBE. These sequences are predicted to encode H-NOX proteins based on the identification of these proteins as belonging to the H-NOX protein family using the Pfam database as described herein.

[0090] Additional H-NOX proteins, H-NOX domains of polymeric H-NOX proteins, and nucleic acids, which may be suitable for use in the pharmaceutical compositions and methods described herein, can be identified using standard methods. For example, standard sequence alignment and/or structure prediction programs can be used to identify additional H-NOX proteins and nucleic acids based on the similarity of their primary and/or predicted protein secondary structure with that of known H-NOX proteins and nucleic acids. For example, the Pfam database uses defined alignment algorithms and Hidden Markov Models (such as Pfam 21.0) to categorize proteins into families, such as the H-NOX protein family (Pfam - A database of protein domain family alignments and Hidden Markov Models, Copyright (C) 1996-2006 The Pfam Consortium; GNU LGPL Free Software Foundation, Inc., 59 Temple Place - Suite 330, Boston, MA 02111-1307, USA). Standard databases such as the swissprot-trembl database (world-wide web at “expasy.org”, Swiss Institute of Bioinformatics Swiss-Prot group CMU - 1 rue Michel Servet CH-1211 Geneva 4, Switzerland) can also be used to identify members of the H-NOX protein family. The secondary and/or tertiary structure of an H-NOX protein can be predicted using the default settings of standard structure prediction programs, such as PredictProtein (630 West, 168 Street, BB217, New York, N.Y. 10032, USA). Alternatively, the actual secondary and/or tertiary structure of an H-NOX protein can be determined using standard methods.

[0091] In some embodiments, the H-NOX domain has the same amino acid in the corresponding position as any of following distal pocket residues in T. tengcongensis H-NOX: Thr4, Ile5, Thr8, Trp9, Trp67, Asn74, Ile75, Phe78, Phe82, Tyrl40, Leul44, or any combination of two or more of the foregoing. In some embodiments, the H-NOX domain has a proline or an arginine in a position corresponding to that of Prol 15 or Argl35 of T. tengcongensis H-NOX, respectively, based on sequence alignment of their amino acid sequences. In some embodiments, the H-NOX domain has a histidine that corresponds to His 105 of R. norvegicus pi H-NOX. In some embodiments, the H-NOX domain has or is predicted to have a secondary structure that includes six alpha-helices, followed by two beta-strands, followed by one alphahelix, followed by two beta-strands. This secondary structure has been reported for H-NOX proteins.

[0092] If desired, a newly identified H-NOX protein or H-NOX domain can be tested to determine whether it binds heme using standard methods. The ability of an H-NOX domain to function as an O2 carrier can be tested by determining whether the H-NOX domain binds O2 using standard methods, such as those described herein. If desired, one or more of the mutations described herein can be introduced into the H-NOX domain to optimize its characteristics as an O2 carrier. For example, one or more mutations can be introduced to alter its O2 dissociation constant, k _O ff for oxygen, rate of heme autoxidation, NO reactivity, NO stability or any combination of two or more of the foregoing. Standard techniques such as those described herein can be used to measure these parameters.

Mutant H-NOX Proteins

[0093] As discussed further herein, an H-NOX protein or an H-NOX domain of a polymeric H-NOX protein may contain one or more mutations, such as a mutation that alters the O2 dissociation constant, the k _O ff for oxygen, the rate of heme autoxidation, the NO reactivity, the NO stability, or any combination of two or more of the foregoing compared to that of the corresponding wild-type protein. In some embodiments, the disclosure provides a polymeric H- NOX protein comprising one or more H-NOX domains that may contain one or more mutations, such as a mutation that alters the O2 dissociation constant, the k _O ff for oxygen, the rate of heme autoxidation, the NO reactivity, the NO stability, or any combination of two or more of the foregoing compared to that of the corresponding wild-type protein. Panels of engineered H- NOX domains may be generated by random mutagenesis followed by empirical screening for requisite or desired dissociation constants, dissociation rates, NO-reactivity, stability, physiocompatibility, or any combination of two or more of the foregoing in view of the teaching provided herein using techniques as described herein and, additionally, as known by the skilled artisan. Alternatively, mutagenesis can be selectively targeted to particular regions or residues such as distal pocket residues apparent from the experimentally determined or predicted three- dimensional structure of an H-NOX protein (see, for example, Boon, E. M. et al. (2005). Nature Chemical Biology 1 :53-59, which is hereby incorporated by reference in its entirety, particularly with respect to the sequences of wild-type and mutant H-NOX proteins) or evolutionarily conserved residues identified from sequence alignments (see, for example, Boon E.M. et al. (2005). Nature Chemical Biology 1 :53-59, which is hereby incorporated by reference in its entirety, particularly with respect to the sequences of wild-type and mutant H-NOX proteins). [0094] In some embodiments of the disclosure, the mutant H-NOX protein or mutant H-NOX domain of a polymeric H-NOX protein has a sequence that differs from that of all H-NOX proteins or domains occurring in nature. In various embodiments, the amino acid sequence of the mutant protein is at least about any of 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 95, 97, 98, 99, or 99.5% identical to that of the corresponding region of an H-NOX protein occurring in nature. In various embodiments, the amino acid sequence of the mutant protein is about 10- 20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-95%, 95-99%, or 99.5% identical to that of the corresponding region of an H-NOX protein occurring in nature. In some embodiments, the mutant protein is a protein fragment that contains at least about any of 25, 50, 75, 100, 150, 200, 300, or 400 contiguous amino acids from a full-length protein. In some embodiments, the mutant protein is a protein fragment that contains 25-50, 50-75, 75-100, 100-150, 150-200, 200-300, or 300-400 contiguous amino acids from a full-length protein. Sequence identity can be measured, for example, using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). This software program matches similar sequences by assigning degrees of homology to various amino acids replacements, deletions, and other modifications.

[0095] In some embodiments of the disclosure, the mutant H-NOX protein or mutant H-NOX domain of a polymeric H-NOX protein comprises the insertion of one or more amino acids (e.g., the insertion of 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids). In some embodiments of the disclosure, the mutant H-NOX protein or mutant H-NOX domain comprises the deletion of one or more amino acids (e.g., a deletion of N-terminal, C-terminal, and/or internal residues, such as the deletion of at least about any of 5, 10, 15, 25, 50, 75, 100, 150, 200, 300, or more amino acids or a deletion of 5-10, 10-15, 15-25, 25-50, 50-75, 75-100, 100-150, 150-200, 200-300, or 300-400 amino acids). In some embodiments of the disclosure, the mutant H-NOX protein or mutant H- NOX domain comprises the replacement of one or more amino acids (e.g., the replacement of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids), or combinations of two or more of the foregoing. In some embodiments, a mutant protein has at least one amino acid alteration compared to a protein occurring in nature. In some embodiments, a mutant nucleic acid sequence encodes a protein that has at least one amino acid alteration compared to a protein occurring in nature. In some embodiments, the nucleic acid is not a degenerate version of a nucleic acid occurring in nature that encodes a protein with an amino acid sequence identical to a protein occurring in nature. [0096] In some embodiments the mutation in the H-NOX protein or H-NOX domain of a polymeric H-NOX protein is an evolutionary conserved mutations (also denoted class I mutations). Examples of class I mutations are listed in Table 1 A. In Table 1 A, mutations are numbered/annotated according to the sequence of human pi H-NOX, but are analogous for all H-NOX sequences. Thus, the corresponding position in any other H-NOX protein can be mutated to the indicated residue. For example, Phe4 of human pi H-NOX can be mutated to a tyrosine since other H-NOX proteins have a tyrosine in this position. The corresponding phenylalanine residue can be mutated to a tyrosine in any other H-NOX protein. In particular embodiments, the one or more mutations are confined to evolutionarily conserved residues. In some embodiments, the one or more mutations may include at least one evolutionarily conserved mutation and at least one non-evolutionarily conserved mutation. If desired, these mutant H- NOX proteins are subjected to empirical screening for NO/O2 dissociation constants, NO- reactivity, stability, and physio-compatibility in view of the teaching provided herein.

Table 1A. Exemplary Class I H-NOX mutations targeting evolutionary conserved residues

F4Y Q30G I145Y

F4L E33P I145H

H7G N61G K151E

A8E C78H I157F

L9W A109F E183F

[0097] In some embodiments, the mutation is a distal pocket mutation, such as mutation of a residue in alpha-helix A, D, E, or G (Pellicena, P. et al. (August 31, 2004). Proc Natl. Acad Sci USA 101(35): 12854-12859). Exemplary distal pocket mutations (also denoted class II mutations) are listed in Table IB. In Table IB, mutations are numbered/annotated according to the sequence of human pi H-NOX, but are analogous for all H-NOX sequences. Because several substitutions provide viable mutations at each recited residue, the residue at each indicated position can be changed to any other naturally or non-naturally-occurring amino acid (denoted “X”). Such mutations can produce H-NOX proteins with a variety of desired affinity, stability, and reactivity characteristics.

Table IB. Exemplary Class II H-NOX mutations targeting distal pocket residues

V8X M73X I145X

L9X F77X I149X

F70X C78X

[0098] In particular embodiments, the mutation is a heme distal pocket mutation. As described herein, a crucial molecular determinant that prevents O2 binding in NO-binding members of the H-NOX family is the lack of an H-bond donor in the distal pocket of the heme. Accordingly, in some embodiments, the mutation alters H-bonding between the H-NOX domain and the ligand within the distal pocket. In some embodiments, the mutation disrupts an H-bond donor of the distal pocket and/or imparts reduced O2 ligand-binding relative to the corresponding wild-type H-NOX domain. Exemplary distal pocket residues include Thr4, Ile5, Thr8, Trp9, Trp67, Asn74, Ile75, Phe78, Phe82, Tyrl40, and Leul44 of T. tengcongensis H-NOX and the corresponding residues in any other H-NOX protein. In some embodiments, the H-NOX protein or H-NOX domain of a polymeric H-NOX protein comprises one or more distal pocket mutations. In some embodiments, the H-NOX protein or H-NOX domain of a polymeric H- NOX protein comprises one, two, three, four, five, six, seven, eight, nine, ten or more than ten distal pocket mutations. In some embodiments, the distal pocket mutation corresponds to a L144F mutation of T. tengcongensis H-NOX. In some embodiments, the distal pocket mutation is a L144F mutation of T. tengcongensis H-NOX. In some embodiments, H-NOX protein or the H-NOX domain of a polymeric H-NOX protein comprises two distal pocket mutations. In some embodiments, the H-NOX protein or H-NOX domain of a polymeric H-NOX protein corresponds to a W9F/ L144F mutation of T. tengcongensis H-NOX. In some embodiments, the H-NOX protein or H-NOX domain of a polymeric H-NOX protein is a W9F/ L144F mutation of T. tengcongensis H-NOX. [0099] Residues that are not in the distal pocket can also affect the three-dimensional structure of the heme group; this structure in turn affects the binding of O2 and NO to iron in the heme group. Accordingly, in some embodiments, the H-NOX protein or H-NOX domain of a polymeric H-NOX protein has one or more mutations outside of the distal pocket. Examples of residues that can be mutated but are not in the distal pocket include Prol 15 and Argl35 of T. tengcongensis H-NOX. In some embodiments, the mutation is in the proximal pocket which includes His 105 as a residue that ligates to the heme iron.

[0100] In some embodiments when two or more mutations are present; at least one mutation is in the distal pocket, and at least one mutation is outside of the distal pocket (e.g., a mutation in the proximal pocket). In some embodiments, all the mutations are in the distal pocket.

[0101] To reduce the immunogenicity of H-NOX protein or H-NOX domains derived from sources other than humans, amino acids in an H-NOX protein or H-NOX domain can be mutated to the corresponding amino acids in a human H-NOX. For example, one or more amino acids on the surface of the tertiary structure of a non-human H-NOX protein or H-NOX domain can be mutated to the corresponding amino acid in a human H-NOX protein or H-NOX domain. In some variations, mutation of one or more surface amino acids may be combined with mutation of two or more distal pocket residues, mutation of one or more residues outside of the distal pocket (e.g., a mutation in the proximal pocket), or combinations of two or more of the foregoing.

[0102] The disclosure also relates to any combination of mutation described herein, such as double, triple, or higher multiple mutations. For example, combinations of any of the mutations described herein can be made in the same H-NOX protein. Note that mutations in equivalent positions in other mammalian or non-mammalian H-NOX proteins are also encompassed by this disclosure. Exemplary mutant H-NOX proteins or mutant H-NOX domains comprise one or more mutations that impart altered O2 or NO ligand-binding relative to the corresponding wildtype H-NOX domain and are operative as a physiologically compatible mammalian O2 blood gas carrier.

[0103] The residue number for a mutation indicates the position in the sequence of the particular H-NOX protein being described. For example, T. tengcongensis I5A refers to the replacement of isoleucine by alanine at the fifth position in T. tengcongensis H-NOX. The same isoleucine to alanine mutation can be made in the corresponding residue in any other H-NOX protein or H-NOX domain (this residue may or may not be the fifth residue in the sequence of other H-NOX proteins). Since the amino acid sequences of mammalian pi H-NOX domains differ by at most two amino acids, mutations that produce desirable mutant H-NOX proteins or H-NOX domains when introduced into wild-type rat pi H-NOX proteins are also expected to produce desirable mutant H-NOX proteins or H-NOX domains when introduced into wild-type pi H-NOX proteins or H-NOX domains from other mammals, such as humans.

[0104] In some embodiments, the H-NOX protein is a trimer comprising three T. tengcongensis L144F H-NOX domains and three foldon domains. In some embodiments, the H- NOX protein is a trimer comprising three T. tengcongensis W9F/L144F H-NOX domains and three foldon domains. In some embodiments, the H-NOX protein is a trimer comprising three T. tengcongensis wild-type H-NOX domains and three foldon domains.

Modifications to H-NOX Proteins

[0105] Any of the wild-type or mutant H-NOX proteins, including polymeric H-NOX proteins, can be modified and/or formulated using standard methods to enhance therapeutic or industrial applications. For example, and particularly as applied to heterologous engineered H- NOX proteins, a variety of methods are known in the art for insulating such agents from immune surveillance, including crosslinking, PEGylation, carbohydrate decoration, etc. (e.g., Rohlfs, R. J. et al. (May 15, 1998). J. Biol. Chem. 273(20): 12128-12134; Migita, R. et al. (June 1997). J. Appl. Physiol. 82(6): 1995-2002; Vandegriff, K. D. et al. (August 15, 2004). Biochem J. 382(Pt 1): 183- 189, which are each hereby incorporated by reference in their entireties, particularly with respect to the modification of proteins) as well as other techniques known to the skilled artisan. Fusing an H-NOX protein, including a polymeric H-NOX protein, with a human protein such as human serum albumin can increase the serum half-life, viscosity, and colloidal oncotic pressure. In some embodiments, an H-NOX protein is modified during or after its synthesis to decrease its immunogenicity and/or to increase its plasma retention time. H-NOX proteins can also be encapsulated (such as encapsulation within liposomes or nanoparticles).

[0106] In some embodiments, the H-NOX protein comprises one of more tags; e.g. to assist in purification of the H-NOX protein. Examples of tags include, but are not limited to Hise, FLAG, GST, and MBP. In some embodiments, the H-NOX protein comprises one of more Hise tags. The one or more Hise tags may be removed prior to use of the polymeric H-NOX protein; e.g. by treatment with an exopeptidase. In some embodiments, the H-NOX protein is a trimer comprising three T. tengcongensis L144F H-NOX domains, three foldon domains, and three Hise tags. In some embodiments, the H-NOX protein is a trimer comprising three T. tengcongensis W9F/L144F H-NOX domains, three foldon domains, and three Hise tags. In some embodiments, the H-NOX protein is a trimer comprising three T. tengcongensis wild-type H-NOX domains, three foldon domains, and three Hise tags.

Polymerization domains

[0107] In some aspects, the disclosure provides polymeric H-NOX proteins comprising two or more H-NOX domains and one or more polymerization domains. Polymerization domains are used to link two or more H-NOX domains to form a polymeric H-NOX protein. One or more polymerization domains may be used to produce dimers, trimers, tetramers, pentamers, etc. of H- NOX proteins. Polymerization domains are known in the art, such as: the foldon of T4 bacteriophage fibritin, Arc, POZ, coiled coil domains (including GCN4, leucine zippers, Velcro), uteroglobin, collagen, 3-stranded coiled coils (matrilin-1), thrombospondins, TRPV1-C, P53, Mnt, avidin, streptavidin, Bcr-Abl, COMP, verotoxin subunit B, CamKII, RCK, and domains from N ethylmaleimide-sensitive fusion protein, STM3548, KaiC, TyrR, Hcpl, CcmK4, GP41, anthrax protective antigen, aerolysin, a-hemolysin, C4b-binding protein, Mi-CK, arylsulfatase A, and viral capsid proteins. The polymerization domains may be covalently or non-covalently linked to the H-NOX domains. In some embodiments, a polymerization domain is linked to an H-NOX domain to form a monomer subunit such that the polymerization domains from a plurality of monomer subunits associate to form a polymeric H-NOX domain. In some embodiments, the C-terminus of an H-NOX domain is linked to the N-terminus of a polymerization domain. In other embodiments, the N-terminus of an H-NOX domain is linked to the N-terminus of a polymerization domain. In yet other embodiments, the C-terminus of an H-NOX domain is linked to the C-terminus of a polymerization domain. In some embodiments, the N-terminus of an H-NOX domain is linked to the C-terminus of a polymerization domain. [0108] Linkers may be used to join a polymerization domain to an H-NOX domain; for example, for example, amino acid linkers. In some embodiments, a linker comprising any one of one, two, three, four, five, six, seven, eight, nine, ten or more than ten amino acids may be placed betweent the polymerization domain and the H-NOX domain. Exemplary linkers include but are not limited to Gly-Ser-Gly and Arg-Gly-Ser linkers. Bacteriophage T4 fibritin trimerization domain

[0109] An exemplary polymerization domain is the foldon domain of bacteriophage T4. The wac gene from the bacteriophage T4 encodes the fibritin protein, a 486 amino acid protein with a C-terminal trimerization domain (residues 457-483) (Efimov, V. P. et al. (1994) J Mol Biol 242:470-486). The domain is able to trimerize fibritin both in vitro and in vivo (Boudko, S. P. et al. (2002) Eur J Biochem 269:833-841; Letarov, A. V., et al., (1999) Biochemistry (A <95C)64:817-823; Tao, Y., et al., (1997) Structure 5:789-798). The isolated 27 residue trimerization domain, often referred to as the “foldon domain,” has been used to construct chimeric trimers in a number of different proteins (including HIV envelope glycoproteins (Yang, X. et al., (2002) J Virol 76:4634-4642), adenoviral adhesins (Papanikolopoulou, K., et al., (2004) J Biol Chem 279:8991-8998; Papanikolopoulou, K. et al. (2004) J Mol Biol 342:219- 227), collagen (Zhang, C., et al. (2009) Biotechnol Prog 25 : 1660-1668), phage P22 gp26 (Bhardwaj, A., et al. (2008) Protein Sci 17: 1475-1485), and rabies virus glycoprotein (Sissoeff, L., et al. (2005) J Gen Virol 86:2543-2552). An exemplary sequence of the foldon domain is shown in Figure 1 and provided by SEQ ID NO:4.

[0110] The isolated foldon domain folds into a single P-hairpin structure and trimerizes into a P-propeller structure involving three hairpins (Guthe, S. et al. (2004) J Mol Biol 337:905-915). The structure of the foldon domain alone has been determined by NMR (Guthe, S. et al. (2004) J Mol Biol 337:905-915) and the structures of several proteins trimerized with the foldon domain have been solved by X-ray crystallography (Papanikolopoulou, K., et al., (2004) J Biol Chem 279:8991-8998; Stetefeld, J. et al. (2003) Structure 11 :339-346; Yokoi, N. et al. (2010) Small 6: 1873-1879). The domain folds and trimerizes rapidly reducing the opportunity for misfolding intermediates or off-pathway oligomerization products (Guthe, S. et al. (2004) J Mol Biol 337:905-915). The foldon domain is very stable, able to maintain tertiary structure and oligomerization in >10% SDS, 6.0M guanidine hydrochloride, or 80° C (Bhardwaj, A., et al. (2008) Protein Sci 17: 1475-1485; Bhardwaj, A., et al. (2007) J Mol Biol 371 :374-387) and can improve the stability of sequences fused to the foldon domain (Du, C. et al. (2008) Appl Microbiol Biotechnol 79: 195-202.

[OHl] In some embodiments, the C-terminus of an H-NOX domain is linked to the N- terminus of a foldon domain. In other embodiments, the N-terminus of an H-NOX domain is linked to the N-terminus of a foldon domain. In yet other embodiments, the C-terminus of an H- NOX domain is linked to the C-terminus of a foldon domain. In some embodiments, the N- terminus of an H-NOX domain is linked to the C-terminus of a foldon domain.

[0112] In some embodiments, linkers are be used to join a foldon domain to an H-NOX domain. In some embodiments, a linker comprising any one of one, two, three, four, five, six, seven, eight, nine, ten or more than ten amino acids may be placed betweent the polymerization domain and the H-NOX domain. Exemplary linkers include but are not limited to Gly-Ser-Gly and Arg-Gly-Ser linkers. In some embodiments, the disclosure provides a trimeric H-NOX protein comprising from N-terminus to C-terminus: a T. tengcongensis H-NOX domain, a Gly- Ser-Gly amino acid linker, and a foldon domain. In some embodiments, the disclosure provides a trimeric H-NOX protein comprising from N-terminus to C-terminus: a T. tengcongensis H- NOX domain, a Gly-Ser-Gly amino acid linker, a foldon domain, an Arg-Gly-Ser amino acid linker, and a Hise tag. In some embodiments, the T. tengcongensis H-NOX domain comprises an L144F mutation. In some embodiments, the T. tengcongensis H-NOX domain comprises a W9F mutation and a L144F mutation. In some embodiments, the T. tengcongensis H-NOX domain is a wild-type H-NOX domain.

Monomeric H-NOX domain subunits

[0113] In one aspect, the disclosure provides recombinant monomeric H-NOX proteins (i.e. monomeric H-NOX subunits of polymeric H-NOX proteins) that can associate to form polymeric H-NOX proteins. In some embodiments, the disclosure provides recombinant H- NOX proteins comprising an H-NOX domain as described herein and a polymerization domain. The H-NOX domain and the polymerization domain may be covalently linked or noncovalently linked. In some embodiments, the C-terminus of an H-NOX domain of the recombinant monomeric H-NOX protein is linked to the N-terminus of a polymerization domain. In other embodiments, the N-terminus of an H-NOX domain of the recombinant monomeric H-NOX protein is linked to the N-terminus of a polymerization domain. In yet other embodiments, the C-terminus of an H-NOX domain of the recombinant monomeric H-NOX protein is linked to the C-terminus of a polymerization domain. In some embodiments, the N-terminus of an H-NOX domain of the recombinant monomeric H-NOX protein is linked to the C-terminus of a polymerization domain. In some embodiments, the recombinant monomeric H-NOX protein does not comprise a guanylyl cyclase domain. [0114] In some embodiments, the monomeric H-NOX protein comprises a wild-type H-NOX domain. In some embodiments of the disclosure, the monomeric H-NOX protein comprises one of more mutations in the H-NOX domain. In some embodiments, the one or more mutations alter the O2 dissociation constant, the k _O ff for oxygen, the rate of heme autooxidation, the NO reactivity, the NO stabilty or any combination of two or more of the foregoing compared to that of the corresponding wild-type H-NOX domain. In some embodiments, the mutation is a distal pocket mutation. In some embodiments, the mutation comprises a mutation that is not in the distal pocket. In some embodiments, the distal pocket mutation corresponds to a L144 mutation of T. tengcongensis (e.g. a L144F mutation). In some embodiments, the recombinant monomeric H-NOX protein comprises two distal pocket mutations corresponding to a W9 and a LI 44 mutation of T. tengcongensis (e.g. a W9F/L144F mutation).

[0115] In some aspects, the disclosure provides recombinant monomeric H-NOX proteins that associate to form trimeric H-NOX proteins. In some embodiments, the recombinant H-NOX protein comprises an H-NOX domain and a trimerization domain. In some embodiments, the trimerization domain is a foldon domain as discussed herein. In some embodiments, the H-NOX domain is a T. tengcongensis H-NOX domain. In some embodiments the C-terminus of the T. tengcongensis H-NOX domain is covalently linked to the N-terminus of the foldon domain. In some embodiments the C-terminus of the T. tengcongensis H-NOX domain is covalently linked to the C-terminus of the foldon domain. In some embodiments, the T. tengcongensis domain is an L144F H-NOX domain. In some embodiments, the T. tengcongensis domain is a W9F/L144F H-NOX domain. In some embodiments, the T. tengcongensis domain is a wildtype H-NOX domain.

[0116] In some embodiments, the H-NOX domain is covalently linked to the polymerization domain using an amino acid linker sequence. In some embodiments, the amino acid linker sequence is one, two, three, four, five, six, seven, eight, nine, ten or more than ten amino acids in length. Exemplary amino acid linker sequences include but are not limited to a Gly-Ser-Gly sequence and an Arg-Gly-Ser sequence. In some embodiments, the polymeric H-NOX protein is a trimeric H-NOX protein comprising three H-NOX domains and three trimerization sequences wherein the H-NOX domain is covalently linked to the trimerization domain via an amino acid linker sequence. In some embodiments, the monomeric H-NOX protein comprises the following from the N-terminus to the C-terminus: an L144F T. tengcongensis H-NOX domain, a Gly-Ser- Gly amino acid linker sequence, and a foldon domain. In some embodiments, the monomeric H- NOX protein comprises the following from the N-terminus to the C-terminus: a W9F/L144F T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker sequence, and a foldon domain. In some embodiments, the monomeric H-NOX protein comprises the following from the N- terminus to the C-terminus: a wild-type T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker sequence, and a foldon domain.

[0117] In some embodiments, the recombinant monomeric H-NOX protein comprises a tag; e.g., a Hise, a FLAG, a GST, or an MBP tag. In some embodiments, the recombinant monomeric H-NOX protein comprises a Hise tag. In some embodiments, the recombinant monomeric H-NOX protein does not comprise a tag. In some embodiments, the tag (e.g. a Hise tag) is covalently linked to the polymerization domain using an amino acid spacer sequence. In some embodiments, the amino acid linker sequence is one, two, three, four, five, six, seven, eight, nine, ten or more than ten amino acids in length. Exemplary amino acid linker sequences include but are not limited to a Gly-Ser-Gly sequence and an Arg-Gly-Ser sequence. In some embodiments, the polymeric H-NOX protein is a trimeric H-NOX protein comprising three H- NOX domains, three trimerization sequences, and three Hise tags, wherein the H-NOX domain is covalently linked to the trimerization domain via an amino acid linker sequence and the trimerization domain is covalently linked to the Hise tag via an amino acid linker sequence. In some embodiments, the monomeric H-NOX protein comprises the following from the N- terminus to the C-terminus: an L144F T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker sequence, a foldon domain, an Arg-Gly-Ser linker sequence, and a Hise tag. In some embodiments, the monomeric H-NOX protein comprises the following from the N-terminus to the C-terminus: a W9F/L144F T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker sequence, a foldon domain, an Arg-Gly-Ser linker sequence, and a Hise tag. In some embodiments, the monomeric H-NOX protein comprises the following from the N-terminus to the C-terminus: a wild-type T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker sequence, a foldon domain, an Arg-Gly-Ser linker sequence, and a Hise tag.

[0118] In some embodiments the recombinant monomeric H-NOX protein comprises the amino acid sequence of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10 or SEQ ID NO: 12. Characteristics of Wild- type and Mutant H-NOX Proteins

[0119] As described herein, a large number of diverse H-NOX mutant proteins, including polymeric H-NOX proteins, providing ranges of NO and O2 dissociation constants, O2 koff, NO reactivity, and stability have been generated. To provide operative blood gas carriers, the H- NOX proteins may be used to functionally replace or supplement endogenous O2 carriers, such as hemoglobin. In some embodiments, H-NOX proteins such as polymeric H-NOX proteins, are used to deliver O2 to hypoxic tumor tissue (e.g. a glioblastoma) as an adjuvant to radiation therapy or chemotherapy. Accordingly, in some embodiments, an H-NOX protein has a similar or improved O2 association rate, O2 dissociation rate, dissociation constant for O2 binding, NO stability, NO reactivity, autoxidation rate, plasma retention time, or any combination of two or more of the foregoing compared to an endogenous O2 carrier, such as hemoglobin. In some embodiments, the H-NOX protein is a polymeric H-NOX protein. In some embodiments, the polymeric H-NOX protein is a trimeric H-NOX protein comprising three monomers, each monomer comprising a T. tengcongensis L144F H-NOX domain and a foldon domain. In some embodiments, the polymeric H-NOX protein is a trimeric H-NOX protein comprising three monomers, each monomer comprising a T. tengcongensis W9F/L144F H-NOX domain and a foldon domain. In some embodiments, the polymeric H-NOX protein is a trimeric H-NOX protein comprising three monomers, each monomer comprising a T. tengcongensis L144F H- NOX domain and a foldon domain.

[0120] In various embodiments, the koff for O2 for an H-NOX protein, including a polymeric H-NOX protein, is between about 0.01 to about 200 s' ¹ at 20 °C, such as about 0.1 to about 200 s' ¹ , about 0.1 to 100 s' ¹ , about 1.0 to about 16.0 s' ¹ , about 1.35 to about 23.4 s' ¹ , about 1.34 to about 18 s' ¹ , about 1.35 to about 14.5 s' ¹ , about 0.21 to about 23. 4 s' ¹ , aboutl.35 to about 2.9 s' ¹ , about 2 to about 3 s' ¹ , about 5 to about 15 s' ¹ , or about 0.1 to about 1 s' ¹ . In some embodiments, the H-NOX protein has a koff for oxygen that is less than or equal to about 0.65 s ^-1 at 20 °C (such as between about 0.21 s' ¹ to about 0.65 s' ¹ at 20 °C).

[0121] In various embodiments, the konfor O2 for an H-NOX protein, including a polymeric H-NOX protein, is between about 0.14 to about 60 pM^s' ¹ at 20 °C, such as about 6 to about 60 pM' ¹ , about 6 to 12 pM' ¹ , about 15 to about 60 pM^s' ¹ , about 5 to about 18 pM' s' ¹ , or about 6 to about 15 pM' ¹ . [0122] In various embodiments, the kinetic or calculated KD for O2 binding by an H-NOX protein, including a polymeric H-NOX protein, is between about 1 nM to 1 mM, about 1 pM to about 10 pM, or about 10 pM to about 50 pM. In some embodiments the calculated KD for O2 binding is any one of about 2 nM to about 2 pM, about 2 pM to about 1 mM, about 100 nM to about 1 pM, about 9 pM to about 50 pM, about 100 pM to about 1 mM, about 50 nM to about 10 pM, about 2 nM to about 50 pM, about 100 nM to about 1.9 pM, about 150 nM to about 1 pM, or about 100 nM to about 255 nM, about 20 nM to about 2 pM, 20 nM to about 75 nM, about 1 pM to about 2 pM, about 2 pM to about 10 pM, about 2 pM to about 9 pM, or about 100 nM to 500 nM at 20 °C. In some embodiments, the kinetic or calculated KD for O2 binding is less than about any of 100 nM, 80 nM, 50 nM, 30 nM, 25 nM, 20 nM, or 10 nM at 20 °C. [0123] In various embodiments, the kinetic or calculated KD for O2 binding by an H-NOX protein, including a polymeric H-NOX protein, is within about 0.01 to about 100-fold of that of hemoglobin under the same conditions (such as at 20 °C), such as between about 0.1 to about 10- fold or between about 0.5 to about 2-fold of that of hemoglobin under the same conditions (such as at 20 °C). In various embodiments, the kinetic or calculated KD for NO binding by an H-NOX protein is within about 0.01 to about 100-fold of that of hemoglobin under the same conditions (such as at 20 °C), such as between about 0.1 to about 10-fold or between about 0.5 to about 2- fold of that of hemoglobin under the same conditions (such as at 20 °C).

[0124] In some embodiments, less than about any of 50, 40, 30, 10, or 5% of an H-NOX protein, including a polymeric H-NOX protein, is oxidized after incubation for about any of 1, 2, 4, 6, 8, 10, 15, or 20 hours at 20 °C.

[0125] In various embodiments, the NO reactivity of an H-NOX protein, including a polymeric H-NOX protein, is less than about 700 s' ¹ at 20 °C, such as less than about 600 s' ¹ , 500 s' ¹ , 400 s' ¹ , 300 s' ¹ , 200 s' ¹ , 100 s' ¹ , 75 s' ¹ , 50 s' ¹ , 25 s' ¹ , 20 s' ¹ , 10 s' ¹ , 50 s' ¹ , 3 s' ¹ , 2 s' ¹ , 1.8 s' ¹ , 1.5 s' ¹ , 1.2 s' ¹ , 1.0 s' ¹ , 0.8 s' ¹ , 0.7 s' ¹ , or 0.6 s' ¹ at 20 °C. In various embodiments, the NO reactivity of an H-NOX protein is between about 0.1 to about 600 s' ¹ at 20 °C, such as between about 0.5 to about 400 s' ¹ , about 0.5 to about 100 s' ¹ , about 0.5 to about 50 s' ¹ , about 0.5 to about 10 s' ¹ , about 1 to about 5 s' ¹ , or about 0.5 to about 2.1 s' ¹ at 20 °C. In various embodiments, the reactivity of an H-NOX protein is at least about 10, 100, 1,000, or 10,000 fold lower than that of hemoglobin under the same conditions, such as at 20 °C. [0126] In various embodiments, the rate of heme autoxidation of an H-NOX protein, including a polymeric H-NOX protein, is less than about 1.0 h ⁴ at 37 °C, such as less than about any of 0.9 h’ ¹ , 0.8 h’ ¹ , 0.7 h’ ¹ , 0.6 h , 0.5 h’ ¹ , 0.4 h’ ¹ , 0.3 h’ ¹ , 0.2 h’ ¹ , 0.1 h’ ¹ , or 0.05 h at 37 C. In various embodiments, the rate of heme autoxidation of an H-NOX protein is between about 0.006 to about 5.0 h' ¹ at 37 °C, such as about 0.006 to about 1.0 h’ ¹ , 0.006 to about 0.9 h' ¹ , or about 0.06 to about 0.5 h' ¹ at 37 °C.

[0127] In various embodiments, a mutant H-NOX protein, including a polymeric H-NOX protein, has (a) an O2 or NO dissociation constant, association rate (k _0n for O2 or NO), or dissociation rate (k _O ff for O2 or NO) within 2 orders of magnitude of that of hemoglobin, (b) has an NO affinity weaker (e.g., at least about 10-fold, 100-fold, or 1000-fold weaker) than that of sGC [Bl, respectively, (c) an NO reactivity with bound O2 at least 1000-fold less than hemoglobin, (d) an in vivo plasma retention time at least 2, 10, 100, or 1000-fold higher than that of hemoglobin, or (e) any combination of two or more of the foregoing.

[0128] Exemplary suitable O2 carriers provide dissociation constants within two orders of magnitude of that of hemoglobin, i.e. between about 0.01 and 100-fold, such as between about 0.1 and 10-fold, or between about 0.5 and 2-fold of that of hemoglobin. A variety of established techniques may be used to quantify dissociation constants, such as the techniques described herein (Boon, E. M. et al. (2005). Nature Chem. Biol. 1 :53-59; Boon, E. M. et al. (October 2005). Curr. Opin. Chem. Biol. 9(5):441-446; Boon, E. M. et al. (2005). J. Inorg. Biochem. 99(4): 892-902), Vandegriff, K. D. et al. (August 15, 2004). Biochem J. 382(Pt 1): 183-189, which are each hereby incorporated by reference in their entireties, particularly with respect to the measurement of dissociation constants), as well as those known to the skilled artisan. Exemplary O2 carriers provide low or minimized NO reactivity of the H-NOX protein with bound O2, such as an NO reactivity lower than that of hemoglobin. In some embodiments, the NO reactivity is much lower, such as at least about 10, 100, 1,000, or 10,000-fold lower than that of hemoglobin. A variety of established techniques may be used to quantify NO reactivity (Boon, E. M. et al. (2005). Nature Chem. Biol. 1 :53-59; Boon, E. M. et al. (October 2005). Curr. Opin. Chem. Biol. 9(5):441-446; Boon, E. M. et al. (2005). J. Inorg. Biochem. 99(4): 892-902), Vandegriff, K. D. et al. (August 15, 2004). Biochem J. 382(Pt 1): 183-189, which are each hereby incorporated by reference in their entireties, particularly with respect to the measurement of NO reactivity) as well as those known to the skilled artisan. Because wild-type T. tengcongensis H-NOX has such a low NO reactivity, other wild-type H-NOX proteins and mutant H-NOX proteins may have a similar low NO reactivity. For example, T. tengcongensis H-NOX Y140H has an NO reactivity similar to that of wild-type T. tengcongensis H-NOX. [0129] In addition, suitable O2 carriers provide high or maximized stability, particularly in vivo stability. A variety of stability metrics may be used, such as oxidative stability (e.g., stability to autoxidation or oxidation by NO), temperature stability, and in vivo stability. A variety of established techniques may be used to quantify stability, such as the techniques described herein (Boon, E. M. et al. (2005). Nature Chem. Biol. 1 :53-59; Boon, E. M. et al. (October 2005). Curr. Opin. Chem. Biol. 9(5):441-446; Boon, E. M. et al. (2005). J. Inorg. Biochem. 99(4): 892-902), as well as those known to the skilled artisan. For in vivo stability in plasma, blood, or tissue, exemplary metrics of stability include retention time, rate of clearance, and half-life. H-NOX proteins from thermophilic organisms are expected to be stable at high temperatures. In various embodiments, the plasma retention times are at least about 2-, 10-, 100- , or 1000-fold greater than that of hemoglobin (e.g. Bobofchak, K. M. et al. (August 2003). Am. J. Physiol. Heart Circ. Physiol. 285(2):H549-H561). As will be appreciated by the skilled artisan, hemoglobin-based blood substitutes are limited by the rapid clearance of cell-free hemoglobin from plasma due the presence of receptors for hemoglobin that remove cell-free hemoglobin from plasma. Since there are no receptors for H-NOX proteins in plasma, wild-type and mutant H-NOX proteins are expected to have a longer plasma retention time than that of hemoglobin. If desired, the plasma retention time can be increased by PEGylating or crosslinking an H-NOX protein or fusing an H-NOX protein with another protein using standard methods (such as those described herein and those known to the skilled artisan).

[0130] In various embodiments, the H-NOX protein, including a polymeric H-NOX protein, has an O2 dissociation constant between about 1 nM to about 1 mM at 20 °C and a NO reactivity at least about 10-fold lower than that of hemoglobin under the same conditions, such as at 20 °C. In some embodiments, the H-NOX protein has an O2 dissociation constant between about 1 nM to about 1 mM at 20 °C and a NO reactivity less than about 700 s' ¹ at 20 °C (e.g., less than about 600 s' ¹ , 500 s' ¹ , 100 s' ¹ , 20 s' ¹ , or 1.8 s ^-1 at 20 °C). In some embodiments, the H-NOX protein has an O2 dissociation constant within 2 orders of magnitude of that of hemoglobin and a NO reactivity at least about 10-fold lower than that of hemoglobin under the same conditions, such as at 20 °C. In some embodiments, the H-NOX protein has a kofr for oxygen between about 0.01 to about 200 s' ¹ at 20 °C and an NO reactivity at least about 10-fold lower than that of hemoglobin under the same conditions, such as at 20 °C. In some embodiments, the H-NOX protein has a k _O ff for oxygen that is less than about 0.65 s ^-1 at 20 °C (such as between about 0.21 s' ¹ to about 0.64 s' ¹ at 20 °C) and a NO reactivity at least about 10-fold lower than that of hemoglobin under the same conditions, such as at 20 °C. In some embodiments of the disclosure, the O2 dissociation constant of the H-NOX protein is between about 1 nM to about 1 pM (1000 nM), about 1 pM to about 10 pM, or about 10 pM to about 50 pM. In particular embodiments, the O2 dissociation constant of the H-NOX protein is between about 2 nM to about 50 pM, about 50 nM to about 10 pM, about 100 nM to about 1.9 pM, about 150 nM to about 1 pM, or about 100 nM to about 255 nM at 20 °C. In various embodiments, the O2 dissociation constant of the H-NOX protein is less than about 80 nM at 20 °C, such as between about 20 nM to about 75 nM at 20 °C. In some embodiments, the NO reactivity of the H-NOX protein is at least about 100-fold lower or about 1,000 fold lower than that of hemoglobin, under the same conditions, such as at 20 °C. In some embodiments, the NO reactivity of the H-NOX protein is less than about 700 s' ¹ at 20 °C, such as less than about 600 s' ¹ , 500 s' ¹ , 400 s' ¹ , 300 s' \ 200 s' ¹ , 100 s' ¹ , 75 s' ¹ , 50 s' ¹ , 25 s' ¹ , 20 s' ¹ , 10 s' ¹ , 50 s' ¹ , 3 s' ¹ , 2 s' ¹ , 1.8 s' ¹ , 1.5 s' ¹ , 1.2 s' ¹ , 1.0 s' 0.8 s' ¹ , 0.7 s' ¹ , or 0.6 s' ¹ at 20 °C. In some embodiments, the k _O ff for oxygen of the H-NOX protein is between 0.01 to 200 s ^-1 at 20 °C, such as about 0.1 to about 200 s' ¹ , about 0.1 to 100 s' ¹ , about 1.35 to about 23.4 s' ¹ , about 1.34 to about 18 s' ¹ , about 1.35 to about 14.5 s' ¹ , about 0.21 to about 23. 4 s' ¹ , about 2 to about 3 s' ¹ , about 5 to about 15 s' ¹ , or about 0.1 to about 1 s' ¹ . In some embodiments, the O2 dissociation constant of the H-NOX protein is between about 100 nM to about 1.9 pM at 20 °C, and the k _O ff for oxygen of the H-NOX protein is between about 1.35 s' ¹ to about 14.5 s' ¹ at 20 °C. In some embodiments, the rate of heme autoxidation of the H-NOX protein is less than about 1 h ^-1 at 37 °C, such as less than about any of 0.9 h' ¹ , 0.8 h' ¹ , 0.7 h' ¹ , 0.6 h' ¹ , 0.5 h' ¹ , 0.4 h' ¹ , 0.3 h' ¹ , 0.2 h' ¹ , or 0.1 h' ¹ . In some embodiments, the k _O ff for oxygen of the H- NOX protein is between about 1.35 s' ¹ to about 14.5 s' ¹ at 20 °C, and the rate of heme autoxidation of the H-NOX protein is less than about 1 h ^-1 at 37 °C. In some embodiments, the k _O ff for oxygen of the H-NOX protein is between about 1.35 s' ¹ to about 14.5 s' ¹ at 20 °C, and the NO reactivity of the H-NOX protein is less than about 700 s' ¹ at 20 °C (e.g., less than about 600 s' ¹ , 500 s' ¹ , 100 s' ¹ , 20 s' ¹ , or 1.8 s ^-1 at 20 °C). In some embodiments, the rate of heme autoxidation of the H-NOX protein is less than about 1 h ^-1 at 37 °C, and the NO reactivity of the H-NOX protein is less than about 700 s' ¹ at 20 °C (e.g., less than about 600 s' ¹ , 500 s' ¹ , 100 s' ¹ , 20 s' ¹ , or 1.8 s at 20 °C).

[0131] In some embodiments, the viscosity of the H-NOX protein solution, including a polymeric H-NOX protein solution, is between 1 and 4 centipoise (cP). In some embodiments, the colloid oncotic pressure of the H-NOX protein solution is between 20 and 50 mm Hg.

Measurement of O2 and/or NO binding

[0132] One skilled in the art can readily determine the oxygen and nitric oxide binding characteristics of any H-NOX protein including a polymeric H-NOX protein such as a trimeric H-NOX protein by methods known in the art and by the non-limiting exemplary methods described below.

Kinetic KD> Ratio of k _o ffto k _on

[0133] The kinetic KD value is determined for wild-type and mutant H-NOX proteins, including polymeric H-NOS proteins, essentially as described by Boon, E.M. et al. (2005). Nature Chemical Biology 1 :53-59, which is hereby incorporated by reference in its entirety, particularly with respect to the measurement of O2 association rates, O2 dissociation rates, dissociation constants for O2 binding, autoxidation rates, and NO dissociation rates. kon (O2 Association Rate)

[0134] O2 association to the heme is measured using flash photolysis at 20 °C. It is not possible to flash off the Fe ⁿ -O2 complex as a result of the very fast geminate recombination kinetics; thus, the Fe ⁿ -CO complex is subjected to flash photolysis with laser light at 560 nm (Hewlett-Packard, Palo Alto, CA), producing the 5-coordinate Fe ¹¹ intermediate, to which the binding of molecular O2 is followed at various wavelengths. Protein samples are made by anaerobic reduction with 10 mM dithionite, followed by desalting on a PD-10 column (Millipore, Inc., Billerica, MA). The samples are then diluted to 20 M heme in 50 mM TEA, 50 mM NaCl, pH 7.5 buffer in a controlled-atmosphere quartz cuvette, with a size of 100 pL to 1 mL and a path-length of 1-cm. CO gas is flowed over the headspace of this cuvette for 10 minutes to form the Fe ⁿ -CO complex, the formation of which is verified by UV-visible spectroscopy (Soret maximum 423 nm). This sample is then either used to measure CO- rebinding kinetics after flash photolysis while still under 1 atmosphere of CO gas, or it is opened and stirred in air for 30 minutes to fully oxygenate the buffer before flash photolysis to watch Ch-rebinding events. O2 association to the heme is monitored at multiple wavelengths versus time. These traces are fit with a single exponential using Igor Pro software (Wavemetrics, Inc., Oswego, OR; latest 2005 version). This rate is independent of observation wavelength but dependent on O2 concentration. UV-visible spectroscopy is used throughout to confirm all the complexes and intermediates (Cary 3K, Varian, Inc. Palo Alto, CA). Transient absorption data are collected using instruments described in Dmochowski, I. J. et al. (August 31, 2000). J Inorg Biochem. 81(3):221-228, which is hereby incorporated by reference in its entirety, particularly with respect to instrumentation. The instrument has a response time of 20 ns, and the data are digitized at 200 megasamples s' ¹ . k _o ff (O2 dissociation rate)

[0135] To measure the k _O ff, Fe ⁿ -O2 complexes of protein (5 pM heme), are diluted in anaerobic 50 mM TEA, 50 mM NaCl, pH 7.5 buffer, and are rapidly mixed with an equal volume of the same buffer (anaerobic) containing various concentrations of dithionite and/or saturating CO gas. Data are acquired on a HI-TECH Scientific SF-61 stopped-flow spectrophotometer equipped with a Neslab RTE- 100 constant-temperature bath set to 20 °C (TGK Scientific LTD., Bradford On Avon, United Kingdom). The dissociation of O2 from the heme is monitored as an increase in the absorbance at 437 nm, a maximum in the Fe ¹¹ - Fe ⁿ -O2 difference spectrum, or 425 nm, a maximum in the Fe ¹¹ - Fe ⁿ -CO difference spectrum. The final traces are fit to a single exponential using the software that is part of the instrument. Each experiment is done a minimum of six times, and the resulting rates are averaged. The dissociation rates measured are independent of dithionite concentration and independent of saturating CO as a trap for the reduced species, both with and without 10 mM dithionite present. Kinetic KD

[0136] The kinetic KD is determined by calculating the ratio of koff to k _on using the measurements of k _O ff and k _on described above.

Calculated KD

[0137] To measure the calculated KD, the values for the k _O ff and kinetic KD that are obtained as described above are graphed. A linear relationship between k _O ff and kinetic KD is defined by the equation (y=mx+b). k _O ff values were then interpolated along the line to derive the calculated KD using Excel: MAC 2004 (Microsoft, Redmond, WA). In the absence of a measured k _on , this interpolation provides a way to relate koff to KD. Rate of Autoxidation

[0138] To measure the rate of autoxidation, the protein samples are anaerobically reduced, then diluted to 5 pM heme in aerobic 50 mM TEA, 50 mM NaCl, pH 7.5 buffer. These samples are then incubated in a Cary 3E spectrophotometer equipped with a Neslab RTE-100 constanttemperature bath set to 37 °C and scanned periodically (Cary 3E, Varian, Inc., Palo Alto, CA). The rate of autoxidation is determined from the difference between the maximum and minimum in the Fe ¹¹¹ - Fe ¹¹ difference spectrum plotted versus time and fit with a single exponential using Excel: MAC 2004 (Microsoft, Redmond, WA).

Rate of reaction with NO

[0139] NO reactivity is measured using purified proteins (H-NOX, polymeric H-NOX, Homo sapiens hemoglobin (Hs Hb) etc.) prepared at 2 pM in buffer A and NO prepared at 200 pM in Buffer A (Buffer A: 50 mM Hepes, pH 7.5, 50 mM NaCl). Data are acquired on a HI-TECH Scientific SF-61 stopped-flow spectrophotometer equipped with a Neslab RTE-100 constanttemperature bath set to 20 °C (TGK Scientific LTD., Bradford On Avon, United Kingdom). The protein is rapidly mixed with NO in a 1 : 1 ratio with an integration time of 0.00125 sec. The wavelengths of maximum change are fit to a single exponential using the software that is part of the spectrometer, essentially measuring the rate-limiting step of oxidation by NO. The end products of the reaction are ferric-NO for the HNOX proteins and ferric-aquo for Hs Hb. p50 measurements

[0140] If desired, the p50 value for mutant or wild-type H-NOX proteins can be measured as described by Guarnone, R. et al. (September/October 1995). Haematologica 80(5):426-430, which is hereby incorporated by reference in its entirety, particularly with respect to the measurement of p50 values. The p50 value is determined using a HemOx analyzer. The measurement chamber starts at 0% oxygen and slowly is raised, incrementally, towards 100% oxygen. An oxygen probe in the chamber measures the oxygen saturation %. A second probe (UV-Vis light) measures two wavelengths of absorption, tuned to the alpha and beta peaks of the hemoprotein’s (e.g., a protein such as H-NOX complexed with heme) UV-Vis spectra. These absorption peaks increase linearly as hemoprotein binds oxygen. The percent change from unbound to 100% bound is then plotted against the % oxygen values to generate a curve. The p50 is the point on the curve where 50% of the hemoprotein is bound to oxygen. [0141] Specifically, the Hemox-Analyzer (TCS Scientific Corporation, New Hope, PA) determines the oxyhemoprotein dissociation curve (ODC) by exposing 50 pL of blood or hemoprotein to an increasing partial pressure of oxygen and deoxygenating it with nitrogen gas. A Clark oxygen electrode detects the change in oxygen tension, which is recorded on the x-axis of an x-y recorder. The resulting increase in oxyhemoprotein fraction is simultaneously monitored by dual -wavelength spectrophotometry at 560 nm and 576 nm and displayed on the y- axis. Blood samples are taken from the antemedial vein, anti coagulated with heparin, and kept at 4°C on wet ice until the assay. Fifty pL of whole blood are diluted in 5 pL of Hem oxsolution, a manufacturer-provided buffer that keeps the pH of the solution at a value of 7.4±0.01. The sample-buffer is drawn into a cuvette that is part of the Hemox -Analyzer and the temperature of the mixture is equilibrated and brought to 37°C; the sample is then oxygenated to 100% with air. After adjustment of the pCh value the sample is deoxygenated with nitrogen; during the deoxygenation process the curve is recorded on graph paper. The P50 value is extrapolated on the x-axis as the point at which O2 saturation is 50% using the software that is part of the Hemox-Analyzer. The time required for a complete recording is approximately 30 minutes.

H-NOX Nucleic Acids

[0142] The disclosure also features nucleic acids encoding any of the mutant H-NOX proteins, polymeric H-NOX, or recombinant monomer H-NOX protein subunits as described herein. [0143] In particular embodiments, the nucleic acid includes a segment of or the entire nucleic acid sequence of any of nucleic acids encoding an H-NOX protein or an H-NOX domain. In some embodiments, the nucleic acid includes at least about 50, 100, 150, 200, 300, 400, 500, 600, 700, 800, or more contiguous nucleotides from a H-NOX nucleic acid and contains one or more mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mutations) compared to the H-NOX nucleic acid from which it was derived. In various embodiments, a mutant H-NOX nucleic acid contains less than about 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 mutations compared to the H-NOX nucleic acid from which it was derived. The disclosure also features degenerate variants of any nucleic acid encoding a mutant H-NOX protein.

[0144] In some embodiments, the nucleic acid includes nucleic acids encoding two or more H- NOX domains. In some embodiments, the nucleic acids including two or more H-NOX domains are linked such that a polymeric H-NOX protein is expressed from the nucleic acid. In further embodiments, the nucleic acid includes nucleic acids encoding one or more polymerization domains. In some embodiments, the nucleic acids including the two or more H-NOX domains and the one or more polymerization domains are linked such that a polymeric H-NOX protein is expressed from the nucleic acid.

[0145] In some embodiments, the nucleic acid includes a segment or the entire nucleic acid sequence of any nucleic acid encoding a polymerization domain. In some embodiments the nucleic acid comprises a nucleic acid encoding an H-NOX domain and a polymerization domain. In some embodiments, the nucleic acid encoding an H-NOX domain and the nucleic acid encoding a polymerization domain a linked such that the produced polypeptide is a fusion protein comprising an H-NOX domain and a polymerization domain.

[0146] In some embodiments, the nucleic acid comprises nucleic acid encoding one or more Hise tags. In some embodiments the nucleic acid further comprised nucleic acids encoding linker sequences positioned between nucleic acids encoding the H-NOX domain, the polymerization domain and/or a Hise tag.

[0147] In some embodiments, the disclosure provides a nucleic acid encoding an H-NOX domain and a foldon domain. In some embodiments, the H-NOX domain is a T. tencongensis H-NOX domain. In some embodiments, the H-NOX domain is a wild-type T. tencongensis H- NOX domain. In some embodiments, the H-NOX domain is a T. tencongensis L144F H-NOX domain. In some embodiments, the H-NOX domain is a T. tencongensis W9F/L144F H-NOX domain.

[0148] In some embodiments, the disclosure provides nucleic acids encoding the following 5’ to 3’ : a L144F T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker sequence, and a foldon domain. In some embodiments, the disclosure provides nucleic acids encoding the following 5’ to 3’: a W9F/L144F T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker sequence, and a foldon domain. In some embodiments, the disclosure provides nucleic acids encoding the following 5’ to 3’: a wild-type T. tengcongensis H-NOX domain, a Gly-Ser- Gly amino acid linker sequence, and a foldon domain.

[0149] In some embodiments, the disclosure provides nucleic acids encoding the following 5’ to 3’: a L144F T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker sequence, a foldon domain, an Arg-Gly-Ser linker sequence, and a Hise tag. In some embodiments, the disclosure provides nucleic acids encoding the following 5’ to 3’ : a W9F/L144F T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker sequence, a foldon domain, an Arg-Gly-Ser linker sequence, and a Hise tag. In some embodiments, the disclosure provides nucleic acids encoding the following 5’ to 3’ : a wild-type T. tengcongensis H-NOX domain, a Gly-Ser-Gly amino acid linker sequence, a foldon domain, an Arg-Gly-Ser linker sequence, and a Hise tag.

[0150] In some embodiments, the nucleic acid comprises the nucleic acid sequence set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9 or SEQ ID NO: 11.

[0151] The disclosure also includes a cell or population of cells containing at least one nucleic acid encoding a mutant H-NOX protein described herein. Exemplary cells include insect, plant, yeast, bacterial, and mammalian cells. These cells are useful for the production of mutant H- NOX proteins using standard methods, such as those described herein.

[0152] In some embodiments, the disclosure provides a cell comprising a nucleic acid encoding an H-NOX domain and a foldon domain. In some embodiments, the H-NOX domain is a T. tencongensis H-NOX domain. In some embodiments, the H-NOX domain is a wild-type T. tencongensis H-NOX domain. In some embodiments, the H-NOX domain is a T. tencongensis L144F H-NOX domain. In some embodiments, the H-NOX domain is a T. tencongensis W9F/L144F H-NOX domain. In some embodiments, the disclosure provides a cell comprising a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, or SEQ ID NO: 11.

Formulations of H-NOX Proteins

[0153] Any wild-type or mutant H-NOX protein, including polymeric H-NOX proteins, described herein may be used for the formulation of pharmaceutical or non-pharmaceutical compositions. In some embodiments, the formulations comprise a monomeric H-NOX protein comprising an H-NOX domain and a polymerization domain such that the monomeric H-NOX proteins associate in vitro or in vivo to produce a polymeric H-NOX protein. As discussed further below, these formulations are useful in a variety of therapeutic and industrial applications.

[0154] In some embodiments, the pharmaceutical composition includes one or more wild-type or mutant H-NOX proteins described herein including polymeric H-NOX proteins and a pharmaceutically acceptable carrier or excipient. Examples of pharmaceutically acceptable carriers or excipients include, but are not limited to, any of the standard pharmaceutical carriers or excipients such as phosphate buffered saline solutions, water, emulsions such as oil/water emulsion, and various types of wetting agents. Exemplary diluents for aerosol or parenteral administration are phosphate buffered saline or normal (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000, which are each hereby incorporated by reference in their entireties, particularly with respect to formulations). In some embodiments, the formulations are sterile. In some embodiments, the formulations are essentially free of endotoxin.

[0155] While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this disclosure, the type of carrier will vary depending on the mode of administration. Compositions can be formulated for any appropriate manner of administration, including, for example, intravenous, intra-arterial, intravesicular, inhalation, intraperitoneal, intrapulmonary, intramuscular, subcutaneous, intra-tracheal, transmucosal, intraocular, intrathecal, or transdermal administration. For parenteral administration, such as subcutaneous injection, the carrier may include, e.g., water, saline, alcohol, a fat, a wax, or a buffer. For oral administration, any of the above carriers or a solid carrier, such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, or magnesium carbonate, may be employed. Biodegradable microspheres (e.g., polylactate polyglycolate) may also be used as carriers.

[0156] In some embodiments, the pharmaceutical or non-pharmaceutical compositions include a buffer (e.g., neutral buffered saline, phosphate buffered saline, etc), a carbohydrate (e.g., glucose, mannose, sucrose, dextran, etc.), an antioxidant, a chelating agent (e.g, EDTA, glutathione, etc.), a preservative, another compound useful for binding and/or transporting oxygen, an inactive ingredient (e.g, a stabilizer, filler, etc.), or combinations of two or more of the foregoing. In some embodiments, the composition is formulated as a lyophilizate. H-NOX proteins may also be encapsulated within liposomes or nanoparticles using well known technology. Other exemplary formulations that can be used for H-NOX proteins are described by, e.g., U.S. Pat. Nos. 6,974,795, and 6,432,918, which are each hereby incorporated by reference in their entireties, particularly with respect to formulations of proteins.

[0157] The compositions described herein may be administered as part of a sustained release formulation e.g., a formulation such as a capsule or sponge that produces a slow release of compound following administration). Such formulations may generally be prepared using well known technology and administered by, for example, oral, rectal or subcutaneous implantation, or by implantation at the desired target site. Sustained-release formulations may contain an H- NOX protein dispersed in a carrier matrix and/or contained within a reservoir surrounded by a rate controlling membrane. Carriers for use within such formulations are biocompatible, and may also be biodegradable. In some embodiments, the formulation provides a relatively constant level of H-NOX protein release. The amount of H-NOX protein contained within a sustained release formulation depends upon the site of implantation, the rate and expected duration of release, and the nature of the condition to be treated or prevented.

[0158] In some embodiments, the pharmaceutical composition contains an effective amount of a wild-type or mutant H-NOX protein. In some embodiments, the pharmaceutical composition contains an effective amount of a polymeric H-NOX protein comprising two or more wild-type or mutant H-NOX domains. In some embodiments, the pharmaceutical composition contains an effective amount of a recombinant monomeric H-NOX protein comprising a wild-type or mutant H-NOX domain and a polymerization domain as described herein. In some embodiments, the formulation comprises a trimeric H-NOX protein comprising three monomers, each monomer comprising a T. tengcongensis L144F H-NOX domain and a foldon domain. In some embodiments, the formulation comprises a trimeric H-NOX protein comprising three monomers, each monomer comprising a T. tengcongensis W9F/L144F H-NOX domain and a foldon domain. In some embodiments, the formulation comprises a trimeric H-NOX protein comprising three monomers, each monomer comprising a T. tengcongensis L144F H-NOX domain and a foldon domain.

[0159] An exemplary dose of hemoglobin as a blood substitute is from about 10 mg to about 5 grams or more of extracellular hemoglobin per kilogram of patient body weight. Thus, in some embodiments, an effective amount of an H-NOX protein for administration to a human is between a few grams to over about 350 grams. Other exemplary doses of an H-NOX protein include about any of 4.4., 5, 10, or 13 G/DL (where G/DL is the concentration of the H-NOX protein solution prior to infusion into the circulation) at an appropriate infusion rate, such as about 0.5 ml/min (see, for example, Winslow, R. Chapter 12 In Blood Substitutes). It will be appreciated that the unit content of active ingredients contained in an individual dose of each dosage form need not in itself constitute an effective amount since the necessary effective amount could be reached by the combined effect of a plurality of administrations. The selection of the amount of an H-NOX protein to include in a pharmaceutical composition depends upon the dosage form utilized, the condition being treated, and the particular purpose to be achieved according to the determination of the ordinarily skilled artisan in the field.

[0160] Exemplary compositions include genetically engineered, recombinant H-NOX proteins, which may be isolated or purified, comprising one or more mutations that collectively impart altered O2 or NO ligand-binding relative to the corresponding wild-type H-NOX protein, and operative as a physiologically compatible mammalian blood gas carrier. For example, mutant H-NOX proteins as described herein. In some embodiments, the H-NOX protein is a polymeric H-NOX protein. In some embodiments, the H-NOX protein is a recombinant monomeric H- NOX protein comprising a wild-type or mutant H-NOX domain and a polymerization domain as described herein. In some embodiments, the composition comprises a trimeric H-NOX protein comprising three monomers, each monomer comprising a T. tengcongensis L144F H-NOX domain and a foldon domain. In some embodiments, the composition comprises a trimeric H- NOX protein comprising three monomers, each monomer comprising a T. tengcongensis W9F/L144F H-NOX domain and a foldon domain. In some embodiments, the composition comprises a trimeric H-NOX protein comprising three monomers, each monomer comprising a T. tengcongensis L144F H-NOX domain and a foldon domain.

[0161] To reduce or prevent an immune response in human subjects who are administered a pharmaceutical composition, human H-NOX proteins or domains (either wild-type human proteins or human proteins into which one or more mutations have been introduced) or other non-antigenic H-NOX proteins or domains (e.g., mammalian H-NOX proteins) can be used. To reduce or eliminate the immunogenicity of H-NOX proteins derived from sources other than humans, amino acids in an H-NOX protein or H-NOX domain can be mutated to the corresponding amino acids in a human H-NOX. For example, one or more amino acids on the surface of the tertiary structure of a non-human H-NOX protein can be mutated to the corresponding amino acid in a human H-NOX protein. Therapeutic Applications of H-NOX Proteins

[0162] Any of the wild-type or mutant H-NOX proteins, including polymeric H-NOX proteins, or pharmaceutical compositions described herein may be used in therapeutic applications.

[0163] Particular H-NOX proteins, including polymeric H-NOX proteins, can be selected for such applications based on the desired O2 association rate, O2 dissociation rate, dissociation constant for O2 binding, NO stability, NO reactivity, autoxidation rate, plasma retention time, or any combination of two or more of the foregoing for the particular indication being treated. [0164] Because the distribution in the vasculature of extracellular H-NOX proteins is not limited by the size of the red blood cells, polymeric H-NOX proteins of the present disclosure can be used to deliver O2 to areas that red blood cells cannot penetrate. These areas can include any tissue areas that are located downstream of obstructions to red blood cell flow, such as areas downstream of one or more thrombi, sickle cell occlusions, arterial occlusions, peripheral vascular occlusions, angioplasty balloons, surgical instruments, tissues that are suffering from oxygen starvation or are hypoxic, and the like. Additionally, all types of tissue ischemia can be treated using H-NOX proteins. Such tissue ischemias include, for example, perioperative ischemia, stroke, emerging stroke, transient ischemic attacks, myocardial stunning and hibernation, acute or unstable angina, emerging angina, and myocardial infarction (e.g., ST- segment elevation myocardial infarction). Other exemplary cardiovascular indications that can be treated using H-NOX proteins include cardioplegia and sickle cell anemia. Exemplary target indications include conditions of functional hemoglobin deficiency, such as where a blood substitute or O2 carrier is indicated, including blood loss, hypoxia, etc.

[0165] In a particular aspect, the present disclosure provides methods of using H-NOX proteins to deliver O2 to for preserving an organ in a donation after brain death of a donor or a donation after cardiac death of a donor. The preservation of the organ is therefore carried out either directly in situ in the deceased donor or ex situ (e.g., on a back table, if necessary). The composition according to the disclosure can be directly perfused in the donor awaiting harvesting of the various transplant organs (heart, lung, liver, kidneys, pancreas, intestine, cornea, etc.). [0166] According to the present disclosure, the organ preservation can be carried out directly in the donor post-mortem. The deceased donor may be in a state of brain death or in cardiac arrest. In the latter case, reference is made to non-heart-beating organ procurement. Preservation solution can also be used as a static preservative, or a perfused preservative kept flowing via pump. Both methods are used or being developed for use, and H-NOX proteins can be used with any of them.

[0167] In the methods described herein, brain death, also known as irreversible coma or stage IV coma, is defined as the complete and definitive irreversible ceasing of brain activity, even though blood circulation persists. A donor is in a state of brain death, or encephalic death, when the encephalon is irreversibly destroyed, despite the temporary persistence of hemodynamic activity and of vascularization of the organs.

[0168] In the methods described herein, a donor is in cardiac arrest if this cardiac arrest is irreversible after resuscitation measures have been ceased. The period of time after which asystole is considered to be irreversible is about one minute, after resuscitation measures have been ceased. However, the recommendations require a period of more than 5 minutes.

[0169] Thus, the present disclosure provides a method for preserving an organ for donation after brain or cardiac death in a donor that involves administering to the donor a composition comprising at least one H-NOX protein, a stabilizing solution and/or an organ preservation solution, wherein the composition is at a temperature of between 0°C and 37° C.

[0170] The present disclosure also provides a method for preserving an organ ex situ in a donation after brain death donor or a donation after cardiac death donor that involves a) perfusion of said deceased donor with a composition as described herein; then b) harvesting of the organ to be transplanted; then c) static or dynamic-perfusion preservation of said organ obtained in b), at a temperature of between 0° C and 37° C, for a time predetermined according to said organ, in the composition or the aqueous solution defined in step a).

[0171] In the methods of the present disclosure, the organ preservation can also be carried out post-mortem after the organ has been removed from the donor. Preservation solution can also be used as a static preservative, or a perfused preservative kept flowing via pump. By way of example, in some embodiments, the H-NOX product can be administered to an organ or organs at the time of their removal from the donor in preparation for storage, transportation and eventual transplantation into a recipient. In other embodiments, the H-NOX product can be administered to an organ or organs that have been removed from the donor for a time period in which the organ or organs have experienced hypoxic injury as a result.

[0172] In one aspect, the present disclosure provides a method for preserving an organ ex situ that involves a) harvesting of the organ to be transplanted; then b) maintaining in static or dynamic perfusion said organ obtained in a), at a temperature of between 0° C and 37° C, for a time predetermined according to said organ, in the composition or the aqueous solution as described herein.

[0173] As described above, the composition according to the disclosure comprises at least one H-NOX protein and a stabilizing solution and/or organ preservation solution. In some embodiments, the organ preservation solution is for use in static-based preservation. In other embodiments, the organ preservation solution is for use in perfusion-based preservation. In some uses, such as normothermic organ preservation using a perfusion pump, the solution could could also be whole blood, supplemented with H-NOX-based product. This could leverage the higher O2 affinity of the H-NOX product to extend the O2 delivery range: if/when the blood became depleted of O2, such as in a highly hypoxic tissue, the H-NOX product would still be bound to O2 and could therefore continue to deliver O2.

[0174] This solution makes it possible to maintain the basal metabolism of the cells constituting the transplant organ. It meets a triple objective: to wash the arterial blood from the transplant organ, to bring the transplant organ homogeneously to the desired preservation temperature, and to protect and prevent damage caused by ischemia and reperfusion and to optimize the resumption of function. The organ preservation solution is therefore clinically acceptable.

[0175] The stabilizing solution, as described herein, can be, for example, an aqueous solution comprising salts, and comprises a pH of between 6.5 and 7.6. In at least one embodiment, the solution is an aqueous solution comprising sodium ions.

[0176] In at least one embodiment, the stabilizing solution is an aqueous solution comprising 20 mM sodium citrate, 250 mM glucose, 10 mM glutathione, and 0.1% pol oxamer 188 at pH 6.8 ± 0.2.

[0177] The organ preservation solution can be an aqueous solution which has a pH of between 6.5 and 7.5, comprising salts, preferably chloride, sulfate, sodium, calcium, magnesium and potassium ions; sugars, preferably mannitol, ramose, sucrose, glucose, fructose, lactobionate (which is an imper-meant), or gluconate; antioxidants, preferably glutathione; active agents, preferably xanthine oxidase inhibitors, such as allopurinol, lactates, amino acids such as histidine, glutamic acid (or glutamate) or tryptophan; and optionally colloids such as hydroxyethyl starch, polyethylene glycol or dextran.

[0178] According to one preferred embodiment of the disclosure, the organ preservation solution is chosen from: University of Wisconsin (UW or Viaspan®) solution, which has an osmolality of 320 mOsmol/kg and a pH of 7.4, having the following formulation for one liter in water:

Potassium lactobionate: 100 mM

KOH: 100 mM

NaOH: 27 mM

KH ₂ PO ₄ : 25 mM

MgSO4: 5 mM

Raffinose: 30 mM

Adenosine: 5 mM

Glutathione: 3 mM

Allopurinol: 1 mM

Hydroxy ethyl starch: 50 g/1;

IGL-1®, which has an osmolality of 320 mOsm/kg and a pH of 7.4, having the following formulation for one liter in water:

NaCl: 125 mM

KH2PO4: 25 mM

MgSO4: 5 mM

Raffinose: 30 mM

Potassium lactobionate: 100 mM

Glutathione: 3 mM

Allopurinol: 1 mM

Adenosine: 5 mM

Polyethylene glycol (molecular weight: 35 kDa): 1 g/1;

Celsior®, which has an osmolality of 320 mOsm/kg and a pH of 7.3, having the following formulation for one liter in water: Glutathione: 3 mM

Mannitol: 60 mM

Lactobionic acid: 80 mM

Glutamic acid: 20 mM

NaOH: 100 mM

Calcium chloride dihydrate: 0.25 mM

MgSO ₄ : 1.2 mM

KC1 : 15 mM

Magnesium chloride hexahydrate: 13 mM

Histidine: 30 mM;

SCOT 15 Multi Organes Abdominaux® and SCOT 30 Greffons Vasculaires® from Macopharma, both comprising in particular high-molecular-weight (20 kDa) polyethylene glycol; BMPS Belzer®, or Belzer machine perfusion solution, or KPS1, comprising in particular 100 mEq/1 of sodium, 25 mEq/1 of potassium, a pH of 7.4 at ambient temperature, and having an osmolarity of 300 mOsm/I; Custodiol® HTK Solution, having the following formulation for one liter in water, the pH being 7.20 at ambient temperature, and the osmolality being 310 mOsm/kg:

NaCl: 18.0 mM

KC1 : 15.0 mM

KH ₂ PO ₄ : 9 mM

Hydrogenated potassium 2-ketoglutarate: 1.0 mM Magnesium chloride hexahydrate: 4.0 mM

Histidine.HCl.H2O: 18.0 mM

Histidine: 198.0 mM

Tryptophan: 2.0 mM

Mannitol: 30.0 mM

Calcium chloride dihydrate: 0.015 mM;

Euro-Collins®, which has an osmolality of 355 mOsm/kg and a pH of 7.0, and having the following formulation for one liter in water:

Sodium: 10 mM

Potassium: 115 mM Chloride: 15 mM

H ₂ PO ₄ ": 15 mM

HPO ₄ ² ": 42.5 mM

HCO ₃ ": 10 mM

Glucose: 194 mM;

Soltran®, which has an osmolality of 486 mOsm/kg and a pH of 7.1, and having the following formulation for one liter in water:

Sodium: 84 mM

Potassium: 80 mM

Magnesium: 41 mM

Sulfate": 41 mM

Mannitol: 33.8 g/1

Citrate: 54 mM

Glucose: 194 mM;

Perfadex®, which has an osmolarity of 295 mOsmol/1 having the following formulation in water:

50 g/1 of dextran 40 (molecular weight: 40 000),

Na: 138 mM,

K+: 6 mM,

Mg ² +: 0.8 mM,

Cl": 142 mM,

SO ₄ ² ": 0.8 mM,

(H ₂ PO ₄ "-HPO ₄ ² "): 0.8 mM and

Glucose: 5 mM;

Ringer Lactate®, having the following formulation in water, the pH being between 6.0 and 7.5 at ambient temperature, and having an osmolarity of 276.8 mOs- mol/1 :

Na+: 130 mM,

K +: 5.4 mM,

Ca ² +: 1.8 mM,

Cl": 111 mM,

Lactates: 27.7 mM; Plegisol®, having the following formulation in water:

KC1: 1.193 g/1,

MgCl ₂ .6H ₂ O: 3.253 g/1,

NaCl: 6.43 g/1,

CaCl ₂ : 0.176 g/1;

Solution of the Edouard Henriot hospital, having the following formulation in water, the pH being equal to 7.4 at ambient temperature, and which has an osmolarity of 320 mOsmol/1 : KOH: 25 mM,

NaOH: 125 mM,

KH ₂ PO ₄ : 25 mM,

MgCh: 5 mM,

MgSO ₄ : 5 mM,

Raffinose: 30 mM,

Lactobionate: 100 mM,

Glutathione: 3 mM,

Allopurinol: 1 mM,

Adenosine: 5 mM,

Hydroxy ethyl starch 50 g/1; and the Steen® solution, comprising human serum albumin, dextran and extracellular electrolytes with a low potassium concentration. All of these organ preservation solutions are commercial products.

[0179] In another embodiment, the organ preservation solution is University of Wisconsin machine perfusion solution, having the following formulation in water:

Calcium chloride (dehydrate): 0.5 mM,

Sodium hydroxide: 18 mM,

HEPES (free acid): 10 mM

Potassium phosphate (monobasic): 25 mM,

Mannitol (USP): 30 mM,

Beta D (+) glucose: 10 mM,

Sodium gluconate: 80 mM,

Magnesium gluconate D (-) gluconic acid, hemimagnesium salt: 5 mM, Ribose, D (-): 5 mM,

Hydroxyethyl starch (HES): 50 g/1, Glutathione, reduced: 3 mM, Adenine (free base): 5 mM.

[0180] According to one preferred embodiment, the temperature of the compositions according to the disclosure is between 0° C and 37° C, preferentially between 2° C and 32° C, preferentially between 4° C and 25° C and more preferentially approximately 4° C.

[0181] The compositions according to the disclosure make it possible to work both under hypothermic conditions and under normothermic conditions (close to physiological temperature).

[0182] Additional compositions are also contemplated herein. In one aspect, the present disclosure is directed to a composition having a pH of 6.5 to 7.6, which comprises: at least one H-NOX protein; calcium ions, preferably in an amount of between 0 and 0.5 mM; KOH, preferably in an amount of between 20 and 100 mM; NaOH, preferably in an amount of between 20 and 125 mM; KH2PO4, preferably in an amount of between 20 and 25 mM; MgCh, preferably in an amount of between 3 and 5 mM; at least one sugar chosen from raffinose and glucose, preferably in an amount of between 5 and 200 mM; adenosine, preferably in an amount of between 3 and 5 mM; glutathione, preferably in an amount of between 2 and 4 mM; allopurinol, preferably in an amount of between 0 and 1 mM; and at least one compound chosen from hydroxyethyl starch, polyethylene glycols of different molecular weights and human serum albumin, preferably in an amount of between 1 and 50 g / 1.

[0183] In another aspect, the present disclosure is directed to a composition having a pH of 6.5 to 7.6, which comprises: at least one H-NOX protein; calcium ions, preferably in an amount of between 0 and 0.5 mM; NaOH, preferably in an amount of between 15 and 30 mM; HEPES, preferably in an amount of between 2 and 10 mM KH2PO4, preferably in an amount of between 20 and 25 mM; mannitol, preferably in an amount of between 20 and 35 mM; glucose, preferably in an amount of between 3 and 10 mM sodium gluconate, preferably in an amount of between 50 and 100 mM magnesium gluconate, preferably in an amount of between 1 and 5 mM ribose, preferably in an amount of between 2 and 5 mM; at least one compound chosen from hydroxyethyl starch, polyethylene glycols of different molecular weights and human serum albumin, preferably in an amount of between 1 and 50 g / 1; glutathione, preferably in an amount of between 2 and 4 mM; and adenine, preferably in an amount of between 3 and 5 mM.

[0184] In various embodiments, the disclosure features a method of delivering O2 to an organ donor (e.g., a mammal, such as a primate (e.g., a human, a monkey, a gorilla, an ape, a lemur, etc. , a bovine, an equine, a porcine, a canine, or a feline) by administering to an donor in need thereof a wild-type or mutant H-NOX protein, including a polymeric H-NOX protein in an amount sufficient to deliver O2 to the donor. In some embodiments, the disclosure provides methods of carrying or delivering blood gas to a donor such as a mammal, comprising the step of delivering (e.g., transfusing, etc.) to the blood of the individual (e.g., a mammal) one or more of H-NOX compositions. Methods for delivering O2 carriers to blood or tissues (e.g., mammalian blood or tissues) are known in the art. In various embodiments, the H-NOX protein is an apoprotein that is capable of binding heme or is a holoprotein with heme bound. The H- NOX protein may or may not have heme bound prior to the administration of the H-NOX protein to the donor. In some embodiments, O2 is bound to the H-NOX protein before it is delivered to the donor. In other embodiments, O2 is not bound to the H-NOX protein prior to the administration of the protein to the donor, and the H-NOX protein transports O2 from one location in the donor to another location in the donor.

[0185] Wild-type and mutant H-NOX proteins, including polymeric H-NOX proteins, with a relatively low KD for O2 (such as less than about 80 nM or less than about 50 nM) are expected to be particularly useful to treat tissues with low oxygen tension (such as tumors, some wounds, or other areas where the oxygen tension is very low, such as a p50 below 1 mm Hg). The high affinity of such H-NOX proteins for O2 may increase the length of time the O2 remains bound to the H-NOX protein, thereby reducing the amount of 02that is released before the H-NOX protein reaches the tissue to be treated.

[0186] In some embodiments for the direct delivery of an H-NOX protein with bound O2 to a particular site in the body (such as the site of an organ needed for transplantation site), the kofr for O2 is more important than the KD value because O2 is already bound to the protein (making the konless important) and oxygen needs to be released at or near a particular site in the body (at a rate influenced by the k _O ff). In some embodiments, the kofr may also be important when H- NOX proteins are in the presence of red cells in the circulation, where they facilitate diffusion of O2 from red cells, and perhaps prolonging the ability of diluted red cells to transport O2 to further points in the vasculature.

[0187] In some embodiments for the delivery of an H-NOX protein that circulates in the bloodstream of an individual, the H-NOX protein binds O2 in the lungs and releases O2 at one or more other sites in the body. For some of these applications, the KD value is more important than the k _O ff since O2 binding is at or near equilibrium. In some embodiments for extreme hemodilution, the KD more important than the koff when the H-NOX protein is the primary O2 carrier because the H-NOX protein will bind and release O2 continually as it travels through the circulation. Since hemoglobin has a p50 of 14 mm Hg, red cells (which act like capacitors) have a p50 of ~30 mm Hg, and HBOCs have been developed with ranges between 5 mm Hg and 90 mm Hg, the optimal KD range for H-NOX proteins may therefore be between -2 mm Hg to -100 mm Hg for some applications.

[0188] H-NOX proteins, including polymeric H-NOX proteins, and pharmaceutical compositions of the disclosure can be administered to an individual by any conventional means such as by oral, topical, intraocular, intrathecal, intrapulmonary, intra-tracheal, or aerosol administration; by transdermal or mucus membrane adsorption; or by injection (e.g., subcutaneous, intravenous, intra-arterial, intravesicular, or intramuscular injection). H-NOX proteins may also be included in large volume parenteral solutions for use as blood substitutes. In exemplary embodiments, the H-NOX protein is administered to the blood (e.g., administration to a blood vessel such as a vein, artery, or capillary), a wound, a tumor, a hypoxic tissue, or a hypoxic organ of the individual.

[0189] In some embodiments, a sustained continuous release formulation of the composition is used. Administration of an H-NOX protein can occur, e.g., for a period of seconds to hours depending on the purpose of the administration. For example, as a blood delivery vehicle, an exemplary time course of administration is as rapid as possible. Other exemplary time courses include about any of 10, 20, 30, 40, 60, 90, or 120 minutes. Exemplary infusion rates for H- NOX solutions as blood replacements are from about 30 mL/hour to about 13,260 mL/hour, such as about 100 mL/hour to about 3,000 mL/hour. An exemplary total dose of H-NOX protein is about 900 mg/kg administered over 20 minutes at 13,260 mL/hour. An exemplary total dose of H-NOX protein for a swine is about 18.9 grams. [0190] The composition according to the disclosure is preferably administered by injection, in particular by intravascular injection. It can also be administered by normothermic extracorporeal membrane oxygenation (normothermic ECMO), /.< ., with an arterial catheter, a venous catheter and a Fogarty catheter, or by hypothermic extracorporeal membrane oxygenation (hypothermic ECMO) for generalized cooling of the donor. It can also be administered using a double-balloon triple-lumen catheter or any other similar technique.

[0191] The normothermic ECMO or hypothermic ECMO system is a technique for extracorporeal circulation of blood fluid.

[0192] The double-balloon triple-lumen catheter enables, for its part, in situ cooling of the abdominal organs. In particular, when it is implanted in the femoral arterial circuit, it makes it possible to isolate renal circulation. A rapid perfusion of the composition according to the disclosure can be carried out through the double-balloon triple-lumen catheter. A discharge route is implanted in the femoral vein, allowing evacuation of the perfused liquid composition.

[0193] Exemplary dosing frequencies include, but are not limited to, at least 1, 2, 3, 4, 5, 6, or 7 times (i.e., daily) a week. In some embodiments, an H-NOX protein is administered at least 2, 3, 4, or 6 times a day. The H-NOX protein can be administered, e.g., over a period of a few days or weeks. In some embodiments, the H-NOX protein is administrated for a longer period, such as a few months or years. The dosing frequency of the composition may be adjusted over the course of the treatment based on the judgment of the administering physician.

[0194] As noted above, the selection of dosage amounts for H-NOX proteins depends upon the dosage form utilized, the frequency and number of administrations, the condition being treated, and the particular purpose to be achieved according to the determination of the ordinarily skilled artisan in the field. In some embodiments, an effective amount of an H-NOX protein for administration to human is between a few grams to over 350 grams.

[0195] In some embodiments, two or more different H-NOX proteins are administered simultaneously, sequentially, or concurrently. In some embodiments, another compound or therapy useful for the delivery of O2 is administered simultaneously, sequentially, or concurrently with the administration of one or more H-NOX proteins.

[0196] Other exemplary therapeutic applications for which H-NOX proteins can be used are described by, e.g., U.S. Pat. Nos. 6,974,795, and 6,432,918, which are each hereby incorporated by reference in their entireties, particularly with respect to therapeutic applications for O2 carriers.

Kits with H-NOX proteins

[0197] Also provided are articles of manufacture and kits that include any of the H-NOX proteins described herein including polymeric H-NOX proteins, and suitable packaging. In some embodiments, the disclosure includes a kit with (i) an H-NOX protein (such as a wild-type or mutant H-NOX protein described herein or formulations thereof as described herein) and (ii) instructions for using the kit to deliver O2 to an individual.

[0198] In some embodiments, kits are provided for use in the methods described herein. In some embodiments, the kit comprises a polymeric H-NOX protein. In some embodiments, the kit comprises an effective amount of a polymeric H-NOX protein comprising two or more wildtype or mutant H-NOX domains. In some embodiments, the kit comprises an effective amount of a recombinant monomeric H-NOX protein comprising a wild-type or mutant H-NOX domain and a polymerization domain as described herein. In some embodiments, the kit comprises a trimeric H-NOX protein comprising three monomers, each monomer comprising a mutation corresponding to a T. tengcongensis L144F H-NOX mutation and a trimerization domain. In some embodiments, the kit comprises a trimeric H-NOX protein comprising three monomers, each monomer comprising a mutation corresponding to a T. tengcongensis W9F/L144F H-NOX mutation and a trimerization domain. In some embodiments, the trimeric H-NOX protein comprises human H-NOX domains. In some embodiments, the trimeric H-NOX protein comprises canine H-NOX domains. In some embodiments, the kit comprises a trimeric H-NOX protein comprising three monomers, each monomer comprising a T. tengcongensis L144F H- NOX domain and a foldon domain. In some embodiments, the kit comprises a trimeric H-NOX protein comprising three monomers, each monomer comprising a T. tengcongensis W9F/L144F H-NOX domain and a foldon domain. In some embodiments, the kit comprises a trimeric H- NOX protein comprising three monomers, each monomer comprising a T. tengcongensis L144F H-NOX domain and a foldon domain.

[0199] Suitable packaging for compositions described herein are known in the art, and include, for example, vials (e.g., sealed vials), vessels, ampules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. These articles of manufacture may further be sterilized and/or sealed. Also provided are unit dosage forms comprising the compositions described herein. These unit dosage forms can be stored in a suitable packaging in single or multiple unit dosages and may also be further sterilized and sealed. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. The instructions relating to the use of H- NOX proteins generally include information as to dosage, dosing schedule, and route of administration for the intended treatment or industrial use. The kit may further comprise a description of selecting an individual suitable or treatment.

[0200] The containers may be unit doses, bulk packages (e.g., multi-dose packages) or subunit doses. For example, kits may also be provided that contain sufficient dosages of H-NOX proteins disclosed herein to provide effective treatment for an individual for an extended period, such as about any of a week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, or more. Kits may also include multiple unit doses of H-NOX proteins and instructions for use and packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies. In some embodiments, the kit includes a dry (e.g., lyophilized) composition that can be reconstituted, resuspended, or rehydrated to form generally a stable aqueous suspension of H- NOX protein.

Exemplary Methods for Production of H-NOX Proteins

[0201] The present disclosure also provides methods for the production of any of the polymeric H-NOX proteins described herein. In some embodiments, the method involves culturing a cell that has a nucleic acid encoding a polymeric H-NOX protein under conditions suitable for production of the polymeric H-NOX protein. In various embodiments, the polymeric H-NOX is also purified (such as purification of the H-NOX protein from the cells or the culture medium). In some embodiments, the method involves culturing a cell that has a nucleic acid encoding a monomer H-NOX protein comprising an H-NOX domain and a polymerization domain. The monomers then associate in vivo or in vitro to form a polymeric H-NOX protein. A polymeric H-NOX protein comprising heterologous H-NOX domains may be generated by cointroducing two or more nucleic acids encoding monomeric H-NOX proteins with the desired H- NOX domains and where in the two or more monomeric H-NOX proteins comprise the same polymerization domain.

[0202] In some embodiments, a polymeric H-NOX protein comprising heterologous H-NOX domains is prepared by separately preparing polymeric H-NOX proteins comprising homologous monomeric H-NOX subunits comprising the desired H-NOX domains and a common polymerization domain. The different homologous H-NOX proteins are mixed at a desired ratio of heterologous H-NOX subunits, the homologous polymeric H-NOX proteins are dissociated (e.g. by heat, denaturant, high salt, etc.), then allowed to associate to form heterologous polymeric H-NOX proteins. The mixture of heterologous polymeric H-NOX proteins may be further purified by selecting for the presence of the desired subunits at the desired ratio. For example, each different H-NOX monomer may have a distinct tag to assist in purifying heterologous polymeric H-NOX proteins and identifying and quantifying the heterologous subunits.

[0203] As noted above, the sequences of several wild-type H-NOX proteins and nucleic acids are known and can be used to generate mutant H-NOX domains and nucleic acids of the present disclosure. Techniques for the mutation, expression, and purification of recombinant H-NOX proteins have been described by, e.g., Boon, E.M. et al. (2005). Nature Chemical Biology 1:53- 59 and Karow, D. S. et al. (August 10, 2004). Biochemistry 43(31): 10203-10211, which is hereby incorporated by reference in its entirety, particularly with respect to the mutation, expression, and purification of recombinant H-NOX proteins. These techniques or other standard techniques can be used to generate any mutant H-NOX protein.

[0204] In particular, mutant H-NOX proteins described herein can be generated a number of methods that are known in the art. Mutation can occur at either the amino acid level by chemical modification of an amino acid or at the codon level by alteration of the nucleotide sequence that codes for a given amino acid. Substitution of an amino acid at any given position in a protein can be achieved by altering the codon that codes for that amino acid. This can be accomplished by site-directed mutagenesis using, for example: (i) the Amersham technique (Amersham mutagenesis kit, Amersham, Inc., Cleveland, Ohio) based on the methods of Taylor, J.W. et al. (December 20, 1985). Nucleic Acids Res. 13(24):8749-8764; Taylor, J.W. et al. (December 20, 1985). Nucleic Acids Res. 13(24):8765-8785; Nakamaye, K. L. et al. (December 22, 1986). Nucleic Acids Res. 14(24):9679-9698; and Dente et al. (1985). in DNA Cloning, Glover, Ed., IRL Press, pages 791-802, (ii) the Promega kit (Promega Inc., Madison, Wis.), or (iii) the Biorad kit (Biorad Inc., Richmond, Calif.), based on the methods of Kunkel, T. A. (January 1985).

Proc. Natl. Acad. Sci. USA 82(2):488-492; Kunkel, T. A. (1987). Methods Enzymol. 154:367- 382; Kunkel, U.S. Pat. No. 4,873,192, which are each hereby incorporated by reference in their entireties, particularly with respect to the mutagenesis of proteins. Mutagenesis can also be accomplished by other commercially available or non-commerci al means, such as those that utilize site-directed mutagenesis with mutant oligonucleotides.

[0205] Site-directed mutagenesis can also be accomplished using PCR-based mutagenesis such as that described in Zhengbin et al. (1992). pages 205-207 in PCR Methods and Applications, Cold Spring Harbor Laboratory Press, New York; Jones, D. H. et al. (February 1990). Biotechniques 8(2): 178- 183 ; Jones, D. H. et al. (January 1991). Biotechniques 10(l):62-66, which are each hereby incorporated by reference in their entireties, particularly with respect to the mutagenesis of proteins. Site-directed mutagenesis can also be accomplished using cassette mutagenesis with techniques that are known to those of skill in the art.

[0206] A mutant H-NOX nucleic acid and/or polymerization domain can be incorporated into a vector, such as an expression vector, using standard techniques. For example, restriction enzymes can be used to cleave the mutant H-NOX nucleic acid and the vector. Then, the compatible ends of the cleaved mutant H-NOX nucleic acid and the cleaved vector can be ligated. The resulting vector can be inserted into a cell (e.g., an insect cell, a plant cell, a yeast cell, or a bacterial cell) using standard techniques (e.g., electroporation) for expression of the encoded H-NOX protein.

[0207] In particular, heterologous proteins have been expressed in a number of biological expression systems, such as insect cells, plant cells, yeast cells, and bacterial cells. Thus, any suitable biological protein expression system can be utilized to produce large quantities of recombinant H-NOX protein. In some embodiments, the H-NOX protein (c.g, a mutant or wildtype H-NOX protein) is an isolated protein.

[0208] If desired, H-NOX proteins can be purified using standard techniques. In some embodiments, the protein is at least about 60%, by weight, free from other components that are present when the protein is produced. In various embodiments, the protein is at least about 75%, 90%, or 99%, by weight, pure. A purified protein can be obtained, for example, by purification (e.g., extraction) from a natural source, a recombinant expression system, or a reaction mixture for chemical synthesis. Exemplary methods of purification include immunoprecipitation, column chromatography such as immunoaffinity chromatography, magnetic bead immunoaffinity purification, and panning with a plate-bound antibody, as well as other techniques known to the skilled artisan. Purity can be assayed by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. In some embodiments, the purified protein is incorporated into a pharmaceutical composition of the disclosure or used in a method of the disclosure. The pharmaceutical composition of the disclosure may have additives, carriers, or other components in addition to the purified protein. [0209] In some embodiments, the polymeric H-NOX protein comprises one or more Hise tags. An H-NOX protein comprising at least one Hise tag may be purified using chromatography; for example, using Ni ²⁺ -affinity chromatography. Following purification, the Hise tag may be removed; for example, by using an exopeptidase. In some embodiments, the disclosure provides a purified polymeric H-NOX protein, wherein the polymeric H-NOX protein was purified through the use of a Hise tag. In some embodiments, the purified H-NOX protein is treated with an exopeptidase to remove the Hise tags.

EXAMPLES

[0210] The examples, which are intended to be purely exemplary of the disclosure and should therefore not be considered to limit the disclosure in any way, also describe and detail aspects and embodiments of the disclosure discussed above. The examples are not intended to represent that the experiments below are all or the only experiments performed. Unless indicated otherwise, temperature is in degrees Centigrade and pressure is at or near atmospheric.

Example 1. Improved Support of Ex Vivo Organ Preservation

[0211] The purpose of this Example is to describe organ preservation studies which will demonstrate improved ex vivo organ preservation for organ transplantation, when using the methods disclosed herein.

[0212] Extracted hearts, kidneys and/or livers from a healthy mini pig will be flushed out to remove blood and then perfused with standard of care University of Wisconsin (UW) cold storage solution alone, or UW solution supplemented with OMX-4.80P (at 0.5-2 mg/ml concentrations) and organs will be bathed in the same solution for up to 24 hours at 4 degrees Celsius. Organ biopsy will be collected at regular time intervals. For example, a subset of time points includes 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 18 hours, and 24 hours. The organ biopsy will be analyzed for energy and metabolic status (e.g., ATP levels, EM assessment of mitochondrial status, lactate production etc.), markers of oxidative stress (e.g., catalase, SOD, lipid peroxidation - MDA etc.), markers of tissue inflammation and integrity (e.g., H&E staining for tissue architecture and structure, presence of cell death, immune infiltration, cytokine expression such as IL6 and TNFa for presence of inflammatory markers etc.).

[0213] It is expected that organs exposed to OMX-4.80P supplemented cold storage solution will show lower levels of tissue structure disintegration and cell death, higher levels of ATP or presence of other indicators of higher energy status and aerobic metabolic rate and mitochondria function, and lower levels of tissue inflammation and/or oxidative stress as compared to organs that were exposed for the same time to standard of care UW cold storage solution alone.

Example 2. Improved Performance of Organs Post-Transplantation and Improved PostTransplantation Outcome

[0214] The purpose of this Example is to describe organ preservation studies which will demonstrate improved performance of transplanted organs post-transplantation, as well as improved post-transplantation patient outcome, when using the methods disclosed herein.

[0215] Extracted hearts, kidneys or livers from a healthy mini pigs or other large animal will be flushed out to remove blood and then perfused with standard of care University of Wisconsin (UW) cold storage solution alone, or UW solution supplemented with OMX-4.80P (at 0.5-2 mg/ml concentrations), and organs will be bathed in the same solution for 4-24 hours at 4°C. Following ex vivo storage in cold solution alone or cold solution supplemented with OMX- 4.80P, organs will be transplanted into recipient animals. Both cohorts of animals will be assessed for organ function and animal survival for up to 7 days post-surgery.

[0216] It is expected that animal recipients of organs that were stored in OMX-4.80P- supplemented preservation solution will exhibit better organ function as assessed by circulatory markers (e.g., ALT, AST for liver, troponin 2 for heart, urea, BUN for kidney), lower inflammatory cytokine levels (e.g, TNFa, IL6, ylFN), and exhibit better tissue integrity and/or lower tissue inflammation as assessed by histological evaluations (e.g., H&E staining, etc.) at terminal necropsy. If overall survival rate in control group is low, improvement in recipient animal survival rate may be seen in the OMX-4.80P cohort.

Example 3. Addition of OMX in Preservation Solution Reduces and/or Defers Hypoxiadependent Dysfunction and Pathophysiology in Machine-perfused Kidneys

[0217] The objective of this ongoing study was to evaluate if OMX-4.80P Drug Product (OMX)-supplemented Cold Storage Solution (CSS) can safely and effectively preserve human deceased donor kidneys placed on perfusion pump better than CSS alone as measured by histological and biochemical markers of tissue quality and function. To date, two matched pairs of kidneys (genetically identical kidneys from the same donor) have been studied; for each pair, one kidney received CSS alone, and the other received CSS + OMX. Functional and histopathological data support the hypothesis that OMX-supplemented CSS provides improved preservation of kidney function during cold storage compared to CSS alone. This study is ongoing, and additional data will be incorporated as it is available.

Experimental Design

[0218] The experimental design is shown in Table 2.

Table 2.

Materials

[0219] OMX is a PEGylated oxygen carrier derived from a member of the heme-nitric oxide/oxygen (H NOX) protein family that is engineered to specifically deliver oxygen to low oxygen (hypoxic) regions of tissues without increasing oxygen levels in normoxic tissues. The OMX Test Article used in this study was produced as a bulk substance prepared using E. coli cell culture and standard procedures for downstream purification and processing and formulated in an aqueous buffer. Product quality and safety were ensured by testing against pre-determined specifications prior to use in animal studies. OMX was stored frozen at -80 °C and thawed before use. [0220] The vehicle for this study was KPS-1 Kidney Perfusion Solution, a version of UW CSS formulated for machine perfusion, delivered using a LifePort Kidney Transporter perfusion system. Both the KPS-1 and LifePort Kidney Transporter are obtained from Organ Recovery Systems. Test article was thawed at room temperature, then held at 2 to 8 °C protected from light. Test article was diluted to the target concentration in cold CSS and stored at 2 to 8 °C until use.

Vehicle/Test Article Administration

[0221] For the organs used to generate the data described herein, the following procedure was carried out. Immediately prior to organ discard arrival, the pump was prepared as follows. The ice chamber was filled with crushed ice and water; the main organ chamber was filled with 1 L of CSS ± OMX; and circulation of solution was started to remove any air bubbles. A 1 mL retain of the CSS ± OMX solution was pulled and frozen (-50 to -90°C) as a test article formulation sample.

[0222] After organ arrival and unpacking, the artery and vein were attached to appropriate cannulas and secured to the main organ chamber lines. Once this was complete, static pressure perfusion with CSS commenced (Time 0) and proceeded for ~10 hours. Each organ was on pump continuously; the organ chamber was intermittently opened for biopsy collections.

Data Collection

[0223] Perfusion system measurements including systolic and diastolic pressure (mmHg), flow (mL/min), resistance (mmHg), and temperature (°C) were recorded at T=0, hourly for the first four hours, and then at 6, 8, and 10 hours. Perfusate retains were collected at 0, 1, 6, and 10 hours, and 6 mm wedge biopsies were taken at T=0 and hourly after that.

[0224] Perfusate retains were stored frozen (-50 to -90°C) for potential future analysis of biochemical markers of organ function. Larger biopsies were taken at T=0, 6 hours, and 10 hours and divided, with half being fixed for histology and the other half frozen for potential evaluation of tissue OMX levels or other biomarkers of organ quality and function.

Histopathology

[0225] Fixed biopsies were processed and blinded for microscopic evaluation. Tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically by a board-certified pathologist. Each biopsy was scored based on the Banff Classification of Allograft Pathology (Roufosse et al, A 2018 Reference Guide to the Banff Classification of Renal Allograft Pathology [published correction appears in Transplantation. 2018 Dec;102(12):e497], Transplantation. 2018; 102(11): 1795-1814).

Results

[0226] Functional analyses. Per protocol, renal blood flow was adjusted to maintain a target resistance of 0 - 0.4 mmHg/mL/min after the first hour. Increased flow/lower resistance is indicative of better kidney function.

[0227] As seen in FIGs. 1 A and 1C, both kidneys from Donor 1 were able to maintain a resistance below 0.4 mmHg/mL/min, with the OMX-treated kidney exhibiting capacity for a slightly higher flow than the CSS-alone kidney early on. For Donor 2, only the OMX-treated kidney was able to maintain a resistance below 0.4 mmHg/mL/min, with the CSS-treated kidney maintaining a resistance of > 0.52 throughout the study. The flow for the OMX-treated kidney was also substantially higher (FIGs. IB and ID).

[0228] Histopathological analyses. Biopsies taken at various time points from each kidney were analyzed for tubular degeneration, tubular necrosis, apoptosis, and cytoskeletal derangement, which are some of the indicators of the impact of hypoxia on kidney function (Chevalier, R. L. The proximal tubule is the primary target of injury and progression of kidney disease: role of the glomerulotubular junction, Am J Physiol Renal Physiol. 2016 Jul 1; 311(1): F145-F161; Shu, S. et al, Hypoxia and Hypoxia-Inducible Factors in Kidney Injury and Repair, Cells. 2019 Mar; 8(3): 207.).

[0229] As seen in FIGs. 2A-2F, OMX-treated machine-perfused kidneys exhibit reduced tubular degeneration, lower levels of tubular necrosis, and lower levels of apoptosis compared to CSS-treated kidneys. Furthermore, as seen in FIG. 3, OMX-treated machine-perfused kidneys show reduced and/or delayed cytoskeletal derangement compared to CSS-treated kidneys.

[0230] Thus, addition of OMX in preservation solution may reduce and/or defer hypoxia-dependent dysfunction and pathophysiology in machine-perfused kidneys.

[0231] Both OMX-treated kidneys were capable of higher renal blood flow rates than the matched CSS-treated kidney, while maintaining the targeted resistance.

[0232] Both OMX-treated kidneys showed lower levels of tubular degeneration, necrosis, and apoptosis relative to their matched CSS-treated kidney. [0233] Both OMX-treated kidneys showed lower levels of cytoskeletal derangement, and the time to increased cytoskeletal derangement was delayed, relative to their matched CSS- treated kidney.

[0234] While the disclosure has been particularly shown and described with reference to specific embodiments (some of which are preferred embodiments), it should be understood by those having skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the present disclosure as disclosed herein.