WO2022256401A1

WO2022256401A1 - Xenogeneic nerve transplants and methods

Info

Publication number: WO2022256401A1
Application number: PCT/US2022/031761
Authority: WO
Inventors: Paul W. HOLZER; Elizabeth J. CHANG; Rodney L. Monroy; Jon ADKINS
Original assignee: Xenotherapeutics, Inc.; Alexis Bio, Inc.
Priority date: 2021-06-01
Filing date: 2022-06-01
Publication date: 2022-12-08

Abstract

A xenogeneic neural or nerve tissue from a non-wildtype, genetically reprogrammed non-human animal for xenotransplantation into a subject in need of neural or nerve tissue replacement, reconstruction, and/or repair, wherein the xenogeneic neural or nerve tissue is biologically active and metabolically active. A method of neural xenogeneic transplantation therapy including nerve tissue derived from a non-wild type, genetically engineered non-human organism, wherein the tissue is configured to be surgically transplanted into a subject.

Description

XENOGENEIC NERVE TRANSPLANTS AND METHODS CROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority benefit of U.S. 63/195,489 filed on June 1, 2021, the disclosures of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

[0002] The subject matter disclosed herein generally relates to the utilization of xenogeneic nerve tissue from non-wildtype, genetically engineered non-human organisms for purposes of transplantation into subjects including mammals such as non-human primates and humans. Specifically, the disclosure relates to utilizing viable, large-caliber, mixed-modal xenogeneic nerve transplants derived from genetically engineered, designated pathogen free porcine donors for the reconstruction of large peripheral nerve gaps, treatment of injuries and ailments, such as neurotmesis, the complete physiological and anatomical transection of both axons and connective tissue, and other therapies.

BACKGROUND

[0003] It is estimated that twenty million Americans suffer from peripheral nerve injury' caused by trauma or medical disorders in the United States. It is estimated that 50,000 surgeries a year are performed to repair peripheral nerve injuries (Zilic, L. (2015) Journal of Anatomy). Peripheral nerve injuries occur in both the upper and lower extremities due to motor vehicle accidents, gunshot and stab wounds, and industrial accidents (Zilic, 2015), with the majority of peripheral nerve injuries occurring in the upper limbs.

[0004] Severe trauma to the extremities frequently results in transection of peripheral nerves, and these injuries have a devastating impact on patients’ quality of life. Regeneration of these nerves, even after surgical repair, is slow and often incomplete. Less than half of patients who undergo nerve repair following an injury regain adequate motor or sensory_' function, and such deficits may result in complete limb paralysis or intractable neuropathic pain,

[0005] The most commonly afflicted are young healthy civilians and military personnel. Severe nerve injury has a devastating impact on patients’ quality of life. Typical symptoms are sensory and motor function defects that success has been shown in situations up to 5cm, the data is extremely limited (Komfeld, 2018; Rinker, 2017; Safa, 2019) and questions remain in the field about whether nerve reconstruction is consistently possible on large gaps of 4cm or more (Komfeld, 2018). The longer the nerve graft, the less predictable the result. This is due in part to the amount of time it takes for regeneration to occur across each anastomosis area (7-14 days) and along the length of the nerve (0.2-3 mm per day). The longer the nerye graft, the more time is required for regeneration to reach the distal anastomosis of the graft, increasing the risk of atrophy and fibrous ingrowth into the distal anastomosis area, resulting in a poorer outcome.

[0006] For the treatment of large gap (>4cm), segmental peripheral nerve deficits, autografts (nerve grafts from a donor site within the same patient) are considered the “gold standard” for treatment (Pi, 2015; Komfeld, 2018; Rinker, 2017). Sources such as the sural or fibular sensory nerves are common candidates in such situations. Despite its effectiveness, the use of autologous grafting is associated with several signifi cant drawbacks. Chief among these is the combination of donor site morbidity, paresthesia, neuroma, scar formation, and subsequent chronic pain and sensor}_' loss, which can be longstanding and debilitating. Adequate amounts of autologous graft material are limited and cannot be relied upon in the most severe injuries with multiple peripheral nerve defects. Autologous nerve candidates, such as the sural nerve, are sensory only and sub optimal to replace peripheral motor nerves. They also have a relatively small diameter and would require surgical "cabling" to be useful in the repair of larger caliber peripheral nerves. Lastly, there is often variability of the cross-sectional area along the nerve and fascicular patterns, which are thought to be of importance for functional recovery (Stewart, 2003), and these may not match those of the nerve in the defect (Zilic, 2015).

[0007] Following transplantation, there are no therapies available to enhance nerve regeneration, so graft reinnervation and functional outcomes are dependent on the recipient’s innate regenerative capacities. This means that functional deficits often remain after surgery.

[0008] As a result of the problems associated with autografts, allografts, and alternative conduits, there remains a need for a high-quality method of graft treatment for large gap (>4crn), segmental peripheral nerve deficits. There is a long-felt, but unmet need to treat this kind of nerve damage with a live-cell product that can facilitate the critical axon regeneration process necessary to restore motor and sensory' function and improve quality of life.

[0009] Successful peripheral nerve regeneration involves improving the rate of nerve regeneration and the reinnervation of composite muscle leading to improved function. Existing treatment options include the use of autologous nerve transplants procured from a donor site from the same patient or deeel lularized human cadaveric nerve allogeneic transplants. Both treatment options have severe shortcomings and thus, there is an unmet need for high-quality nerve transplants for large-gap (>4cm), segmental peripheral nerve defects exists.

[00010] Xenotransplantation, such as the transplantation of a non -hum an animal tissues into a human recipient, has the potential to provide nerve tissue for transplant, potentially helping thousands of people worldwide. However, xenotransplantation using non-human animal-derived live ceil tissue into a human is most often accompanied by rejection of the transplanted tissue. [00011] Such tissues evoke rejection by the human immune system upon transplantation into a human where natural human antibodies target epitopes on the foreign cells, causing rejection and failure of the transplanted tissue. The rejection may be a cellular rejection (lymphocyte mediated) or humoral (antibody mediated) rejection including but not limited to hyperacute rejection, an acute rejection, a chronic rejection, may involve survival limiting thrombocytopenia coagulopathy and an acute humoral xenograft reaction (AHXR). Other roadblocks with respect to swine to human xenotransplantation include risks of cross-species transmission of disease or parasites.

[00012] Many attempts have been made by others to modify animals to serve as a source for xenotransplantation products. Other commercial, academic and other groups have focused on interventions, gene alterations, efforts to induce tolerance through ehimerism, inclusion of transgenes, concomitant use of exogenous immunosuppressive medications aimed to reduce the recipients’ natural immunologic response(s) and other approaches. These groups have sought to create a “one size fits air source animal aiming to create one, standardized source animal for all recipients.

[00013] Specifically, certain groups have focused on creating transgenic swine free of PERV and utilizing transgenic bone marrow' for therapy (see, e,g,, eGenesis, Inc, PCT/US2018/028539); creating transgenic swine utilizing stem cell scaffolding (see, e,g, United Therapeutics/Revivicor [US20190111180A1 ]); mixed ehimerism and utilizing transgenic bone marrow' for therapy to tolerize patient T-celis (see, e,g, Columbia University [US20180070564A1]). These “downstream” approaches - post recognition by the human immune system - have not succeeded in producing swine that produce products suitable for prolonged use in xenotransplantation or that survive the above-referenced transgenic and other alterations. [00014] Thus, there remains a long-felt, but unmet need to treat nerve damage with a livecell product derived from a non-human, non-wild-type animal that can facilitate the critical axon regeneration process necessary' to restore motor and sensory function and improve quality of life. SUMMARY OF THE INVENTION

[00015] In one aspect, the present disclosure includes a method, biological system, tissues, genetically modified non-human animals, tissues, products, vectors, kits, and/or genetic materials for providing a xenogeneic nerve transplant into a subject including mammals such as non-human primates and humans. The xenogeneic nerve transplant may include nerve tissue derived from a non-wild type, genetically engineered non-human organism, wherein the tissue is configured to be surgically grafted into a subject. In some aspects, the xenogeneic nerve transplant tissue is nerve fascicles. In some aspects, the xenogeneic nerve transplant tissue is at least 4 cm in length. [00016] In a first aspect, the present disclosure includes silencing, knocking out, inactivating, or causing the minimal expression of specific proteins, epitopes or molecules in a wild-type non-human animal to create a genetically -modified non-human animal that produces biological products that are tolerogenic when transplanted into subjects including mammals such as non-human primates and humans. In a second aspect, the present disclosure includes humanizing genes encoding specific proteins, epitopes or molecules in a wild-type non-human animal to create a genetically-modified non-human animal that produces biological products that are tolerogenic when transplanted into subjects including mammals such as non-human primates and humans. In a third aspect, the present disclosure includes personalizing genes encoding specific proteins, epitopes or molecules in a wild-type non-human animal to create a genetically- modified non-human animal that produces biological products that are tolerogenic when transplanted into subjects including mammals such as non-human primates and humans. In certain aspects, the first, second, and third aspects are combined to create a genetically-modified nonhuman animal that produces biological products that are tolerogenic when transplanted into subjects including mammals such as non-human primates and humans. In some aspects, one, two, or all three of the described aspects involve minimal manipulation of the non-human animal’s genome. In some aspects, minimal manipulation involves a method of replacing specific lengths (referred to herein as “frames” or “cassettes”) of nucleotide sequences within genes of the wild- type non-human animal’s genome. In some aspects, replacing frames or cassettes involves the use of a standardized length of nucleotide sequences. In some aspects, the mammalian recipient or subject is a monkey. In some aspects, the mammalian recipient or subject is a marmoset. In some aspects, the mammalian recipient or subject is a rhesus monkey. In some aspects, the mammalian recipient or subject is a human.

[00017] In one aspect of the first aspect, the genome of the non-human animal is genetically modified so as to not present, one or more surface glycan epitopes selected from alpha-Gal, NeuSGc, and SDa. In another aspect of the first aspect, MHC class I sequences encoding SLA-1 and SLA-2 are silenced, knocked out, or inactivated in the wild-type non-human animal’s genome. In another aspect of the first aspect, MHC class II sequences encoding SLA- DR are silenced, knocked out or inactivated in the wild-type non-human animal’s genome. In another aspect of the first aspect, MHC class II sequences encoding SLA-DRpl are silenced, knocked out, or inactivated in the wild-type non-human animal’s genome. In another aspect of the first aspect, one of two copies of beta 2 microglobulin (B2m) is silenced, knocked out, or inactivated in the wild-type non-human animal’s genome. In another aspect of the first aspect, both copies of porcine beta 2 microglobulin (pB2.ni} are silenced, knocked out, or inactivated in the wild-type swine’s genome. In some aspects, a stop codon is inserted into the wild-type nonhuman animal’s genome. In other aspects, other genetic modifications discussed in the Detailed Description are included in the invention of the present disclosure. The genetic modification can be made utilizing known genome editing techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), adeno-associated virus (AAV)-mediated gene editing, and clustered regular interspaced palindromic repeat Cas9 (CRISPR-Cas9).In one aspect, a xenogeneic nerve transplant is provided wherein the nerve tissue is configured to be surgically grafted into a subject for nerve reconstruction following various types of axonal and neuronal injuries, including neurotmesis, the complete physiological and anatomical transection of both axons and connective tissue.

[00018] It will be further understood that causing the donor tissues to express a known human MHC genotype or the recipient’s MHC specifically as described herein, combined with the elimination in the donor swine cells of alpha-1, 3-galactosytransferase, NeuSGc, and b1,4-N- acetylgalactosaminyltransferase (B4GALNT2) (e.g., ‘’single knockout,” “double knockout,” or “triple knockout”), presents a swine whose cells will have a decreased immunological rejection as compared to a triple knockout swine that lacks the specific SLA/MHC reprogramming of the present disclosure. [00019] In accordance with another embodiment of the invention, a method of facilitating the reconstruction of neuronal connections across up to large gaps in the peripheral nervous system is provided comprising surgically grafting nerve tissue derived from a non- wild type, genetically engineered non-human organism into a subject.

[00020] In another exemplary' embodiment, a method of facilitating the reconstruction of neuronal connections across up to large gaps in the peripheral nervous system is provided, wherein the xenogeneic nerve transplant facilitates the reconstruction of neuronal connections across up to large gaps in the peripheral nervous system of the subject.

BRIEF DESCRIPTION OF THE DRAWINGS [00021] The accompanying drawings, which are incorporated herein and form part of the disclosure, help illustrate various aspects of the present invention and, together with the description, further serve to describe the invention to enable a person skilled in the pertinent art to make and use the aspects disclosed herein. In the drawings, like reference numbers indicate identical or functionally similar elements.

[00022] FIG. 1 illustrates peripheral nerve anatomy (Grinsell, 2014).

[00023] FIG, 2 illustrates large gap peripheral nerve injuries (Grinsell, 2014).

[00024] FIG. 3 illustrates the regeneration nerves after injury', including after surgical repair (Grinsell, 2014).

[00025] FIG. 4 illustrates an exemplary flow chart of the current state of the art in nerve injuries and repair.

[00026] FIG. 5 illustrates FDA Approved Nerve Tubes for Peripheral Nerve Repair. Modified and Supplemented from FDA Medical Device Database (Kornfeid, 2018).

[00027] FIG. 6A illustrates a schematic experimental study design for harvesting sciatic nerve from a donor and transplantation into a recipient. FIG. 6B illustrates Porcine sciatic nerve in situ and trimmed nerve (4 cm-top). FIG, 6C illustrates reconstructed radial nerve from a limb treated with the xenogeneic transplant, photographed at necropsy (12- months postoperative). [00028] FIG. 7A illustrates Autologous treated limb at necropsy (12-months postoperative) demonstrating neuroma enlargement at anastomotic sites, expected as a result of the nerve repair surgery'. FIG. 7B illustrates Xenogeneic treated limb at necropsy. FIG. 7C illustrates histological image of autologous treated limb. FIG. 7D illustrates histological image of xenogeneic treated limb. FIG. 7E illustrates prominent tertiary' lymhphoid follicles at low' power. FIG. 7F illustrates prominent tertiary lymhphoid follicles at high power. FIG. 7G illustrates qualitative PCR using primate-specific gene target demonstrating the presence of primate cells and the absence of porcine cells at. transplant sites repaired with autologous (lanes 2-6) and xenogeneic transplants (lanes 8- 11). Lane 12 is the negative control, lane 13 is the primate control, and lane 14 is porcine control. [00029] FIG. 8 illustrates PERV and porcine centromeric DNA to confirm the presence of porcine cells at the end of the experiment. 18s vras utilized as an internal control to confirm the validity of the Q-PCR. Ct values were consistent with thepresence of DNA and indicated no inhibition in the analysis.

[00030] FIG. 9 A illustrates white blood cell count (WBC) for the entirety of the study, FIG, 9B illustrates composition of WBC populations. FIG. 9C illustrates total IgM and IgG titer levels. FIG. 9D illustrates Anti-Porcine IgM and IgG responses.

[00031] FIG. 10A illustrates wrist extension assessments performed to evaluate functional recovery following repair of a surgically introduced large-gap (>4em), segmental peripheral nerve defect. FIG. 10B illustrates wrist assessment scores for all subjects (mean), by month, autologous control nerve transplant as compared to experimental xenogeneic nerve transplant.

[00032] FIG, 11 illustrates recovery' of wrist extension function that is qualitatively assessed from monthly video recordings by an analyst blinded to the transplant type, depicting severe impairment to no observable impairment,

[00033] FIG. 12 illustrates Magnitude of recovery of wrist extension function that is illustrated via a heat map.

[00034] FIG. 13A illustrates motor nerve conduction at 0 and 5 months. FIG. 13B illustrates sensory' nerve conduction at 0 and 5 months. FIG. 13C illustrates CMAP Amplitude at 0 and 5 months. FIG. 13D illustrates CMAP duration at 0 and 5 months,

[00035] FIG. 14A illustrates motor nerve conduction at 8 and 12 months. FIG. 14B illustrates sensory nerve conduction at 8 and 12 months. FIG. 14C illustrates CMAP Amplitude at 8 and 12 months. FIG. 14D illustrates CMAP duration at 8 and 12 months.

[00036] FIG. 15A illustrates histology sections (Baseline, Autologous, Xenogeneic) stained by immunohistochemistry for expression of neurofilament H. Arrowheads indicate axonal material. Representative images of native radial nerve at baseline and explanted nerve samples obtained at. necropsy from the regenerated nerve in both autologous and xenogeneic transplanted limbs. FIG. 15B illustrates qualitative assessment of nerve bundle diameter. Nerve bundle diameters of the regenerated nerve from both transplant types were smaller than the diameters of native nerve obtained perioperatively and qualitatively equivalent between the two transplant types. Higher scores indicate larger diameters. FIG. 15C illustrates histology sections (Baseline, Autologous, Xenogeneic) stained by Luxol fast blue for degree of myeiination of regenerated nerve. Arrowheads indicate myelin. FIG. 15D illustrates qualitative assessment of myeiination. FIG. 15E illustrates histology sections (Baseline, Autologous, Xenogeneic) with hematoxylin and eosin staining for necrosis and fibrosis. Arrowheads indicate nerve bundles. FIG, 15F illustrates qualitative assessment of necrosis and fibrosis.

[00037] FIG. 16 illustrates the exposed radial nerve anatomy in non-human primate (rhesus monkey) at necropsy, postoperative month 12.

[00038] FIG. 17 illustrates the exposed resected radial nerve, proximal and distal anastomotic sites at necropsy, postoperative month 12,

DETAILED DESCRIPTION OF THE INVENTION

[00039] While aspects of the subject matter of the present disclosure may be embodied in a variety of forms, the following description is merely intended to disclose some of these forms as specific examples of the subject mater encompassed by the present disclosure. Accordingly, the subject matter of this disclosure is not intended to be limited to the forms or aspects so described. [00016] The disclosures of US2020/0108I75A1 (Holzer et al.) and US2020/0283737A1 (Holzer et al.) are incorporated herein by reference in its entirety for all purposes as if expressly- recited herein.

[00017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[00018] “Best alignment” or “optimum alignment” means the alignment for which the identity percentage determined as described below- is the highest. Comparisons of sequences between two nucleic acid sequences are traditionally made by comparing these sequences after aligning them optimally, the said comparison being made by segment or by “comparison window” to identify and compare local regions for similar sequences. For the comparison, sequences may be optimally aligned manually, or by using alignment software, e.g., Smith and Waterman local homology algorithm (1981), the Neddleman and Wunsch local homology algorithm (1970), the Pearson and Li pm an similarity search method (1988), and computer software using these algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA and IF ASIA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.). In some aspects, the optimum alignment is obtained using the BLAST program with the BLOSUM 62 matrix or software having similar functionality. The “identity percentage” between two sequences of nucleic acids or amino acids is determined by comparing these two optimally aligned sequences, the sequence of nucleic acids or amino acids to be compared possibly including additions or deletions from the reference sequence for optimal alignment between these two sequences. The identity percentage is calculated by determining the number of positions for which the nucleotide or the amino acid residue is identical between the two sequences, by dividing this number of identical positions by the total number of compared positions and multiplying the result obtained by 100 to obtain the identity percentage between these two sequences.

[00019] “Conservative,” and its grammatical equivalents as used herein include a conservative amino acid substitution, including substitution of an amino acid residue by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). Conservative amino acid substitutions may be achieved by modifying a nucleotide sequence so as to introduce a nucleotide change that will encode the conservative substitution. In general, a conservative amino acid substitution will not substantially change the functional properties of interest of a protein, for example, the ability of MHC I to present a peptide of interest. Examples of groups of amino acids that have side chains with similar chemical properties include aliphatic side chains such as glycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxyl side chains such as serine and threonine; amide-containing side chains such as asparagine and glutamine; aromatic side chains such as phenylalanine, tyrosine, and tryptophan; basic side chains such as lysine, arginine, and histidine, acidic side chains such as aspartic acid and glutamic acid; and, sulfur-containing side chains such as cysteine and methionine. Conservative amino acids substitution groups include, for example, valine/ieucine/isoleucine, phenylalanine/tyrosine, lysine/arginine, alanine/valine, glutamate/aspartate, and asparagine/glutamine. One skilled in the art would understand that in addition to the nucleic acid residues encoding a human or humanized MHC I polypeptide, MHC II polypeptide, and/or b2 microglobulin described herein, due to the degeneracy of the genetic code, other nucleic acid sequences may encode the polypeptide(s) of the invention. Therefore, in addition to a genetically modified non-human animal that comprises in its genome a nucleotide sequence encoding MHC I, MHC II, and/or b2 microglobulin polypeptide(s) with conservative amino acid substitutions, a non- human animal whose genome comprises a nucleotide sequence(s) that differs from that described herein due to the degeneracy of the genetic code is also provided.

[00020] “Conserved'’ and its grammatical equivalents as used herein include nucleotides or amino acid residues of a polynucleotide sequence or amino acid sequence, respectively, that are those that occur unaltered in the same position of two or more related sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences. Herein, two or more sequences are said to be “completely conserved” if they are 100% identical to one another. In some embodiments, two or more sequences are said to be “highly conserved” if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical, but less than 100% identical, to one another. In some embodiments, two or more sequences are said to be “conserved” if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least. 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical, but less than 100% identical, to one another. In some embodiments, two or more sequences are said to be “conserved” if they are about 30% identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% identical, about 98% identical, or about 99% identical to one another.

[00021] “Designated pathogen free,” and its grammatical equivalents as used herein include reference to animals, animal herds, animal products derived therefrom, and/or animal facilities that are free of one or more specified pathogens. Preferably, such “designated pathogen free” animals, animal herds, animal products derived therefrom, and/or animal facilities are maintained using well-defined routines of testing for such designated pathogens, utilizing proper standard operating procedures (SOPs) and practices of herd husbandry and veterinary' care to assure the absence and/or destruction of such designated pathogens, including routines, testing, procedures, husbandry, and veterinary care disclosed and described herein. It will be further understood that as used herein the term “free,” and like terms when used in connection with “pathogen free” are meant to indicate that the subject pathogens are not present, not alive, not active, or otherwise not detectable by standard or other testing methods for the subject pathogens. [00022] “Alter,” “altering,” “altered” and grammatical equivalents as used herein include any and/or all modifications to a gene including, but not limited to, deleting, inserting, silencing, modifying, reprogramming, disrupting, mutating, rearranging, increasing expression, knocking-in, knocking out, and/or any or all other such modifications or any combination thereof.

[00023] “Endogenous loci” and its grammatical equivalents as used herein include the natural genetic loci found in the animal to be transformed into the donor animal.

[00024] “Functional,” e.g., in reference to a functional polypeptide, and its grammatical equivalents as used herein include a polypeptide that retains at least one biological activity normally associated with the native protein. For example, in some embodiments of the invention, a replacement at an endogenous locus (e.g., replacement at an endogenous non-human MHC I, MHC II, and/or b2 microglobulin locus) results in a locus that fails to express a functional endogenous polypeptide. Likewise, the term “functional” as used herein in reference to functional extracellular domain of a protein, can refer to an extracellular domain that retains its functionality, e.g., in the case of MHC I, ability to bind an antigen, ability to bind a T cell co-receptor, etc. In some embodiments of the invention, a replacement at the endogenous MHC locus results in a locus that fails to express an extracellular domain (e.g., a functional extracellular domain) of an endogenous MHC while expressing an extracellular domain (e.g., a functional extracellular domain) of a human MHC.

[00025] “Genetic or molecular marker,” and their grammatical equivalents as used herein include polymorphic locus, i.e. a polymorphic nucleotide (a so-called single nucleotide polymorphism or SNP) or a polymorphic DNA sequence at a specific locus. A marker refers to a measurable, genetic characteristic with a fixed position in the genome, which is normally inherited in a Mendelian fashion, and which can be used for mapping of a trait of interest. Thus, a genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change, i.e. a single nucleotide polymorphism or SNP, or a long DNA sequence, such as microsatellites or Simple Sequence Repeats (SSRs). The nature of the marker is dependent on the molecular analysis used and can be detected at the DNA, RNA or protein level. Genetic mapping can be performed using molecular markers such as, but not limited to, RFLP (restriction fragment length polymorphisms; Botstein et al. (1980), Am J Hum Genet. 32:314-331; Tanksley et al. (1989), Bio/Technology 7:257-263), RAPD [random amplified polymorphic DMA; Williams et al. (1990), NAR 18:6531-6535], AFLP [Amplified Fragment Length Polymorphism; Vos et al. (1995) NAR 23:4407-4414], SSRs or microsatellites [Tautz et al. (1989), NAR 17:6463-6471], Appropriate primers or probes are dictated by the mapping method used.

[00026] “Improving” and its grammatical equivalents as used herein include any improvement recognized by one of skill in the art. For example, improving transplantation can mean lessening hyperacute rejection, which can encompass a decrease, lessening, or diminishing of an undesirable effect or symptom. In some aspects, a clinically relevant improvement is achieved.

[00027] “Locus” (loci plural) or “site” and their grammatical equivalents as used herein include a specific place or places on a chromosome where, for example, a gene, a genetic marker or a QTL is found,

[00028] “Minimally altered” and its grammatical equivalents as used herein include alteration of a donor animal genome including removing and replacing certain distinct sequences of native base pairs appearing on the donor animal’s genome and replacing each such sequence with a synthetic sequence comprising the same number of base pairs, with no net change to the number of base pairs in the donor animal’s genome, while not disturbing other aspects of the donor animal’s native genome including, for example, introns and other codons naturally existing in the donor animal genome. For example, in the case of a swine as donor animal, a minimally altered swine can include specific alterations silencing, removing or deactivating certain SLA exons to regulate the donor swine cell’s extracellular expression or non-expression of Ml iC Class II, la, and/or lb; reprogramming certain native, naturally occurring swine cell SLA exons to regulate the swine cell’s extracellular expression or non-expression ofMHC Class II; conserving or otherwise not removing swine introns existing in or in the vicinity of the otherwise engineered sequences; increasing the expression of swine CTLA4 and PD- 1; and removing or deactivating alpha- 1,3 galactosyltransferase, cytidine monopbosphate-N-acetylneuraminic acid hydroxylase, and b!,4~ N-aeetylgalactosaminyltransferase according to the first aspect.

[00029] “Minimally manipulated” and its grammatical equivalents as used herein include treatment of source animals, biological products derived from those source animals, and other biological products with minimal physical alteration of the related tissues such that such animals and products are substantially in their natural state.

[00030] ‘Ortholog,” “orthologous,” and their grammatical equivalents as used herein include a polynucleotide from one species that corresponds to a polynucleotide in another species, which has the same function as the gene or protein or QTL, hut is (usually) diverged in sequence from the time point on when the species harboring the genes or quantitative trait loci diverged (i.e. the genes or quantitative trait loci evolved from a common ancestor by speciation),

[00031] “Quantitative trait locus (QTL)” and its grammatical equivalents as used herein include a stretch of DNA (such as a chromosome arm, a chromosome region, a nucleotide sequence, a gene, and the like) that is closely linked to a gene that underlies the trait in question. “QTL mapping” involves the creation of a map of the genome using genetic or molecular markers, like AFLP, RAPD, RFLP, SNP, S8R, and the like, visible polymorphisms and aliozymes, and determining the degree of association of a specific region on the genome to the inheritance of the trait of interest. As the markers do not necessarily involve genes, QTL. mapping results involve the degree of association of a stretch of DNA with a trait rather than pointing directly at the gene responsible for that trait. Different statistical methods are used to ascertain whether the degree of association is significant or not. A molecular marker is said to be “linked” to a gene or locus, if the marker and the gene or locus have a greater association in inheritance than would be expected from independent assortment, i.e. the marker and the locus co-segregate in a segregating population and are located on the same chromosome. “Linkage” refers to the genetic distance of the marker to the locus or gene (or two loci or two markers to each other). The closer the linkage, the smaller the likelihood of a recombination event taking place, vriiich separates the marker from the gene or locus. Genetic distance (map distance) is calculated from recombination frequencies and is expressed in centiMorgans (cM) [Kosambi (1944), Ann. Eugenet. 12:172-175],

[00032] “Capture sequence” or “reference sequence” and their grammatical equivalents as used herein include a nucleic acid or amino acid sequence that has been obtained, sequenced or otherwise become known from a sample, animal (including humans), or population. For example, a capture sequence from a human patient is a “human patient capture sequence.” A capture sequence from a particular human population is a “human population-specific human capture sequence.” And a capture sequence from a human allele group is an “al!e!e-group-specific human capture sequence.” [00033] “Humanized” and its grammatical equivalents as used herein include embodiments wherein all or a portion of an endogenous non-human gene or allele is replaced by a corresponding portion of an ortho!ogous human gene or allele. For example, in some embodiments, the term “humanized” refers to the complete replacement of the coding region (e.g., the exons) of the endogenous non-human MHC gene or allele or fragment thereof with the corresponding capture sequence of the human MHC gene or allele or fragment thereof, while the endogenous non-coding region(s) (such as, but not limited to, the promoter, the 5' and/or 3' untranslated region(s), enhancer elements, etc.) of the non-human animal is not replaced.

[00034] “Personalized" or "individualized," and their grammatical equivalents as used herein, include a gene, allele, genome, proteome, cell, cell surface, tissue, or organ from a non- human animal which is adapted to the needs or special circumstances of an individual human recipient or a specific human recipient subpopulation.

[00035] “Reprogram,” “reprogrammed,” including in reference to “immunogenomic reprogramming,” and their grammatical equivalents as used herein, refer to the replacement or substitution of endogenous nucleotides in the donor animal with orthologous nucleotides based on a separate reference sequence, wherein frameshift mutations are not introduced by such reprogramming. In addition, reprogramming results in no net loss or net gain in the total number of nucleotides in the donor animal genome, or results in a net loss or net gain in the total number of nucleotides in the donor animal genome that is equal to no more than 1%, no more than 2%, no more than 3%, no more than 4%, no more than 5%, no more than 6%, no more than 7%, no more than 8%, no more than 9%, no more than 10%, no more than 12%, no more than 15%, or no more than 20% of the number of nucleotides in the separate reference sequence. In one example of “reprogramming,” an endogenous non-human nucleotide, codon, gene or fragment thereof is replaced with a corresponding synthetic nucleotide, codon, gene or fragment thereof based on a human capture sequence, through which the total number of base pairs in the donor animal sequence is equal to the total number of base pairs of the human capture sequence.

[00036] “Tolerogenic” and its grammatical equivalents as used herein include characteristics of an organ, cell, tissue, or other biological product, that are tolerated by the reduced response by the recipient’s immune system upon transplantation.

[00037] “Transgenic” and its grammatical equivalents as used herein, include donor animal genomes that have been modified to introduce non-native genes from a different species into the donor animal’s genome at a non-orthologous, non-endogenous location such that the homologous, endogenous version of the gene (if any) is retained in whole or in part. “Transgene,” “transgenic,” and grammatical equivalents as used herein do not include reprogrammed genomes, knock-outs or other modifications as described and claimed herein. By way of example, “transgenic” swine include those having or expressing hCD46 (“human membrane cofactor protein,” or “MCP”), hCD55 (“human decay-accelerating factor,” “DAF”), human B2M (beta-2-microgiobulin), and/or other human genes, achieved by insertion of human gene sequences at a non-orthologous, non- endogenous location in the swine genome without the replacement of the endogenous versions of those genes.

[00038] In some embodiments, as described herein, a non-human non-wild-type animal can be utilized as a source animal. It will be understood that any number of source animals could be utilized in accordance with the present invention (containing modifications to reduce hyperacute rejection), including, but not limited to, pigs, monkeys, sheep, goats, mice, cattle, deer, horses, dogs, cats, rats, mules, and any other mammals. Source animals could also include any other animals including, but not limited to, birds, fish, reptiles, and amphibians. As used herein, the terms "swine," "pig" and "porcine" are generic terms referring to the same type of animal without regard to gender, size, or breed.

[00039] In some non-limiting embodiments, the source animals from which the subject nerve grafts are derived may include animals with genetic modifications as described in US2020/0108175AI (Holzer et al.) and US2020/0283737AI (Holzer et al.) and references cited therein, the entire disclosures of which are incorporated herein by reference.

[00040] It will be further understood that any animal serving as a source animal hereunder, including swine, regardless of how such swine may be configured, engineered, or otherwise altered and/or maintained, may be created, bred, propagated and/or maintained in accordance with the present disclosure to create and maintain animals and resulting biological products to be used in or in preparation or pursuit of clinical xenotransplantation.

[00041] For example, the present disclosure includes non-human animals, e.g., swine, having certain combinations of specific genetic characteristics, breeding characteristics and pathogen-free profile. Such animals may include, as described above and herein, immunogenomic reprogrammed swine having a biologically reprogrammed genome such that it does not express one or more extracellular surface glycan epitopes, e.g., genes encoding alpha-1,3 galactosyJtransferase, cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and b 1 ,4-N-acetylgalactosaminyltransferase are disrupted such that surface glycan epitopes encoded by said genes are not expressed, as well as other modifications to the swine’s SLA to express MHC-I or MHC-II, and regulation of PD- 1 and CTLA4, as described above and herein. Resulting from the process described herein, the swine is free of at least the following zoonotic pathogens:

(i) Ascaris species, Cryptosporidium species, Echinococcus, Strongyloids sterocoUs , and Toxoplasma gondii in fecal matter;

(ii) Leptospira species, Mycoplasma hyopneumoniae , porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies, transmissible gastroenteritis virus (TGE) / Procine Respiratory Coronavirus, Toxoplasma Gondii in antibody titers;

(iii) Porcine Influenza;

(iv) the following bacterial pathogens as determined by bacterial culture: Bordetella bronchisceplica, Coagulase-positive staphylococci, Coagul ase-negative staphylococci, Livestock-associated methicillin resistant Staphylococcus aureus (LA MRSA), Microphyton and Trichophyton spp.;

(v) Porcine cytomegalovirus; and

(vi) Brucella suis; is raised and maintained according to a bioburden-reducing procedure, the procedure comprising maintaining the swine in an isolated closed herd, wherein all other animals in the isolated closed herd are confirmed to be free of said zoonotic pathogens; wherein the swine is isolated from contact, with any non-human animals and animal housing facilities outside of the isolated closed herd. [00042] In accordance with one aspect of the invention a method is provided for producing a biological nerve product for xenotransplantation into a human recipient, said biological product comprising live cells and tissues that vascularize after xenotransplantation, the method including: producing a non-wild type, biologically engineered swine, wherein said swine has a biologically engineered genome such that it does not express one or more extracellular surface glycan epitopes; confirming that said swine is free of at. least the following zoonotic pathogens:

(i) Ascaris species, Cryptosporidium species, Echinococcus, Strongyloids slerocolis , and Toxoplasma gondii in fecal matter; (ii) Leptospira species, Mycoplasma hyopneumoniae , porcine reproductive and respirator syndrome virus (PRRSV), pseudorabies, transmissible gastroenteritis virus (TGE) / Procine Respiratory Coronavims, Toxoplasma Gondii in antibody titers,

(iii) Porcine Influenza;

(iv) the following bacterial pathogens as determined by bacterial culture: Bordetella bronchisceptica , Coagulase-positive staphylococci, Coagulase-negative staphylococci, Livestock-associated methicillin resistant Staphylococcus aureus (LA AIRS A), Microphyton and Trichophyton spp.;

(v) Porcine cytomegalovirus; and

(vi) Brucella suis; maintaining the swine according to a bioburden-reducing procedure, said procedure comprising maintaining the swine in an isolated closed herd, wherein all other animals in the isolated closed herd are confirmed to be free of said zoonotic pathogens, wherein the swine is isolated from contact with any non-human animals and animal housing facilities outside of the isolated closed herd; harvesting a biological product from said swine, wherein said harvesting comprises euthanizing the swine and aseplically removing the biological product from the swine; processing said biological product comprising sterilization after harvesting using a sterilization process that does not reduce cell viability to less than 50% cell viability in a 3-(4,5-dimethylthiazol-2-yl)-2,5- dipheny!tetrazolium bromide (MTT)-reduction assay and does not reduce mitochondrial activity to less than 50% mitochondrial activity; and storing said biological product in a sterile container. [00043] In accordance with one aspect of the invention, a method is provided for producing a biological nerve product suitable for xenotransplantation into a human recipient, the method including : producing a non-wild type, biologically engineered swine, wherein said swine is produced through natural breeding and natural birthing, wherein said swine has a biologically engineered genome such that it does not express one or more extracellular surface glycan epitopes, and wherein said swine is free of at least the following pathogens: Ascaris species, Cryptosporidium species, Echnococcus, Strongyioids sterocolis, Toxoplasma gondii, Brucella suis, Leptospira species, mycoplasma hyopneumoniae, pseudorabies, Toxoplasma Gondii, staphylococcus species, Microphyton species, and Trichophyton species, porcine influenza, cytomegalovirus, arterivirus, and a coronavims; rearing the swine and maintaining the swine according to a bioburden-redueing procedure, said procedure comprising maintaining the swine in a closed herd, wherein all other animals in the closed herd are confirmed to be free of at least the following pathogens: Ascaris species, Cryptosporidium species, Echnococcus, Strongyloids sterocolis, Toxoplasma gondii. Brucella suis, Leptospira species, mycoplasma hyopneumoniae, pseudorabies, Toxoplasma Gondii, staphylococcus species, Microphylon species, and Trichophyton species, porcine influenza, cytomegalovirus, arteri virus, and a coronavirus, wherein the swine is isolated from contact with any non-human animals and animal housing facilities outside of the closed herd; harvesting a biological product from said swine, wherein said harvesting comprises euthanizing the swine and asepticaily removing the biological nerve product from the swine; processing said biological nerve product comprising sterilization within 15 hours of harvesting and storing said biological product in a sterile container, wherein said biological product does not contain one or more extracellular surface glycans, wherein said product is free of Ascaris species, Cryptosporidium species, Echnococcus, Strongyloids sterocolis, Toxoplasma gondii, Brucella suis, Leptospira species, mycoplasma hyopneumoniae, pseudorabies, Toxoplasma Gondii, staphylococcus species, Microphylon species, and Trichophyton species, porcine influenza, cytomegalovirus, arterivirus, and a coronavirus, and wherein said product is biologically active and comprises live cells and tissues, wherein cellular mitochondrial activity of said product is greater than 50% as measured by MTT assay; wherein said nerve product is less immunogenic when transplanted into a human xenotransplant recipient as compared to a biological product obtained a xenotransplantation nerve product made from conventional Gal-T knockout swine, from conventional triple knockout swine, from transgenic swine, from wild-type animals, and/or allograft, wherein said nerve product is less antigenic when transplanted into a human xenotransplant recipient as compared to a biological nerve product obtained from a xenotransplantation nerve product made from conventional Gal-T knockout swine, from conventional triple knockout swine, from transgenic swine, from wild-type animals, and/or allograft, and wherein said nerve product is resistant to rejection by the human xenotransplant recipient in the absence of administration of immunosuppressant drugs or other immunosuppressant therapies to the human xenotransplant recipient.

[00044] In a further aspect of the invention, following the clinical xenotransplantation of the product into a human recipient, the nerye product exhibits a clinical benefit that is on par or enhanced above an allograft product. In some aspects, clinical benefits may include decreased graft, dislocation, increased graft adherence, reduced bacterial Infection compared to allograft, e.g., colony counts of less than !0⁵ g/tissue, reduced cellulitis, reduced erythema, reduced edema, reduced hyperesthesia, reduced induration, reduced tenderness, reduced itching, reduced abscesses, reduced incidence of toxic shock syndrome, reduced colonization of toxin-1 producing S. aureus, reduced incidence of sepsis and septic shock, reduced colonization by E. coli , P. aeruginosa , Klebsiella spp., Providencia spp., enterobacteriaceae, and yeasts such as C. albicans. In a further aspect, the product may incorporate into the transplantation site or healing site. In a further aspect, the product may reduce or prevent infection at the transplantation site or the healing site, increase or retain fluids at the transplantation site or the healing site, increase or retain electrolytes at the transplantation site or the healing site, increase or retain temperature homeostasis at the transplantation site or the healing site, reduce scarring at the transplantation site or the healing site, reduce or eliminate sepsis, reduce or eliminate protein losses, provide, improve or facilitate restoration of normal bodily functions, or a combination thereof. In a further aspect of the invention, the nerve product is capable of being transplanted in the absence of immunosuppressant drugs or other immunosuppressant therapies. In some aspects, immunosuppressants are not used according to the present disclosure prior to, during, and/or after transplantation of the xenotransplantation nerve product of the present disclosure. In some aspects, immunosuppressants may be used according to the present disclosure prior to, during, and/or after transplantation of the xenotransplantation nerve product of the present disclosure. In some aspects, immunosuppressants are used according to the present disclosure after transplantation of the xenotransplantation nerve product of the present disclosure to prolong the adherence of an already transplanted xenotransplantation product.

[00045] In accordance with another exemplary' aspect of the invention, the present disclosure provides a method of preparing biological nerve product for clinical xenotransplantation into a human comprising selecting a known human major histocompatibility complex gene sequence or sequencing a human recipient’s major histocompatibility complex gene, genetically modifying ceils of a swine to replace a portion of the swine’s major histocompatibility complex gene sequence with a corresponding portion of the known human major histocompatibility complex gene sequence or a corresponding portion of the human recipient’s major histocompatibility complex gene sequence such that the swine’s cells express the corresponding portion of the known human major histocompatibility complex gene sequence or the corresponding portion of the human recipient’s major histocompatibility' complex gene sequence, isolating one or more tissues from the swine that express the corresponding portion of the known human major histocompatibility complex gene sequence or the corresponding portion of the human recipient’s major histocompatibility complex gene sequence, wherein the isolated tissues are the biological product.

[00046] In accordance with another exemplary aspect of the invention, the present disclosure provides a method of preparing a genetically reprogrammed swine comprising a nuclear genome having disrupted alpha-1,3 galactosyl transferase gene, and genetically modified such that it expresses a major histocompatibility complex of a known human sequence or a human recipient of a cell, a tissue, and/or an organ isolated from said genetically reprogrammed swine, the method comprising selecting a known human major histocompatibility complex sequence or sequencing the human recipient’s major histocompatibility complex gene, obtaining a swine comprising a nuclear genome having at. least one disrupted swine surface glycan gene, genetically modifying ceils of the swine to replace portions of the swine’s major histocompatibility' complex gene with corresponding portions of the known human major histocompatibility complex gene or corresponding portions of the human recipient’s major histocompatibility complex gene such that the swine’s cells express portions of the human recipient’s major histocompatibility complex. [00047] In accordance with another exemplary' aspect of the invention, the present disclosure provides a method of delaying, reducing, or preventing rejection, separation, or adverse reactions to xenotransplanted tissues in a human recipient, comprising selecting a known human major histocompatibility complex gene sequence or sequencing the human recipient’s major histocompatibility complex gene, obtaining a swine comprising a nuclear genome having disrupted alpha- 1,3 galactosyltransferase gene, genetically modifying cells of the swine to replace a portion of the swine’s major histocompatibility complex gene with a corresponding portion of the known human major histocompatibility complex gene sequence or the corresponding portion of the human recipient’s major histocompatibility complex gene sequence such that the swine’s cells express the corresponding portion of the known human major histocompatibility' complex gene sequence or the corresponding portion of the human recipient’s major histocompatibility complex gene sequence, isolating cells, tissue, and/or an organ from the genetically reprogrammed swine that express the corresponding portion of the known human major histocompatibility complex gene sequence or the corresponding portion of the human recipient’s major histocompatibility complex gene sequence, and transplanting the isolated cells, tissue, and/or an organ from the genetically reprogrammed swine into the human recipient.

[00048] In accordance with another exemplary aspect of the invention, the present, disclosure provides a biological product for clinical xenotransplantation derived from a non-wild type, biologically engineered, non-human organism, wherein said organism from which said biological product is derived is produced through natural breeding and/or assisted reproductive technologies, and wherein said organism has a biologically engineered genome such that it does not express one or more extracellular surface glycan epitopes, and wherein said organism is free of at least the following pathogens: Ascaris species, Cryptosporidium species, Echnoeoccus, Strongyloids sterocolis, Toxoplasma gondii, Brucella suis, Leptospira species, mycoplasma hyopneumoniae, pseudorabies, Toxoplasma Gondii, staphylococcus species, Microphyton species, and Trichophyton species, porcine influenza, cytomega!ovims, arterivirus, and a coronavirus, wherein said organism is not transgenic, wherein said product does not contain one or more extracellular surface glycans, wherein said product is free of: Ascaris species, Cryptosporidium species, Echnococcus, Strongyloids sterocolis, Toxoplasma gondii, Brucella suis, Leptospira species, mycoplasma hyopneumoniae, pseudorabies. Toxoplasma Gondii, staphylococcus species, Microphyton species , and Trichophyton species , porcine influenza, cytomegalovirus, arterivirus, and a coronavirus, wherein said product has not been terminally sterilized, wherein said product is less immunogenic compared to biological product obtained from a xenotransplantation product made from conventional Gal-T knockout swine, from conventional triple knockout swine, from transgenic swine, from wild-type animals, and/or allograft, wherein said product is less antigenic when transplanted into a human xenotransplant recipient as compared to a biological product obtained from a xenotransplantation product made from conventional Gal- T knockout swine, from conventional triple knockout swine, from transgenic swine, from wild- type animals, and/or allograft, and wherein said product is biologically active and comprises live ceils and tissues capable of vascularizing after xenotransplantation.

[00049] It is therefore understood that multiple source animals, with an array of biological properties including, but not limited to, genome modification and/or other genetically engineered properties, can be utilized to reduce immunogenicity and/or immunological rejection (e.g., acute, hyperacute, and chronic rejections) in humans resulting from xenotransplantation. In certain aspects, the present disclosure can be used to reduce or avoid thrombotic microangiopathy by transplanting the biological product of the present disclosure into a human patient. In certain aspects, the present disclosure can be used to reduce or avoid glomerulopathy by transplanting the biological product of the present disclosure into a human patient. It will be further understood that the listing of source animals set forth herein is not limiting, and the present invention encompasses any other type of source animal with one or more modifications (genetic or otherwise) that serve(s) to reduce immunogenicity and/or immunological rejection, singularly or in combination.

Immunogenomic Reprogrammed Source Animals ! 00050] As disclosed herein, tolerogenic non-human animal nerve tissues for several human Class I and/or Class II Vi I iC molecules are provided. One non-limiting type of source animal is swine.

[00051] The human immune response system is a highly complex and efficient defense system against invading organisms. T-cells are the primary effector cells involved in the cellular response. Just as antibodies have been developed as therapeutics, T-Cell Receptors (TCRs), the receptors on the surface of the T-cells, which give them their specificity, have unique advantages as a platform for developing therapeutics. While antibodies are limited to recognition of pathogens in the blood and extracellular spaces or to protein targets on the cell surface, TCRs recognize antigens displayed by MHC molecules on the surfaces of cells (including antigens derived from intracellular proteins). Depending on the subtype of T-cells that recognize displayed antigen and become activated, TCRs and T-cells harboring TCRs participate in controlling various immune responses. For instance, helper T-cells are involved in regulation of the humoral immune response through induction of differentiation of B ceils into antibody secreting cells. In addition, activated helper T-cells initiate cell-mediated immune responses by cytotoxic T-cells. Thus, TCRs specifically recognize targets that are not normally seen by antibodies and also trigger the T-cells that bear them to initiate wide variety of immune responses.

[00052] It will be understood that T-cell recognizes an antigen presented on the surfaces of cells by means of the TCRs expressed on their cell surface. TCRs are disulfide-linked heterodimers, most consisting of a and b chain glycoproteins. T-cells use recombination mechanisms to generate diversity in their receptor molecules similar to those mechanisms for generating antibody diversity operating in B cells (Janeway and Travers, Immunobiology 1997). Similar to the immunoglobulin genes, TCR genes are composed of segments that rearrange during development of T-cells. TCR polypeptides consist of variable, constant, transmembrane and cytoplasmic regions. While the transmembrane region anchors the protein and the intracellular region participates in signaling when the receptor is occupied, the variable region is responsible for specific recognition of an antigen and the constant region supports the variable region-binding surface. The TCR a chain contains variable regions encoded by variable (V) and joining (J) segments only, while the b chain contains additional diversity (D) segments.

[00053] Major histocompatibility complex Class I (MHO) and Class II (MHCII) molecules display peptides on antigen -presen ting cell surfaces for subsequent T-cell recognition. Within the human population, allelic variation among the classical MHCI and II gene products is the basis for differential peptide binding, thymic repertoire bias and allograft rejection, MHC molecules are cell-surface glycoproteins that are central to the process of adaptive immunity, functioning to capture and display peptides on the surface of antigen-presenting cells (APCs). MHC Class I (MHCI) molecules are expressed on most cells, bind endogenously derived peptides with sizes ranging from eight to ten amino acid residues and are recognized by CDS cytotoxic T-lymphocytes (CTL). On the other hand, MHC Class II (MHCII) are present only on specialized APCs, bind exogenously derived peptides with sizes varying from 8 to 26, e.g., 9 to 22, residues, and are recognized by CD4 helper T-cells. These differences indicate that MHCI and MHCII molecules engage two distinct arms of the T-cell-mediated immune response, the former targeting invasive pathogens such as viruses for destruction by CDS CTLs, and the latter inducing cytokine-based inflammatory' mediators to stimulate CD4 helper T-cell activities including B-cell activation, maturation and antibody production. In some aspects, the biological product of the present disclosure is not recognized by CD8+ T cells, do not bind anti-HLA antibodies, and are resistant, to NK-mediated lysis.

[00054] The human leukocyte antigen (HLA) system or complex is a gene complex encoding the major histocompatibility complex (MHC) proteins in humans. These cell-surface proteins are responsible for the regulation of the immune system in humans. The HLA gene complex resides on a 3 Mbp stretch within chromosome 6p21. HLA genes are highly polymorphic, which means that they have many different alleles, allowing them to fine-tune the adaptive immune system. The proteins encoded by certain genes are also known as antigens, as a result of their historic discovery' as factors in nerve tissue transplants. Different classes have different functions. [00055] The HLA segment, is divided into three regions (from centromere to telomere), Class II, Class III and Class I. Classical Class I and Class II HLA genes are contained in the Class I and Class II regions, respectively, whereas the Class III locus bears genes encoding proteins involved in the immune system but not structurally related to MHC molecules. The classical HLA Class I molecules are of three types, HLA- A, HLA-B and HLA-C. Only the a chains of these mature HLA Class I molecules are encoded within the Class I HLA locus by the respective HLA- A, HLA-B and HLA-C genes. In contrast, the beta-2 microglobulin b^'2 m chain encoded by the b2 m gene is located on chromosome 15. The classical HLA Class II molecules are also of three types (HLA-DP, HLA-DQ and HLA-DR), with both the a and b chains of each encoded by a pair of adjacent loci. In addition to these classical HLA Class I and HLA Class II genes, the human MHC locus includes a long array of HL A pseudogenes as well as genes encoding non-classical MHO and MHCII molecules. HLA-pseudogenes are an indication that gene duplication is the main driving force for HL A evolution, whereas non-classical MHO and MHCII molecules often serve a restricted function within the immune system quite distinct from that of antigen presentation to ab TCRs.

[00056] Aside from the genes encoding the antigen-presenting proteins, there are a large number of other genes, many involved in immune function, located on the HLA complex. Diversity ofHLAs in the human population is one aspect of disease defense, and, as a result, the chance of two unrelated individuals with identical HLA molecules on all loci is extremely low. [00057] Class I MHC molecules are expressed on all nucleated cells, including tumor cells. They are expressed specifically on T and B lymphocytes, macrophages, dendritic cells and neutrophils, among other cells, and function to display peptide fragments (typically 8-11 amino acids in length) on the surface to CD8+ cytotoxic T lymphocytes (CTLs). CTLs are specialized to kill any cell that bears an MHC I-bound peptide recognized by its own membrane-bound TCR. When a cell displays peptides derived from cellular proteins not normally present (e.g., of viral, tumor, or other non-self origin), such peptides are recognized by CTLs, which become activated and kill the cell displaying the peptide.

[00058] MHC Class I protein comprises an extracellular domain (which comprises three domains: cu, 012 and as), a transmembrane domain, and a cytoplasmic tail. The ouand a?, domains form the peptide-binding cleft, while the 0.3 interacts with b2-ihΐVk¾1oI>hϋh. Class I molecules consist of two chains: a polymorphic a-chain (sometimes referred to as heavy chain) and a smaller chain called p2-microglobulin (also known as light chain), which is generally not polymorphic. These two chains form a non-covalent heterodimer on the cell surface. The a-chain contains three domains (aΐ, a2 and a3). Exon 1 of the a-chain gene encodes the leader sequence, exons 2 and 3 encode the al and a2 domains, exon 4 encodes the a3 domain, exon 5 encodes the transmembrane domain, and exons 6 and 7 encode the cytoplasmic tail. The a-chain forms a peptide-binding cleft involving the al and a2 domains (which resemble Ig-iike domains) followed by the a3 domain, which is similar to p2-microglobulin.

[00059] b2 microglobulin is a non-glycosylated 12 kDa protein; one of its functions is to stabilize the MHC Class I a-chain. Unlike the a-chain, the b2 microglobulin does not span the membrane. The human b2 mieroglobulin locus is on chromosome 15 and consists of 4 exons and 3 introns. Circulating forms of b2 mieroglobulin are present in serum, urine, and other body fluids; non-covalent!y MHC I-associated b2 mieroglobulin can be exchanged with circulating b2 mieroglobulin under physiological conditions.

[00060] MHC Class II protein comprises an extracellular domain (which comprises three domains: ai, a? , bΐ, and b 1), a transmembrane domain, and a cytoplasmic tail. The ai and bΐ domains form the peptide-binding cleft, while the ai and bΐ interacts with the transmembrane domain.

[00061] In addition to the aforementioned antigens, the Class I antigens include other antigens, termed non-classical Class I antigens, in particular the antigens HLA-E, HLA-F and HLA-G; this latter, in particular, is expressed by the extravillous trophoblasts of the normal human placenta in addition to HLA-C.

Cell Phenotype

[00062] The source animals to be used in the present invention include genetic modifications to remove or deactivate certain SLA exons to regulate the swine cell’s extracellular expression or non-expression of MHC Class II, la, and/or lb; reprogramming certain native, naturally occurring swine cell SLA exons to regulate the swine cell’s extracellular expression or non-expression of MHC Class II; conserving or otherwise not removing swine introns existing in or in the vicinity of the otherwise engineered sequences; increasing the expression of swine CTLA4 and PD- 1; and removing or deactivating alpha-1,3 galactosyltransferase, cytidine monophosphate-N-acety!neuraminic acid hydroxylase, and b!,4~N~ acetylgalactosaminyltransferase according to the first aspect. Such removal, reprogramming, and modification to cause such increase of expression, and other engineered aspects of a swine genome, to create a tolerogenic xenotransplantation swine cell that mimics the extracellular configuration of a human trophoblast, is described as follows.

[00063] The former and current attempts to this unmet clinical need has precisely followed the classic medical dogma of ‘"one-size fits all”. We refer to this as the “downstream” approach - which must contend with addressing all of the natural immune processes in sequence. Instead of adopting this limited view, the present invention takes a “patient-specific” solution to dramatically improve clinical outcome measures. The latter, our approach, we term the “upstream” approach - one winch represents the culmination of unfilled scientific effort into a coordinated translational effort. The central theorem of our approach is countervailing to the existing and previous dogmatic approaches. The “downstream” approach accepts the innate and immovable disparity between donor and recipient, and focuses on interventions, gene alterations, and/or concomitant exogenous immunosuppressive medications used as a method of reducing/eiiminating/negatively-altering the recipients' naturally resulting immunologic response. In contrast, we intentionally choose to reverse the focus of the otherwise area of fundamental scientific dogma. Rather than accept the immunological incompatibilities between the donor and recipient, specifically (but not limited to) those mismatches of the Major Histocompatibility Complex(es), we alter these catalytic antigens at the source, thereby eliminating all of the precipitating mechanisms that are the causative effectors of rejection between donor and recipient. This approach applies beyond the field of xenotransplantation including, but not limited to, the fields of genetics, obstetrics, infectious disease, oncology, agriculture, animal husbandry, food industry and other areas.

[00064] The present disclosure embodies the above modification in creating a non- transgenie genetically reprogrammed swine for xenotransplantation, wherein the MHC surface characterization of the swine mimic that of the recipient’s trophoblast, wherein the immune response from the xenotransplantation is significantly reduced. The human extravillous trophoblast cells express HLA-C, HLA-E, HLA-F, and HLA-G, but not HLA-A, HLA-B, HLA- DQ and HLA-DR. As such, the current embodiment combines the unique MHC surface characterization of human trophoblast with site-directed mutagenic substitutions to minimize or remove the immune response associated with xenotransplantation while minimizing off target effects on the native donor swine’ s SLA/MHC gene.

[00065] The human immune response system is a highly complex and efficient defense system against invading organisms. T-cells are the primary effector cells involved in the cellular response. Just as antibodies have been developed as therapeutics, (TCRs), the receptors on the surface of the T-cells, which give them their specificity, have unique advantages as a platform for developing therapeutics. While antibodies are limited to recognition of pathogens in the blood and extracellular spaces or to protein targets on the cell surface, TCRs recognize antigens displayed by MHC molecules on the surfaces of cells (including antigens derived from intracellular proteins). Depending on the subtype of T-cells that recognize displayed antigen and become activated, TCRs and T-cells harboring TCRs participate in controlling various immune responses. For instance, helper T-cells are involved in regulation of the humoral immune response through induction of differentiation of B cells into antibody secreting ceils. In addition, activated helper T-ceils initiate cell-mediated immune responses by cytotoxic T-cells. Thus, TCRs specifically recognize targets that are not normally seen by antibodies and also trigger the T-cells that bear them to initiate wide variety of immune responses.

[00066] With respect to the MHC-I proteins, the current disclosure either inactivate, or where necessary to retain the function of the “find and replace” orthologous SLA proteins with HLA analogs that would result in minimal immune recognition. Such genetic modifications may be referred to herein as “selectively silencing” (and grammatical variants thereof) according to the first aspect. In some aspects, silencing the genes which encode and are responsible for the expression of 8LA-1 removes the highly-problematic and polymorphic HLA- A analog. Similarly, inactivation or complete removal of genes associated with SLA-2 would reduce the burden imposed by mismatched HLA-B proteins. This would, at the cell surface interface, appear to the human recipient’s T cells as a HLA-A and HLA-B negative cell. With respect to the last of the classical MHC Class I proteins, HLA-C, site-directed mutagenesis of genes that encode for SLA- 3 using a reference HLA-C sequence would mimic an allo-transplant with such a disparity. Given the “less- polymorphic” nature of HLA-C, as compared to HLA-A and HLA-B, this would be further improved by the replacement of SLA-3 with a reference replacement sequence based on the subclass of HLA-C that is naturally prevalent in nature, and also invoking mechanisms that would allow for the minimal but requisite level of expression that would afford functionality and non-interruption of the numerous known and also those unknown MHC-I dependent processes. [00067] With respect to the MHC-I proteins, the current disclosure either inactivate, or where necessary to retain the function of the “find and replace” orthologous SLA proteins with HLA analogs that would result in minimal immune recognition. In some aspects, silencing the genes which encode and are responsible for the expression of SLA-1 removes the highly- probiematic and polymorphic HLA-A analog. Similarly, inactivation or complete removal of genes associated with SLA-2 would reduce the burden imposed by mismatched HLA-B proteins. This would, at the cell surface interface, appear to the human recipient’s T cells as a HLA-A and HLA- B negative cell. With respect to the last of the classical MHC Class I proteins, HLA-C, site-directed mutagenesis of genes that encode for SLA-3 using a reference HLA-C sequence would mimic an alio-transplant with such a disparity. Given the ‘dess- polymorphic” nature of HLA-C, as compared to HLA-A and HLA-B, this would he further improved by the replacement of SLA-3 with a reference replacement sequence based on the subclass of HLA-C that is naturally prevalent in nature, and also invoking mechanisms that would allow for the minimal but requisite level of expression that would afford functionality and non-interruption of the numerous known and also those unknown MHC-i dependent processes.

[00068] Furthermore, the expression of non-classical MHC proteins - those included in the I-b category, which include HLA-E, F, and G are vitally important to both the survival of the fetus and synergistic existence of the trophobiast(s). Fortunately, these are significantly less polymorphic than the “classical” MHC-Ia variety. Without expression of these, heightened upregulation of ceil lysis is a direct result of NK cell recognition and activation is observed. In an identical manner as described to the MHC-Ia components, the orthoiogous SLA proteins with HLA analogs are either inactivated, or where necessary, to “find and replace.”

[00069] HLA-G can be a potent immuno-inhibitory and tolerogenic molecule. HLA-G expression in a human fetus can enable the human fetus to elude the maternal immune response. Neither stimulator}_' functions nor responses to allogeneic HLA-G have been reported to date. HLA-G can be a non-classical HLA Class I molecule. It can differ from classical MHC Class I molecules by its genetic diversity, expression, structure, and function. HLA-G can be characterized by a low allelic polymorphism. Expression of HLA-G can be restricted to trophoblast cells, adult thymic medulla, and stem cells. The sequence of the HLA-G gene (HLA-6.0 gene) has been described by GERAGHTY et ah, (Proc. Natl. Acad. Sci. USA, 1987, 84, 9145-9149): it comprises 4,396 base pairs and exhibits an intron/exon organization which is homologous to that of the HLA- A, HLA-B and HLA-C genes. More precisely, this gene comprises 8 exons and an untranslated, 3'UT, end, with the following respective correspondence: exon 1: signal sequence, exon 2: al domain, exon 3: o2 domain, exon 4: a3 domain, exon 5: transmembrane region, exon 6: cytoplasmic domain I, exon 7: cytoplasmic domain II, exon 8: cytoplasmic domain III and 3' untranslated region (GERAGHTY et al., mentioned above, ELLIS et a!., I. Immunol., 1990, 144, 731-735). However, the HLA-G gene differs from the other Class I genes in that the in-frame translation termination codon is located at the second codon of exon 6; as a result, the cytoplasmic region of the protein encoded by this gene HLA-6.0 is considerably shorter than that of the cytoplasmic regions of the HLA-A, HLA-B and HLA-C proteins.

[00070] Natural killer (NK) cell-mediated immunity, comprising cytotoxicity and cytokine secretion, plays a major role in biological resistance to a number of autologous and allogeneic ceils. The common mechanism of target ceil recognition appears to be the lack or modification of self MHC Class I-peptide complexes on the cell surface, which can lead to the elimination of viraliy infected cells, tumor cells and major histocompatibility' MHC-incompatible grafted cells. Kill's, members of the Ig superfamily which are expressed on NK cells, have recently been discovered and cloned. KIR's are specific for polymorphic MHC Class I molecules and generate a negative signal upon ligand binding which leads to target cell protection from NK cell-mediated cytotoxicity in most systems. In order to prevent NK cell autoimmunity, i.e., the lysis of normal autologous cells, it is believed that every given NK cell of an individual expresses at least on KIR recognizing at least one of the autologous HLA-A, B, C, or G alleles.

[00071] According to the present disclosure, in the context of swine-to-human xenotransplantation, each human recipient will have a major histocompatibility complex (MHC) (Class I, Class II and/or Class III) that is unique to that individual and will not match the MHC of the donor swine. Accordingly, wihen a donor swine graft is introduced to the recipient, the swine MHC molecules themselves act as antigens, provoking an immune response from the recipient, leading to transplant rejection.

[00072] According to this aspect of the present disclosure (i.e., reprogramming the SLA/MHC to express specifically selected human MHC alleles), when applied to swine tissues for purposes of xenotransplantation will decrease rejection as compared to tissues derived from a wild- type swine or otherwise genetically modified swine that lacks this reprogramming, e.g., transgenic swine or swine with non-specific or different genetic modifications.

[00073] With the previous modifications incorporated, insertion or activation of additional extracellular ligands that would create a protective, localized immune response as seen with the maternal-fetal symbiosis, would be an additional step to minimize deleterious eellular-mediated immunological functions that may remain as a result of minor-antigen disparities. Therefore, porcine ligands for SLA-MIC2 is orthologously reprogrammed with human counterparts, MICA. Human Major Histocompatibility Complex Class I Chain-Related gene A (MICA) is a cell surface glycoprotein expressed on endothelial cells, dendritic cells, fibroblasts, epithelial ceils, and many tumours. It is located on the short arm of human chromosome 6 and consists of 7 exons, 5 of which encodes the transmembrane region of the MICA molecule. MICA protein at normal states has a low level of expression in epithelial tissues but is upregulated in response to various stimuli of cellular stress. MICA is classified as a non-classical MHC Class I gene, and functions as a ligand recognized by the activating receptor NKG2D that is expressed on the surface of NK cells and CD8+ T cells.

[00074] In addition, porcine ligands for PD-LI, CTLA-4, and others are overexpressed and/or otherwise orthologously reprogrammed with human counterparts. PD-LI is a transmembrane protein that has major role in suppressing the adaptive immune system in pregnancy, allografts, and autoimmune diseases. It is encoded by the CD274 gene in human and is located in chromosome 9. PD-LI hinds to PD-1, a receptor found on activated T cells, B cells, and myeloid cells, to modulate activation or inhibition. Particularly, the binding of PD-LI to receptor PD-1 on T cells inhibits activation of IL-2 production and T cell proliferation. CTLA4 is a protein receptor that also functions as an immune checkpoint that downregulates immune responses. It is encoded by the CTLA4 gene and is located in chromosome 2 in human. It is constitutively expressed on regulator)' T cells but are upregulated in activated T cells. Gene expression for CTLA-4 and PD-LI is increased, for example, based on reprogramming promoters thereof. There is a relationship between genotype and CTLA-4 or PD-LI expression. For example, individuals carrying thymine at position 318 of the CTLA4 promoter (T318)) and homozygous for adenine at position 49 in exon 1 showed significantly increased expression both of cell-surface CTLA-4 after cellular stimulation and of CTLA-4 mRNA in non-stimulated cells in Ligers A, et al. CTLA-4 gene expression is influenced by promoter and exon 1 polymorphisms, Genes Immun. 2001 May;2(3): 145-52, which is incorporated herein by reference in its entirety for all purposes. A similar upregulation can be achieved to overexpress PD-LI using a PD-LI promoter reprogramming.

[00075] Further, anti-coagulant porcine ligands for Endothelial protein C receptor (EPCR), Thrombomodulin (TBM), Tissue Factor Pathway Inhibitor (TFPI), and others are orthologously reprogrammed with human counterpans. Endothelial protein C receptor is endothelial cell-specific transmembrane glycoprotein encoded by PRGCR gene that is located in chromosome 20 in human. It enhances activation of Protein C, an anti-coagulant serine protease, and has crucial role in activated protein C mediated signaling. Thrombomodulin is an integral membrane glycoprotein present on surface of endothelial cells. It is encoded by THBD gene that is located in chromosome 20 in human. In addition to functioning as cofactor in the thrombin-induced activation of protein C in the anticoagulant pathway, it also functions in regulating C3b inactivation. Tissue Factor Pathway Inhibitor (TFPI) is a glycoprotein that functions as natural anticoagulant by inhibiting Factor Xa. It encoded by TFPI gene located in chromosome 2 in human and the protein structure consists of three tandemly linked Kunitz domains. In human, two major isoforms of TFPi exists, TFPIa and TRRIb. TFPIa consists of three inhibitor}' domains (Kl, K2, and K3) and a positively charged C terminus while TRRIb consists of two inhibitory domains (Kl and K2) and C terminus. While Kl and K2 domains are known to bind and inhibit Factor VII and Factor Xa, respectively, the inhibitory function of K3 is unknown. In certain aspects, the present disclosure centralizes (predicates) the creation of hypoimmunogenic and/or tolerogenic tissues that do not necessitate the transplant recipients’ prevalent and deleterious use of exogenous immunosuppressive drugs (or prolonged immunosuppressive regimens) following the transplant procedure.

[00076] It is therefore understood that multiple source animals, with an array of biological properties including, but not limited to, genome modification and/or other genetically engineered properties, can be utilized to reduce immunogenicity and/or immunological rejection (e.g., acute, hyperacute, and chronic rejections) in humans resulting from xenotransplantation. In certain aspects, the present disclosure can be used to reduce or avoid thrombotic microangiopathy by transplanting the biological product of the present disclosure into a human patient. In certain aspects, the present disclosure can be used to reduce or avoid glomerulopathy by transplanting the biological product of the present disclosure into a human patient. It will be further understood that the listing of source animals set forth herein is not limiting, and the present invention encompasses any other type of source animal with one or more modifications (genetic or otherwise) that serve(s) to reduce immunogenicity and/or immunological rejection, singularly or in combination.

Gene Editing Schema to Create Multiple, Independent, Single-Variable Humanized Pilot

Porcine Cell Lines by Genetic Modification [00077] The genetic modification can be made utilizing known genome editing techniques, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), adeno-associated virus (AAV)-mediated gene editing, and clustered regular interspaced palindromic repeat Cas9 (CRISPR-Cas9). These programmable nucleases enable the targeted generation of DNA double- stranded breaks (DSB), which promote the upregulation of cellular repair mechanisms, resulting in either the error-prone process of non -homologous end joining (NHEJ) or homology -directed repair (HDR), the latter of which is used to integrate exogenous donor DNA templates. CRISPR-Cas9 may also be used to perform precise modifications of genetic material. For example, the genetic modification via CRI8PR-Cas9 can be performed in a manner described in Kelton, W. et. al, “Reprogramming MHC specificity by CRJSPR-Cas9-assisted cassette exchange/’ Nature, Scientific Reports, 7:45775 (2017) (“Kelton”), the entire disclosure of which is incorporated herein by reference. Accordingly, the present disclosure includes reprogramming using CRI8PR-Cas9 to mediate rapid and scarless exchange of entire alleles, e.g., MHC, HI.. A, SLA, etc.

[00078] According to the present disclosure, genetic modification techniques may be used to mediate rapid and scarless exchange of entire MHC alleles at specific native locus in swine cells. Multiplex targeting of Cas9 with two gRNAs is used to introduce single or double-stranded breaks flanking the MHC allele, enabling replacement with the template HLA/MHC sequence (provided as a single or double-stranded DNA template).

[00079] In some aspects, the expression of polymorphic protein motifs of the donor animal’ s

MHC can be further modified by knock-out methods known in the art. For example, knocking out one or more genes may include deleting one or more genes from a genome of a non-human animal. Knocking out may also include removing all or a part of a gene sequence from a non-human animal. It is also contemplated that knocking out can include replacing all or a part of a gene in a genome of a non-human animal with one or more nucleotides. Knocking out one or more genes can also include substituting a sequence in one or more genes thereby disrupting expression of the one or more genes. Knocking out one or more genes can also include replacing a sequence in one or more genes thereby disrupting expression of the one or more genes without frameshifts or frame disruptions in the native donor swine’s 8LA/MHC gene. For example, replacing a sequence can generate a stop codon in the beginning of one or more genes, which can result in a nonfunctional transcript or protein. For example, if a stop codon is created within one or more genes, the resulting transcription and/or protein can be disrupted, silenced and rendered nonfunctional.

[00080] In another aspect, the present invention utilizes alteration by nucleotide replacement of STOP codon at exon regions of the wild-type swine’s SLA-DR to avoid provocation of natural cellular mediated immune response (CD8+ T Cell) by the recipient, including making cells that lack functional expression of SLA-DR, SLA-1, SLA-2. For example, the present invention utilizes TAA . In other embodiments, the invention utilizes TAG. In other embodiments, the invention utilizes TGA.

[00081] In one aspect; the present invention utilizes insertion or creation (by nucleotide replacement) of STOP codon at exons regions of the wild-type swine’s second, identical duplication B2-microglobulin gene to reduce the B2-microglobulin mRNA expression level in pigs. It will be understood that B2-microglobulin is a predominant immunogen, specifically a non- gal xeno-antigen. In one aspect, the present invention utilizes insertion or creation (by nucleotide replacement) of STOP codon at exons regions of the wild-type swine’s B2-microglobulin gene to reduce the porcine B2-microglobulin mRNA expression level in pigs. In some aspects, the present invention utilizes reprogramming of regions of the wild-type swine’s B2~microgl obulin gene to reprogram with human B2-microglobulin gene sequences. The disclosures of PCT/US21/63931 are incorporated herein by reference in their entireties.

[00082] in one aspect, the present disclosure includes: a) obtaining a multipotent or pluripotent cell from a wild-type non-human donor animal, wherein said multipotent or pluripotent cell comprise a porcine genome comprising copy 1 and copy 2 of a porcine p2-microglobulin (rb2M) gene; b) inactivating copy 1 and copy 2 of the rb2M gene in the porcine genome; c) forming a genomic sequence construct that comprises a) a nucleic acid sequence encoding a 5’ UTR upstream region of a hp2M gene or a rb2M gene including its promoter sequence and b) a nucleic acid sequence encoding a 1ib2M protein; d) inserting the genomic sequence construct of step c) into a safe harbor locus of the porcine genome to form a genetically reprogrammed cell; e) generating an embryo from the genetically reprogrammed cell; f) transferring the embryo into a surrogate animal; and g) growing the transferred embryo to produce the genetically reprogrammed non-human donor animal as offspring of said surrogate animal.

[00083] In one aspect, the present disclosure includes: a) obtaining a multi potent or p!uripotent cell from a wild-type non-human donor animal, wherein said multipotent or pluripotent cell comprise a porcine genome comprising copy 1 and copy 2 of a porcine p2-microglobulin (rb2M) gene; b) inactivating copy 1 and/or copy 2 of the rb2M gene in the porcine genome; c) forming a genomic sequence construct that comprises a) a nucleic acid sequence encoding a 5’ UTR upstream region of a IibZM gene or a rbZM gene including its promoter sequence and b) a nucleic acid sequence encoding a 1ib2M protein; d) performing scarless exchange of a nucleotide acid sequence encoding copy 1 and/or copy 2 of the rb2M gene in the porcine genome with the genomic sequence construct of step c) to form a genetically reprogrammed cell, e) generating an embryo from the genetically reprogrammed cell; f) transferring the embryo into a surrogate animal; and g) growing the transferred embryo to produce the genetically reprogrammed non-human donor animal as offspring of said surrogate animal.

[00084] In one aspect, the preesnt disclosure includes a) genes encoding alpha- 1,3 galactosyltransferase (GalT), cytidine monophosphate-N- acety!neuraminic acid hydroxylase (CMAH), and beta-1, 4-N- acety!galactosaminyltransferase (B4GALNT2) are disrupted such that the genetically reprogrammed porcine donor lacks functional expression of surface glycan epitopes encoded by said genes; b) reprogramming the porcine genome such that endogenous exon and/or intron regions of the wild-type porcine donors’ Major Histocompatibility Complex corresponding to exon regions of 83LA-1, SLA-2, SLAG, 8LA-6, SLAG, SLAG, SLA-DRA, SLA-DRB, SLA-DQA, and/or 8LA- DQB and any combination thereof, that are disrupted, silenced or otherwise not functionally expressed on (95%) of extracellular surfaces achieved through specific combinations of precise, site-directed mutagenic substitutions or modifications; c) reprogramming the porcine genome such that endogenous exon and/or intron regions of the wild-type porcine donor’s PD-L1, CTLA-4, EPCR, TBM, TFPI, and/or MIC-2, and any combination thereof, that are humanized via reprogramming through specific combinations of precise, site-directed mutagenic substitutions or modifications with synthetic nucleotides from ortho! ogous exons of a known human PD-Ll, CTLA-4, EPCR, TBM, TFPi, and MIC-2 from the human captured reference sequence, designed from the human captured reference sequence and which minimizes collateral genomic disruptions and ideally results in no net gain or loss of total numbers of nucleotides and avoids genomic organizational disruption and that render the donor animal’s cells, tissues, and organs tolerogenic when transplanted into a human without sacrificing the natural immune function of the PD-Ll, CTLA-4, EPCR, TBM, TFPI, and MIC-2 proteins; or d) reprogramming the porcine genome such that endogenous exon and/or intron regions of the wild-type porcine donor’s Major Histocompatibility Complex corresponding to exon regions of SLA-1, SLA-2, SLA-3, SLA-6, SLA-7, SLA-8, SLA-DRA, SLA-DRB, SLA-DQA, and/or SLA- DQB, and any combination thereof, that are reprogrammed through specific combinations of precise, site-directed mutagenic substitutions or modifications with synthetic nucleotides from orthologous exons of a known human HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA- DRA, HLA-DRB, HLA-DQA, and/or HLA-DQB from the human captured reference sequence, designed from the human captured reference sequence and which minimizes collateral genomic disruptions and ideally results in no net gain or loss of total numbers of nucleotides and avoids genomic organizational disruption and that, render the donor animal’s cells, tissues, and organs tolerogenic when transplanted into a human without sacrificing the natural immune function of the SLA-1, SLA-2, SLA-3, SLA-6, SLA-7, SLA-8, SLA-DRA, SLA-DRB, SLA-DQA, and/or SLA-DQB proteins.

[00085] In one aspect, the recipient’s HLA/MHC gene is sequenced and template HLA/MHC sequences are prepared based on the recipient’s HLA/MHC genes. In another aspect, a known human HLA/MHC genotype from a World Health Organization (WHO) database may be used for genetic reprogramming of swine of the present disclosure.

[00086] CRISPR-Cas9 plasmids are prepared, e.g., using polymerase chain reaction and the recipient’s HLA/MHC sequences are cloned into the plasmids as templates. CRJSPR cleavage sites at the SLA/MHC locus in the swine cells are identified and gRNA sequences targeting the cleavage sites and are cloned into one or more CRISPR-Cas9 plasmids. CRISPR-Cas9 plasmids are then administered into the swine cells and CRIP8R/Cas9 cleavage is performed at the MHC locus of the swine cells.

[00087] The SLA/MHC locus in the swine cells are precisely replaced with one or more template HLA/MHC sequences matching the known human HLA/MHC sequences or the recipient’s sequenced HLA/MHC genes. Cells of the swine are sequenced after performing the SLA/MHC reprogramming steps in order to determine if the SLA/MHC sequences in the swine cells have been successfully reprogrammed. One or more neural tissues from the HLA/MHC sequence-reprogrammed swine are transplanted into a human recipient.

[00088] The modification to the donor SLA/MHC to match recipient HLA/MHC causes expression of specific MHC molecules in the new swine cells that are identical, or virtually identical, to the MHC molecules of a known human genotype or the specific human recipient. In one aspect, the present disclosure involves making modifications limited to only specific portions of specific SLA regions of the swine’s genome to retain an effective immune profile in the swine while biological products are tolerogenic when transplanted into human recipients such that use of immunosuppressants can be reduced or avoided. In contrast to aspects of the present disclosure, xenotransplantation studies of the prior art required immunosuppressant use to resist rejection [00089] In one aspect, the swine genome is reprogrammed to disrupt, silence, cause nonfunctional expression of swine genes corresponding to HLA-A, HLA-B, DR, and one of the two copies of the swine B2m (first aspect), and to reprogram via substitution ofHLA-C, HLA-E, HLA-F, HLA-G, HLA-DQ-A, and HLA-DQ-B (third aspect). Further, according to the second aspect, the swine genome is reprogrammed to humanize the other copy of the swine B2m, PD-L1 , CTLA-4, EPCR, IBM, TFPI, and MIC2.

[00090] In certain aspects, HLA-C expression is reduced in the reprogrammed swine genome. By reprogramming the swine cells to be invisible to a human’s immune system, this reprogramming thereby minimizes or even eliminates an immune response that would have otherwise occurred based on swine MHC molecules otherwise expressed from the donor swine cells.

[00091] Various cellular marker combinations in swine cells are made and tested to prepare biologically reprogrammed swine cells for acceptance by a human patient's body for various uses. For these tests, Porcine Aorta Endothelial Cells, fibroblast, or a transformed porcine macrophage ceil line available from ATCC® (3D4/21) are used. [00092] The knockout only and knockout plus knock in cell pools are generated by designing and synthesizing a guide RNA for the target gene. Each guide RNA is composed of two components, a CRISPR RNA (crRNA) and a trans-activating RNA (tracrRNA). These components may be linked to form a continuous molecule called a single guide RNA (sgRNA) or annealed to form a two-piece guide RNA.

[00093] CRISPR components (gRNA and Cas9) can be delivered to cells in DNA, RNA, or ribonucleoprotein (RNP) complex formats. The DNA format involves cloning gRNA and Cas9 sequences into a plasmid, which is then introduced into cells. If permanent expression of gRN A and/or Cas9 is desired, then the DNA can be inserted into the host cell’s genome using a ientivirus. Guide RNAs can be produced either enzymatically (via in vitro transcription) or synthetically. Synthetic RNAs are typically more pure than IVT-derived RNAs and can be chemically modified to resist degradation. Cas9 can also be delivered as RNA. The ribonuc!eoproteins (RNP) format consists of gRNA and Cas9 protein. The RNPs are pre-complexed together and then introduced into cells. This format is easy to use and has been shown to be highly effective in many cell types, [00094] After designing and generating the guide RNA, the CRISPR components are introduced into cells via one of several possible transfection methods, such as lipofection, electroporation, nueleofection, or mieroinjection. After a guide RNA and Cas9 are introduced into a ceil culture, they produce a DSB at the target site within some of the cells. The NHEJ pathway then repairs the break, potentially inserting or deleting nucleotides (indels) in the process. Because NHEJ may repair the target site on each chromosome differently, each cell may have a different set of indels or a combination of indels and unedited sequences.

[00095] For knock in cells, the desired sequences are knocked into the cell genome through insertion of genomic material using, e.g., homology-directed repair (HDR).

[00096] It will be further understood that disruptions and modifications to the genomes of source animals provided herein can be performed by several methods including, but not limited to, through the use of clustered regularly interspaced short palindromic repeats (“CRISPR”), which can be utilized to create animals having specifically tailored genomes. See, e.g., Niu et ah, “Inactivation of porcine endogenous retrovirus in pigs using CRISPR-Cas-9,” Science 357:1303- 1307 (22 September 2017). Such genome modification can include, but not be limited to, any of the genetic modifications disclosed herein, and/or any other tailored genome modifications designed to reduce the bioburden and immunogenicity of products derived from such source animals to minimize immunological rejection.

[00097] CRISPR/CRISPR-associated protein (Cas), originally known as a microbial adaptive immune system, has been adapted for mammalian gene editing recently. The CRISPR/Cas system is based on an adaptive immune mechanism in bacteria and archaeato defend the invasion of foreign genetic elements through DNA or KNA interference. Through mammalian codon optimization, CRISPR/Cas has been adapted for precise DNA/RNA targeting and is highly efficient in mammalian ceils and embryos. The most commonly used and intensively characterized CRISPR/Cas system for genome editing is the type II CRISPR system from Streptococcus pyogenes, this system uses a combination of Cas9 nuclease and a short guide RNA (gRNA) to target specific DNA sequences for cleavage. A 20-nucleotide gRNA complementary to the target DNA that lies immediately 5' of a PAM sequence (e.g., NGG) directs Cas9 to the target DNA and mediates cleavage of double-stranded DNA to form a DSB. Thus, CRISPR/Cas9 can achieve gene targeting in any N20-NGG site.

[00098] Thus, also encompassed by the invention is a genetically modified non-human animal whose genome comprises a nucleotide sequence encoding a human or humanized MHC I polypeptide, MHC II polypeptide and/or b2 microglobulin polypeptide, wherein the polypeptide(s) comprises conservative amino acid substitutions of the amino acid sequence(s) described herein. [00099] One skilled in the art would understand that in addition to the nucleic acid residues encoding a human or humanized MHC I polypeptide, MHC II polypeptide, and/or b2 microglobulin described herein, due to the degeneracy of the genetic code, other nucleic acids may encode the polypeptide(s) of the invention. Therefore, in addition to a genetically modified nonhuman animal that comprises in its genome a nucleotide sequence encoding MHC I, MHC II polypeptide and/or b2 microglobulin polypeptide(s) with conservative amino acid substitutions, a non-human animal whose genome comprises a nucleotide sequenee(s) that differs from that described herein due to the degeneracy of the genetic code is also provided.

[000100] In an additional or alternative approach, the present disclosure includes reprogramming, or leveraging the inhibitory and co- stimulatory' effects of the MHC-I (Class B) molecules. Specifically, the present disclosure includes a process that “finds and replaces” portions of the donor animal genome corresponding to portions of the HLA gene, e.g., to overexpress HLA-G where possible, retaining and overexpressing portions corresponding to HLA- E, and/or “finding and replacing” portions corresponding to HLA-F. As used herein, the term “find and replace” includes identification of the homologous/analogous/orthologous conserved genetic region and replacement of the section or sections with the corresponding human components through gene editing techniques.

[000101] Another aspect includes finding and replacing the beta-2 microglobulin protein which is expressed in HLA -A, -B, -C, -E, -F, and -G. Homologous/analogous/orthologous conserved cytokine mediating complement inhibiting or otherwise immunomodulatory cell markers, or surface proteins, that would enhance the overall immune tolerance at donor-recipient cellular interface,

[000102] In an additional or alternative approach, the present invention utilizes immunogenomic reprogramming to reduce or eliminate MHC-I (Class A) components to avoid provocation of natural cellular mediated immune response by the recipient. In another aspect, exon regions in the donor animal (e.g., swine) genome corresponding to exon regions of HLA- A and HLA-B are disrupted, silenced or otherwise nonfun ctiona!iy expressed on the donor animal. In another aspect, exon regions in the donor animal (e.g., swine) genome corresponding to exon regions of HLA- A and HLA-B are disrupted, silenced or otherwise nonfunctionally expressed in the genome of the donor animal and exon regions in the donor animal (e.g., swine) genome corresponding to exon regions of HLA-C may be modulated, e.g., reduced. In one aspect, the present disclosure includes silencing, knocking out, or causing the minimal expression of source animal’s orthologous HLA-C (as compared to how such would be expressed without such i m mun ogenom i c reprogramm ing) .

[000103] Further, the beta-2-microglobulin protein which comprises the heterodimer structure of each of the MHC-I proteins is species-specific. Thus, in one embodiment of the present disclosure, it is reprogrammed. In contrast to its counterparts, the genetic instructions encoding for this prevalent, building-block protein is not located in the MHC-gene loci. Thus, in one embodiment of the present disclosure includes a genetic modification in addition to those specific for the respective targets as described herein.

[000104] The immune response of the modified swine cells is evaluated through Mixed Lymphocyte Reaction (MLR) study. The impact of the modification or non-expression of MHC la polypeptides on the immune response are measured through the immune response of CD8+ T Ceils. The impact of the modification of MHC lb polypeptides on the immune response are measured through the immune response of NK Cells. The impact of the modification or nonexpression of MHC II polypeptides on the immune response are measured through the immune response of CD4+ T Cells. The MLR study, herein, not only measures the efficacy of the site- directed mutagenic substitution, but also evaluates and identifies the impact of individual modifications, individually and as a whole, as measurements are taken iteratively as additional site-directed mutagenic substitutions are made.

[000105] For knock in cells, the desired sequences are knocked into the cell genome through insertion of genomic material using, e.g., homology-directed repair (HDR). To optimize expression of Class II molecules, the cells are incubated in porcine interferon gamma (IFN-g) for 72 hours which stimulates expression. Expression is then measured by flow cytometry using target specific antibodies. Flow cytometry' may include anti-HLA-C, HLA-E, HLA-G, or other HLA antibodies, or pan anti -HLA Class I or Class II antibodies. According to the present, disclosure, cell surface HLA expression after knock-in is confirmed.

[000106] Complement Dependent Cytotoxicity (CDC) assays may be performed to determine if anti-HLA antibodies recognize the cells from the biological product of the present disclosure. Assay plates prepared by adding a specific human serum containing previously characterized anti-HLA antibodies (or control serum) can be used. IFN-g treated donor cells are resuspended and added to the assay plates, incubated with a source of complement, e.g., rabbit serum. After at least 1 hour of incubation at. room temperature, acridine orange/ethidium bromide solution is added. Percent cytotoxicity is determined by counting dead and live cells visualized on a. fluorescent, microscope, subtracting spontaneous lysis values obtained in the absence of anti- HLA antibodies, and scoring with a scale.

[000107] When knocking out or otherwise silencing surface sugar g!ycans, a cell line that does not express the sugar moieties is obtained, so there is no binding of natural preformed antibodies found in human serum. This is detected using flow cytometry and human serum and a labeled goat anti human IgG or IgM antibody; or specific antibodies directed against sugars. The result is no binding of the antibodies to the final cell line. Positive control is the original cell line (WT) without genetic modifications, in addition, a molecular analysis demonstrates changes in those genes.

[000108] In knocking out or otherwise silencing expression of SLA Class I molecules using CRISPR technologies, the resulting cell line lacks the above sugar moieties as well as SLA Class I expression. Analysis by flow cytometry and molecular gene are performed to demonstrate no surface expression and changes made at the gene level. Cellular reactivity is assessed using a mixed lymphocyte reaction (MLR) with human PBMCs and the irradiated cell line. In comparison to the WT line, there is a reduction in the T cell proliferation, predominantly in the CD8+ T cells. [000109] Reactivity against expression of SLA Class II molecules, DR and DQ is also minimized or eliminated (there is no porcine DP). Analysis is performed at the molecular level, cell surface expression, and in vitro reactivity with human PBMC. There is a significant downward modulation of reactivity against the resulting ceil line.

[000110] To test for cellular reactivity, all cells are incubated with porcine IFN-g for 72 hours then human CD4+ T cells are added to porcine cell lines and cultured for 7 days. The readout is a form of activation/proliferation depending on the resources available.

[000111] To observe a specific response to DQ, human antigen presenting cells (APCs) are absent from the culture such that the cellular response is not the result of pig antigens presented by the APCs.

Creation of a Humanized, “Bespoke”, Designated-Pathogen Free, (Non-Homan) Donor of

Tissues for Transplantation

[000112] Others have attempted to develop homozygous transgenic pigs, which is a slow process, requiring as long as three years using traditional methods of homologous recombination in fetal fibroblasts followed by somatic ceil nuclear transfer (SCNT), and then breeding of heterozygous transgenic animals to yield a homozygous transgenic pig. The attempts at. developing those transgenic pigs for xenotransplantation has been hampered by the lack of pluripotent stem cells, relying instead on the fetal fibroblast as the cell upon which genetic engineering was carried out. For instance, the production of the first live pigs lacking any functional expression of a(l,3) galactosyltransferase (GTKO) was first reported in 2000. In contrast to such prior attempts, the present disclosure provides a faster and fundamentally different process for making non-transgenie reprogrammed swine as disclosed herein. In some aspects, porcine fetal fibroblast cells are reprogrammed using gene editing, e.g., by using CRISPR/Cas for precise reprogramming and transferring a nucleus of the genetically modified porcine fetal fibroblast cell to a porcine enucleated oocyte to generate an embryo; and d) transferring the embryo into a surrogate pig and growing the transferred embryo to the genetically modified pig in the surrogate pig. [000113] Upon confirmation of study results, genetically reprogrammed pigs are bred so that several populations of pigs are bred, each population having one of the desirable human cellular modifications determined from the above assays. The pigs’ cellular activity after full growth is studied to determine if the pig expresses the desired traits to avoid rejection of the pigs’ ceils and tissues after xenotransplantation. Thereafter, further genetically reprogrammed pigs are bred having more than one of the desirable human cellular modifications to obtain pigs expressing cells and tissues that will not be rejected by the human patient’s body after xenotransplantation. [000114] The generation of an induced pluripotent stem cell (iPSC) from pigs offers an opportunity beyond the use of primary cells from fetal fibroblasts. The ability ofiPSC to proliferate almost indefinitely, which contrasts with the limited number of cell divisions that primary' somatic cells can undergo before they senesce, likely means that the iPSC will tolerate the multiple selection steps needed to accommodate directed changes in several genes, especially for gene knock-outs and knock-ins, before nuclear transfer. Another advantage of iPSC over somatic cells is that it has been predicted that cloning efficiency should be inversely correlated with differentiation state and associated epigenetic state. The PAM cells presented in this disclosure are a transformed cell line but the genetic engineering schema can be transferred to porcine iPSC. The specific genetically modified IPSC line would then be used for somatic cell nuclear transfer (SCNT), transferring a nucleus of the genetically modified porcine fetal fibroblast cell to a porcine enucleated oocyte to generate an embryo; and transferring the embryo into a surrogate pig and growing the transferred embryo to the genetically modified pig in the surrogate pig. This has the advantage in that the transferred nucleus contains the specific genome, hence the piglets do not need to go through breeding to obtain a homozygous offspring. The genotype and phenotype of the piglets are identical to the iPSC,

[000115] Specific populations of gene modified iPSC can be cryopreserved as a specific cell line and used as required for development of pigs needed for that genetic background. Thawed iPSCs are cultured and nucleus Is transferred Into enucleated oocytes to generate blastocysts/embryos for implantation into surrogate pig. This creates a viable bank of genetically modified iPSC for generation of pigs required for patient specific tissue, organ, or cell transplantation.

[000116] Restated, the former/previous approach to this unmet clinical need has precisely followed the classic medical dogma of “one-size fits all”. Instead of following this limited approach, we pragmatically demonstrate the ability to harness present technological advances and fundamental principles to achieve a “patient-specific” solution which dramatically improves clinical outcome measures. The former, we refer as the “downstream” approach - which must contend with addressing ail of the natural immune processes in sequence. The latter, our approach, we optimistically term the “upstream” approach - one which represents the culmination of unfilled scientific effort into a coordinated translational effort.

[000117] In another aspect, disclosed herein is a method for making a genetically modified animal described in the application, comprising: a) obtaining a cell with reduced expression of one or more of a component of a MI 1C I-specific enhanceosome, a transporter of a MHC I-binding peptide, and/or C3; b) generating an embryo from the cell; and c) growing the embryo into the genetically modified animal. In some cases, the cell is a zygote.

[000118] In certain aspects, HLA/MHC sequence-reprogrammed swine are bred for at least one generation, or at least two generations, before their use as a source for live tissues, used in xenotransplantation. In certain aspects, the CRISPR/Cas9 components can also be utilized to inactivate genes responsible for PERV activity', e.g., the pol gene, thereby simultaneously completely eliminating PERV from the swine donors.

[000119] In certain aspects, the present disclosure includes embryogenesis and live birth of SLA-free and HLA-expressing biologically reprogrammed swine. In certain aspects, the present disclosure includes breeding SLA-free and HLA-expressing biologically reprogrammed swine to create SLA-free and HLA-expressing progeny. In certain aspects, the CRISPR/Cas9 components are injected into swine zygotes by intracytoplasmic microinjection of porcine zygotes. In certain aspects, the CRISPR/Cas9 components are injected into swine prior to selective breeding of the CRISPR/Cas9 genetically modified swine. In certain aspects, the CRISPR/Cas9 components are injected into donor swine prior to harvesting cells, tissues, zygotes, and/or organs from the swine. In certain aspects, the CRISPR/Cas9 components include all necessary' components for controlled gene editing including self-inactivation utilizing governing gRNA molecules as described in U.S. Pat. No. 9,834,791 (Zhang), which is incorporated herein by reference in its entirety.

[000120] Upon confirmation of study results, genetically reprogrammed pigs are bred so that several populations of pigs are bred, each population having one of the desirable human cellular modifications determined from the above assays. The pigs’ cellular activity after full growth is studied to determine if the pig expresses the desired traits to avoid rejection of the pigs’ cells and tissues after xenotransplantation. Thereafter, further genetically reprogrammed pigs are bred having more than one of the desirable human cellular modifications to obtain pigs expressing cells and tissues that will not be rejected by the human patient’s body after xenotransplantation. [000121] Any of the above protocols or similar variants thereof can be described in various documentation associated with a medical product. This documentation can include, without limitation, protocols, statistical analysis plans, investigator brochures, clinical guidelines, medication guides, risk evaluation and mediation programs, prescribing information and other documentation that may be associated with a pharmaceutical product. It is specifically contemplated that such documentation may be physically packaged with cells, tissues, reagents, devices, and/or genetic material as a kit, as may be beneficial or as set forth by regulatory authorities.

[000122] In another aspect, disclosed herein is a method for making a genetically modified animal described in the application, comprising: a) obtaining a cell with reduced expression of one or more of a component of a MI if I -specific enhanceosome, a transporter of a MHC I-bi tiding peptide, and/or C3; b) generating an embryo from the cell; and c) growing the embryo into the genetically modified animal. In some cases, the cell is a zygote,

THERAPEUTIC APPLICATIONS

[000123] In therapeutic applications, in one embodiment, the subject xenotransplantation tissue can be utilized to reconstruct human nerve gaps and/or peripheral nerve injuries. Various source animals according to the present disclosure may be used. In some aspects, the recipient or subject, has been injured, crushed, has a disease, disorder, and/or genetic defect. In some aspects, the injury is from physical trauma. In some aspects, the recipient or subject has had a mastectomy. In some aspects, the recipient or subject has an autoimmune, e.g., multiple sclerosis or Guillain- Barre syndrome, or a or nervous system disorder, e.g., amyotrophic lateral sclerosis. In some aspects, the recipient or subject has cancer, carpal tunnel syndrome, a repetitive strain injury, a neurodegenerative disease, polyneuropathy, infection, neuritis, diabetes, or other disease or disorder that damages neural or nervous tissue.

[000124] In one aspect, such xenogeneic transplants be derived from non-wildtype, genetically engineered, designated pathogen free porcine donors. Such porcine nerves of the present disclosure share many physiological characteristics to human motor and sensory nerves, including size, length, extracellular matrix, and architecture. The xenogeneic nerve transplantation tissue of the present disclosure provides, viable, xenogeneic nerve transplants may be used for successful reconstruction and treatment of large-gap (>4cm) peripheral nerve injuries including neurotmesis, the complete physiological and anatomical transection of both axons and connective tissue. The xenogeneic nerve product of the present disclosure may be provided in a length of, for example, 2 to 20 cm, 3 to 15 cm, 4 to 12 cm, 4, 5, 6, 7, 8, 9, 10, 11, or 12 cm, with proximal and/or distal diameters of, for example, between 1 mm and 25 mm, e.g., 5, 10, 15, or 20 mm. An exemplary' xenogeneic nerve may have a length of 4 to 10 cm, a proximal diameter of 5 mm and a distal diameter of 1 mm. An exemplary' xenogeneic nerve may have a length of 4-10 cm, a proximal diameter of 10 mm and a distal diameter of 5 mm. An exemplary xenogeneic nerve may have a length of 4-10 cm, a proximal diameter of 15 mm and a distal diameter of 10 mm. An exemplary' xenogeneic nerve may have a length of 4-10 cm, a proximal diameter of 20 mm and a distal diameter of 15 mm.

[000125] The xenogeneic nerve tissues harvested from a genetically engineered, designated pathogen free porcine donor may be stored as frozen (e.g., cry opre served) and/or fresh prior to utilization in the surgical repair of peripheral nerve injuries, including neurotmesis, the complete physiological and anatomical transection of both axons and connective tissue. The term “cryopreserved” refers to tissue that has been maintained at a temperature below' the freezing point of the tissue or medium in which it is stored. For example, the term “cryopreserved” may include tissue that has been stored at. ~2()°C, -40°C, or ~80°C or other suitable subzero temperature. Critically, the inventive xenogeneic nerye tissues are viable and live cell tissues that perform the same function as the human subject’s original nerve tissue. As used herein, the term “fresh” may refer to tissue that has been harvested and has not been frozen. The term “fresh” may include tissue that has been placed in a medium, e.g., a nutritive medium used to support cell viability in biological samples. The term “fresh” may include tissue that has been stored in a refrigerator, e.g., at 2 to 8°C or 4°C.

[000126] The xenogeneic nerve tissues of the present disclosure may include a combination of three primary components: a scaffold, cells, and signaling factors. Scaffolds can provide a temporary' structure necessary' for Schwann cell migration and axon outgrowth and are eventually replaced with host cells and extracellular matrix.

[000127] As shown in FIG. 1, the peripheral nervous system is composed of motor and sensory' neurons with their cell bodies in the spinal cord and long cytoplasmic extensions called axons, which signal with a distant target organ. Axons are grouped together in spatially arranged motor or sensory bundles called fascicles. Individual axons are surrounded by a connective tissue layer, the endoneurium, and fascicles are separated by perineurium. Motor or sensor)-’ fascicle patterns within major limb nerves are most prominent distally in a peripheral nerve where it is important, to correctly match fascicles during nerve repair for optimal regeneration. Groups of fascicles are contained within a peripheral nerve surrounded by a connective tissue layer called the epineurium. The internal epineurium separates fascicles and the external epineurium surrounds all the fascicles and defines the nerve anatomically. The epineurium is sutured in nerve repair and nerve grafting and comprises 50% of the total cross-sectional area of a peripheral nerve. External to this layer is the mesoneurium, containing the blood supply to the nerve. A fine network of capillaries exists at the endoneurial level. This fragile blood supply is easily disrupted due to trauma or tension at the nerve repair. Therefore, nerve grafts have better outcomes than direct repair under tension, due to devascularization of the nerve. According to the method of the present disclosure, a complete nerve or any part thereof can be used for xenotransplantation. Thus, the methods of the present disclosure provide the ability to use a complete, custom, or modular procedure, depending on the needs of the recipient. For example, a full nerve as shown in FIG. 1 may be used. In another example, an axon may be used. In another example, myelin and an axon may be used. In another example, endoneurium may be used. In another example, perineurium may be used. In another example, epineurium may be used.

[000128] Surprisingly, according to the present disclosure, the number and size of fascicles do not need to correlate between the xenotransplant donor and the human subject’s nerves. [000129] The live cell nerve tissue of the present disclosure vascularizes at the xenotransplantation site.

[000130] As shown in FIG. 2, axonotmesis from a crush mechanism is axonal injury' wiiere the connective tissue and nerve continuity remain intact. Wallerian degeneration ensues and slow axonal regeneration follows at a rate of lmm/day. Incomplete recover)- is common, depending on the distance for regeneration between the injury' and target tissue. Neurotmesis is complete physiological and anatomical transection of both axons and connective tissue. A neuroma may form but no spontaneous regeneration occurs without surgical intervention. Experiments are now indicating that it is the progressive failure of the neurons and Schwann cells to sustain axon regeneration over distance and time. Thus, a long-felt and unmet need in the field includes large gap peripheral nerye injuries.

[000131 j As shown in FIG. 3, conventionally, regeneration of large gap peripheral nerve injuries, even after surgical repair, has been slow and often incomplete. According to conventional treatments, less than half of patients who underwent nerve repair following an injury' regained adequate motor or sensory' function, and such deficits often resulted in complete limb paralysis or intractable neuropathic pain,

[000132] FIG. 4 show's the current state of the art/the unmet clinical need. Successful peripheral nerve regeneration involves improving the rate of nerve regeneration and the reinnervation of composite muscle leading to improved function. Existing treatment options include the use of autologous nerve transplants procured from a donor site from the same patient or deeellu!arized human cadaveric nerve allogeneic transplants. Both treatment, options have severe shortcomings and thus, there is a need for high-quality nerve transplants for large-gap (>4cm), segmental peripheral nerve defects. As explained in Grinsell et ah, human autografts are preferred as the literature is clear that autografting is superior to nerve conduits for longer gaps (>3 cm), more proximal injuries, and critical nerves. A single graft joins nerve gaps with a segment of a donor nerve of similar diameter. To span gaps between large diameter nerves, cable grafts are used, comprising multiple lengths of a smaller diameter donor nerve to approximate the diameter of the injured nerve.

[000133] FIG. 5 show's the FDA Approved Nerve Tubes for Peripheral Nerve Repair. Modified and Supplemented from FDA Medical Device Database (Kornfeld et al .). These conduits include autologous, biological, and nonbiological materials. Nerve Guide Conduits (NGCs) have been largely replaced with the use of decell ulari zed Peripheral Nerve Allograft (PNA), such as Avanee™ Such a product provides a surgical alternative and consists of human nerves that have been prepared via enzymatic decellularization, which retains the epineuriurn and fascicular architecture. PNAs have demonstrated improved functional recovery', but are indicated and FDA approved for the treatment of nerve gaps of 3cm or less (Whitlock, E. L. et al. Muscle & Nerve 39, (2009); Moore, A. M., (2011 ) Muscle & Nerve). The logistical and supply constraints of human tissue makes allograft material very' costly (Grinsell, et al.).

[000134] FIG. 6A schematically shows the experimental study design. Porcine sciatic nerve w'as trimmed to 4 cm and used to repair the complete transection of the radial nerve in 10 Rhesus Macaque recipients. In the contralateral arm, the transected, autologous radial nerve is rotated 180°and reimplanted as a control. FIG. 6B: Visualization of porcine sciatic nerve in situ during procurement surgery. Inset shows trimmed nerve (4 cm). FIG. 6C: Representative image of the reconstructed radial nerve from a limb treated with the xenogeneic transplant, photographed at necropsy (12-months postoperative).

[000135] FIG. 7A: Autologous treated limb at necropsy (12-months postoperative) demonstrating neuroma enlargement at anastomotic sites, expected as a result of the nerve repair surgery. FIG. 7B: Xenogeneic treated limb with similar presentation as seen in limbs treated with the autologous transplant. FIG, 7C: Representative histological images from sectioned regenerated nerve from autologous treated limb. Arrowheads indicate nerve bundles. FIG. 7D: Representative histological images from sectioned regenerated nerve from xenogeneic treated limb. Arrowheads indicate nerve bundles.

[000136] FIG. 8: PERV and porcine centromeric DNA confirm presence of porcine cells. Autografts and Xenografts were tested for the presence of PERV and porcine centromeric DNA to confirm the presence of porcine cells at the end of the experiment. 18s was utilized as an internal control to confirm the validity of the Q-PCR. Ct values were consistent with the presence of DNA and indicated no inhibition in the analysis.

[000137] FIG. 9A: Overall, white blood cell count (WBC) remained at target values for the entirety of the study. FIG. 9B: Composition of WBC populations remained within target proportional ranges for the entirety of the study. FIG. 9C: Total IgM and IgG titer levels did not exceed variations of greater than +/-50% of baseline levels for all recipients over the course of the entire study. Data not available at 12-months for recipients in Group 2. FIG. 9D: Anti-Porcine IgM and IgG responses increased the greatest at 1 -month postoperative and demonstrated a gradual decrease by 8 and 12-months, respectively. Anti-porcine IgG levels remained elevated compared to preoperative levels.

[000138] FIG. 10A: Wrist extension assessments were performed monthly to evaluate functional recovery' following repair of a surgically introduced large-gap (>4cm), segmental peripheral nerve defect. In this configuration, collateral limb movement is minimized and permits focused evaluation of extensor carpi radialis longus and extensor carpi radiaiis brevis muscles reinnervation as subjects attempt to grasp reward visibly placed on a shelf. LEFT^': Illustration of wrist extension measurement scale, divided evenly into three main categorical scores by assigning a numerical value of 1-3 for every 30° of wrist extension from neutral (inline with the forearm, 0°), defined as: angles less than 31° (Score 1), 31°-60° (Score 2), 61°-90° (Score 3), respectively. Wrist extension was video-recorded and freeze-frame analyzed monthly for each limb by two blinded observers. CENTER: An in situ example of a wrist extension measurement, receiving a score of 1 . RIGHT: An in situ example of a waist extension measurement, receiving a score of 2. FIG. 10B: Wrist assessment scores for ail subjects (mean), by month, autologous control nerve transplant as compared to experimental xenogeneic nerve transplant. By postoperative month four, subjects began demonstrating varying degrees of functional recovery. By the end of the respective observation periods, all ten limbs repaired with the autologous nerve transplant had demonstrated functional recovery values equal to baseline values, whereas seven limbs treated with the xenogeneic nerve transplant had recovered to pre-operative levels. In these successful cases, the rate of recover)_'’ appeared to be equivalent between the autologous and xenogeneic nerve transplants. (Preoperative values reflected in the graph corresponding to the xenogeneic transplants (blue) reflect the baseline values in the arm ultimately transplanted with the xenogeneic nerve during surgery.). *Mean and SD calculations w'ere performed using GraphPad Prism version 9.1.1 for Mac OS, GraphPad Software, San Diego, California USA,

[000139] FIG. 11: Recovery' of wrist extension function was qualitatively assessed from monthly video recordings by an analyst blinded to the transplant type, top image depicting severe impairment and lower image depicting no observable impairment.

[000140] FIG. 12: Magnitude of recovery¹ of wrist extension function is illustrated via a heat map. Month 0 indicates baseline values for all graphs and heat maps. Phase 1 and 2 are separated by a line indicating variation in tacrolimus treatment. Numbers in the boxes to the left indicate specific subjects and their respective genders. Numbers in the boxes to the left indicate specific subjects and their respective genders.

[000141] FIG, 13 A: Motor nerve conduction velocity showed partial recovery in all subjects, with the limbs treated with the xenogenic transplant exhibiting greater recovery. FIG. 13B: Sensory nerve conduction velocity values w'ere similar between limbs treated with xeno-or-allo nerve transplants and demonstrated only partial recovery' compared to preoperative levels. FIG. 13C: At 5-months, compound muscle action potential (CMAP) amplitudes remained below the critical firing threshold in all limbs. FIG. 13D: CMAP duration values were widely varied between subjects; however, mean CMAP durations for each transplant type returned to baseline by month 5.

[000142] FIG. 14A: Motor nerve conduction velocities increased from Phase 1 values and continued to increase between 8 and 12-months. FIG. 14B: Sensory nerve conduction velocities increased from Phase 1, but appeared to plateau at 8-months. FIG. 14C: Compound muscle action potential (CMAP) amplitudes increased from 5-months and continued to increase in magnitude from 8 to 12-months, approaching preoperative values, in all limbs regardless of transplant type. FIG. 14D: Negligible changes in CMAP duration values were observed between 8 and 12 months. [000143] FIG. 15 A: Histology sections stained by immunohistocbemistry for expression of neurofilament H. Arrowheads indicate axonal material. Representative images of native radial nerve at baseline and explanted nerve samples obtained at necropsy from the regenerated nerve in both autologous and xenogeneic transplanted limbs. FIG. 15B: Nerve bundle diameters of the regenerated nerve from both transplant types were smaller than the diameters of native nerve obtained perioperatively and qualitatively equivalent between the two transplant types. Higher scores indicate larger diameters. FIG. 15C: Luxol fast blue (LFB) staining was performed to assess the degree of myelination of regenerated nerve. Arrowheads indicate myelin. FIG. 15D; A reduction in the presence of myelin was observed in ail regenerated nerves compared to baseline levels, favoring limbs treated with the autologous transplant. Higher score indicates a greater presence of myelin. FIG. 15E; Necrosis and fibrosis were assessed using hematoxylin and eosin staining. Arrowheads indicate nerve bundles. FIG. 15F: Qualitative assessment of necrosis and fibrosis was equivalent, in regenerated nerve tissue from both types of transplants. A higher score indicates a greater degree of necrosis or fibrosis observed.

[000144] FIG. 16: Exposed radial nerve anatomy in non-human primate (rhesus monkey) at necropsy, postoperative month 12. Proximal surgical plane-oriented left in the photo showing the isolation of the radial nerve after reconstruction and 12 month recovery' following surgically introduced large-gap (>4cm), segmental peripheral nerve defect.

[000145] FIG. 17: Examination of the exposed resected radial nerve, proximal and distal anastomotic sites. Images taken at. necropsy at postoperative month 12. LEFT: Macroscopic view of autologous nerve transplant in situ showing neuroma formation at both the proximal and distal anastomotic site. RIGHT: Macroscopic view of the successful xenogeneic nerve transplant in situ showing neuroma formation at both the proximal and distal anastomotic site. Both reconstruction surgeries successfully achieved reinnervation and resulted in subject recovery of wrist extension function.

[000146] Different growth factors can be incorporated directly (in solution), into the tube’s lumen or through a delivery system. Because the effect of growth factors is often dose-dependent and requires their release over extended periods, delivery' systems are generally preferred (

P. Konofaos and J. P. ver Halen, “Nerve repair by means of tubulization: past, present, future,” Journal of Reconstructive Microsurgery, vol. 29, no. 3, pp. 149-164, 2013; Grinsell et ah). [000147] In one aspect, a single graft joins nerve gaps with a segment of a donor nerve of similar diameter. To span gaps between large diameter nerves, cable grafts may be used, comprising multiple lengths of a smaller diameter donor nerve to approximate the diameter of the injured nerve. Donor nerve grafts may be harvested from sensory' nerves including the sural and medial antebrachial. In some aspects, harvested nerve tissue is xenotranspl anted in a reverse orientation in the human subject. In some aspects, the present disclosure includes maximizing the number of axons successfully regenerating through the graft by funneling them distally. This prevents loss of regenerating axons down side branches of the donor nerve graft.

[000148] The subject invention provide a viable Schwann cells to promote axonal regeneration. Viable xenogeneic nerve transplants include living Schwann ceils and a matrix-rich scaffold, as well as offer the potential for greater clinical availability, thereby eliminating the necessity and comorbidity associated with an additional surgical procurement procedure.

[000149] The method of the present disclosure includes peripheral nerve regeneration equal to or greater than peripheral nerve regeneration effected by allograft nerve implantation; peripheral nerve regeneration at least 90% of peripheral nerve regeneration effected by autograft nerve implantation, peripheral nerve regeneration persists after withdrawal of immunosuppressant; peripheral nerve regeneration persists after withdrawal of immunosuppressant; wherein fastconducting nerve fibers are recovered and remyelinated at 8 months after the xenotransplantation, wherein motor conduction velocity' of the xenotranspl anted nerve product recovers to at least 90% of the subject’s healthy motor conduction velocity at 12 months after the xenotransplantation; wherein motor conduction velocity of the xenotransplanted nerve product is at least 50 m/s; w'herein restoration of at least 60% of the subject’s baseline wrist extension is observed within 4 months of the xenotransplantation; wherein restoration of at least 80% of the subject’s baseline wrist extension is observed within 6 months of the xenotransplantation; w'herein restoration of at least 90% of the subject’s baseline wrist extension is observed within 12 months of the xenotransplantation; wherein restoration of at least 60% of the subject’s baseline wrist extension is observed within 4 months of the xenotransplantation; wherein restoration of at least 80% of the subject’s baseline wrist extension is observed within 6 months of the xenotransplantation; wherein restoration of at least 90% of the subject’s baseline wrist extension is observed within 12 months of the xenotransplantation; wherein the compound muscle action potential (CMAP) amplitude of the xenograft is at least 90% of the CMAP amplitude of autograft; wherein the CMAP amplitude of the xenograft continues to increase after withdrawal of immunosuppressant; wherein the CMAP amplitude of the xenograft post transplantation is at least 50% of that of the subject’s baseline peripheral nerve; wherein the xenotransplantation of a nerve product exhibit restoration of CMAP amplitude of at least 50% of that of the subject’s baseline peripheral nerve; wherein radial motor nerve conduction velocity (NCV) of the xenograft is at least 90% that of the radial motor NVC of autograft; wherein radial motor NCV of the xenograft continues to increase after withdrawal of the immunosuppressants; wherein radial motor NCV of the xenograft post transplantation is at least 90% of a baseline peripheral nerve; wherein radial motor NCV of the xenograft post transplantation is at least 90% of a baseline peripheral nerve; wherein sensory? nerve conduction velocity (NCV) of the xenograft is at least 90% that of the sensory⁷ NVC of the subject’s baseline sensory⁷ NCV; wherein sensory' nerve conduction velocity (NCV) of the xenograft is at least 90% that of the sensory NVC of the subject’s baseline sensory NCV, wherein recovery of peripheral nerve is characterized by at least one of: 50% restoration of CMAP amplitude; 90% restoration of Motor Nerve NCV; and 90% restoration of wrist, extension, within 12 months of the xenotransplantation compared to the subject’s native peripheral nerve.

[000150] Nerve injury/regeneration may be evaluated non-invasively on anesthetized animals by testing the ability of the nerve to conduct an electrical impulse. The nerve was stimulated at one or more sites along its course, and the electrical response of the nerve was recorded. Locations for stimulation and recording were chosen relative to anatomical landmarks (e.g., elbow', spiral groove, axilla). For sensory? branches, the recording electrode was positioned directly over the distal branches of the nerve. For radial motor branches, the recording electrode was positioned over the belly of the Extensor Digitorum Communis muscle (EDC; innervated by the radial nerve). The recording location was at the junction of the upper third and middle third of the forearm. The Nerve Conduction Velocity (NOV) was expected to vary? across the length of the nerve with progressive recovery, and therefore stimulation was performed at four locations proximal and distal from the graft (e.g., lateral from the ulna or biceps tendon in the antecubital fossa; at the spiral groove; across the graft; and at the axilla between the coracobrachialis and the long head of the biceps).

[000151] The method of the present disclosure includes recovery of peripheral nerve is characterized by at least one of: 50% restoration of CMAP amplitude; 90% restoration of Motor Nerve NCV; and 90% restoration of wrist extension, within 12 months of the xenotransplantation compared to the subject’s native peripheral nerve.

[000152] The porcine donor is especially promising because it shares many physiological characteristics with human nerve. Porcine nerve tissue has great potential to address large gap peripheral nerve defects. As a result of the physiologic similarities with swine, the size, length, and architecture of motor and sensory nerves, especially the sciatic nerve, xenotransplantation nerve grafts according to the present disclosure provide are an excellent clinical alternative to the current standard of care. Retaining live cellular components beyond just the Extracellular Matrix (ECM), as is the case in PNAs or NGCs, is highly beneficial, with improved outcomes over PNAs. Retention of fibronectin, collagen, Giycosaminoglycans (GAGs) such as hyaluronic acid, and laminin will promote axonal regeneration akin to that observed with autologous grafts, without the aforementioned detractors. Examples of some nerve types that may be used include sciatic nerve, brachial plexus (radial nerve, median nerve, ulnar nerves), peroneal nerve (foot drop), femoral nerve, lateral femoral cutaneous nerve, spinal accessory nerve, or tibia! nerve.

[000153] The xenogeneic neural or nerve tissue of the present invention is configured for xenophagy in the human subject such that no xenogeneic ceils are detectable in the subject within 1, 2, 3, 4, 6, 8, 10, or 12 months of surgical graft into the human subject. As used herein, the term “xenophagy” refers to phagocytosis and autophagy of the xenogeneic neural or nerve tissue by cells in the human recipient, e.g., including Schwann cells. As demonstrated by the experimental results in the following Examples, it was unexpectedly found that after transplantation, the xenogeneic neural or nerve tissue of the present invention culminated in the complete, macrophage-mediated clearance of porcine cellular material and elimination of immunogenic porcine antigens. This was supported by the lack of detectable porcine DNA using Q-PCR in both the xenogeneic and autologous nerve tissue at necropsy. In addition, assessment of blood and tissue from the recipients indicated no circulating porcine cells or replication of PER V elements. [000154] EXAMPLES

[000155] Example 1 : Long-term immunological response to viable xenogeneic nerye transplants in non -hum an primates - a precfinicai study.

[000156] In this 12-month study, the safety and efficacy of viable, large-caliber, mixed modal xenogeneic nerve transplants derived from genetically engineered, designated pathogen-free porcine donors, was evaluated as a potential treatment in the reconstruction of large-gap (>4em), segmental peripheral nerve defects in non-human primates.

[000157] Methods/Sample Preparation: All xenogeneic nerve transplants used in this study were sourced from one genetically engineered a-l,3-galactosyitransferase knockout (GalT- KO), designated pathogen free (DPF) porcine donor. Five male and five female naive Rhesus Macaques (Macaca mulatta) served as xenogeneic nerve transplant recipients. Bilateral, 4 cm radial nerve defects were surgically introduced in ten Rhesus monkeys and repaired with an autologous transplant in one limb and a xenogeneic nerve transplant in the contralateral limb. Samples of nerve, spleen, liver, kidney, lung and heart were evaluated for various macro-and- microscopic histomorphological qualities. Subjects were iteratively assessed for anti-GalT-KO porcine IgG and IgM antibodies and the presence of porcine cells by quantitative PCR.

[000158] Method/Cryopreservation: Following procurement, nerve xenotransplants were prepared via standardized institutional protocol where the nerve was packaged in cryovials (Simport, T31G-1 A, Beloeil, QC) and cryoprotective media (5 mL, CryoStor CSS media, BioLife Solutions, Bothwell, WA) was added to each vial before it was sealed. Cryopreservation was achieved via a controlled rate, phase freezer at a rate of 1°C per minute to -40°C, and then rapidly cooled to a temperature of -8Q°C. Fresh nerve xenotransplants were stored in RPMI 1640 media and maintained at 4°C until use (24-48 hours).

[000159] Method/Surgical Procedures: The porcine donor was euthanized and prepared for surgery as previously described (Holzer, P. W. et ai. Immunological response in cynomolgus macaques to porcine a-1,3 galactosyltransferase knockout viable skin xenotransplants — A pre- clinica! study. Xenotransplantation (2020)). To isolate the sciatic nerve prior to harvesting, a linear incision was made midway between the sacrum and the ischium and extended ventral ly along the posterior aspect of the femur, longitudinally dissecting the gluteus medius, gluteus maxim us, piriformis, and biceps femoris muscles, to the proximal tibiofibular joint (FIGs. 6A-6C and 7A-7B). The sciatic nerve w^?as visualized and harvested by radial transections distal to the nerve origin and proximal to the bifurcation into the tihial and common peroneal nerves. This process was repeated on the bilateral side.

[000160] One unmodified sciatic nerve segment was stored in RPMI media and maintained at 4°C until surgical use 48 hours later, which was segmented into five xenogeneic nerve transplants, used as the source of the donor nerve to repair the radial nerve defect in 5 Rhesus Macaque recipients. The bilateral porcine sciatic nerve was cryopreserved per protocol, and stored at -80°C for a period of 7 days, after which it was thawed and used as the source of the donor nerve to repair the radial nerve defects in the remaining 5 Rhesus Macaque recipients. [000161] To avoid potential necrosis in the central portion of a nonvascularized, large- caliber nerve transplant, the porcine nerve transplants were selected during surgery from regions of the naturally tapered sciatic nerve, which closely matched the caliber and diameter of the proximal and distal radial nerve end in the nonhuman primate. Prior to transplantation, xenogeneic nerves were trimmed to 4 cm to fit the defect size.

[000162] Large-gap (> 4 cm) peripheral nerve defects were surgically introduced bilaterally in all ten Rhesus Macaque recipients. Recipients, under anesthesia, were positioned in lateral recumbency with the shoulder at 90° flexion, full internal rotation, and neutral abduction. The subcutaneous tissue and deep fascia w^?ere dissected for anatomical orientation. A 6-8 cm skin incision was made along the posterolateral margin of the proximal arm towards the antecubital fossa. This procedure exposed the long and lateral heads of the triceps, which converged to form the triceps aponeurosis. The intramuscular plane between the long and lateral head of the triceps was developed approximately 2.5 cm proximal to the apex of the aponeurosis, where the radial nerve and accompanying vessels were observed against the humerus in the radial groove. The surgical plane was extended proximally and dista!iy to minimize unintended injury. The radial nerve was distaily transected approximately 1 cm proximal to the origin of the deep branch. A 4 cm segment was removed to create the defect and saved for reattachment or subsequent analysis, [000163] Nerve transplants w^ere attached proximally and distaily with four to eight equidistant 8-0 nylon monofilament sutures at each neurorrhaphy site. The incision was then closed in layers using subcuticular, absorbable sutures. This process was performed bilaterally per each of the ten recipients; both xenogeneic and autologous neryes were transplanted in the same surgical procedure. Limb designation (right/left) for xenogeneic or autologous transplants was randomly assigned and blinded from observers for analysis. The ten recipients were randomly, evenly divided between two surgical series, one week apart. Five fresh xenogeneic transplants were used in the first series, and five thawed, previously frozen viable porcine xenogeneic transplants were used in the second.

[000164] The porcine sciatic nerve was selected as the source of the xenogeneic transplant due to its superstructural similarity to human and primate nerves (Zific, L. et a! ). The radial nerve was selected as the transplantation recipient site because there are minimal neighboring nerves. Those in close proximity may reinnervate downstream muscle fibers and complicate electrophysiology and functional analysis of the extensor digitalis muscles. Transplantation at the radial nerve also allowed for ethical loss of function and clearly articulated return of function in an observable and isolated movement. The maximum practical gap size possible was 4 cm based on the measured lengths of the recipients’ limbs. The mean distance from the recipients’ proximal neurorrhaphy site to the site of innervation of the extensor carpi radialis longus and extensor carpi radialis brevis muscles measured 15.7 ± 0.17 cm. Bilateral, 4 cm complete transections of radial nerves were surgically introduced in a total of ten Rhesus Macaque recipients. Two porcine sciatic nerves were harvested from one donor (FIG. 6A), trimmed into ten 4 cm segments (FIG. 6B), and transplanted into one of the radial nerve gaps in each recipient, randomized to the left or right limb. In the contralateral arm, excised Rhesus Macaque radial nerve segments were rotated 180°and reimplanted as a surgical control (FIG. 6A) (Grinsell et al.). Surgeries were performed synchronously, and the surgical personnel, sterile field, surgical technique, and uniformity of the transplant procedure were independently assessed for quality control at. each step.

[000165] Due to the large number of surgeries, five sciatic nerve segments were transplanted fresh one to two days after donor harvesting, while the remaining five were cryopreserved and subsequently thawed for transplantation over two days the w¾ek following. [000166] Method/Immunosuppression: Intramuscular injection of tacrolimus, at a dosage of 0.15 mg/kg/day, began 10 days before surgery and was continued until 6-months for subjects in Group 2 and 8-months for Group 1. Trough levels were maintained at 20 to 30 ng/niL. [000167] Method/Pathology: At the designated necropsy time point, the animals were sedated with Ketamine (10 - 15 mg/kg, IM). An IV catheter was placed and euthanasia performed by administration of Euthasof (> 50 mg/kg or to effect, IV). Explants of the entire autologous or xenogeneic transplant, including proximal and distal nerve, as well as samples of spleen, liver, kidney, lung, and heart were collected, fixed in 10% neutral buffered formalin, and transferred to 70% ethanol after approximately 72 hours. Nerve explants were trimmed longitudinally, routinely processed, and embedded in paraffin blocks. Resulting blocks were sectioned, and stained with either hematoxylin and eosin (H&E), Luxol Fast Blue (LFB), or immunohistochemically stained for neurofilament H (NF-H). Spleen, liver, kidney, and heart were trimmed, processed, embedded in paraffin, sectioned and stained with H&E. All tissues were evaluated in a manner blinded to treatment. Nerve explants were evaluated for morphologic changes and underwent semi -quantitative scoring according to the criteria in Table 1. All measurements of axon diameter were made by the pathologi st using an ocular micrometer. [000168] At necropsy, samples of the spleen, liver, kidney, and other organs w^rere collected. PERV copy number and expression were analyzed by Q-PCR to assess the presence of PER V DNA and mixed chimerism. Samples analyzed included xenogeneic and autologous nerve tissues harvested at 8- and 12-months postoperative, sera and PBMCs from the nine subjects obtained at various time points over the 12-month study, and spleen, kidney, liver, heart, and lung samples obtained at necropsy.

[000169] Method/Biodistribution: To confirm whether porcine or primate ti ssue was present, qualitative PCR using a primate-specific target gene demonstrated the presence of primate cells in both autologous and xenogeneic transplants (FIG. 7A). All samples were positive for either the internal positive control (sera) or the control reference genes indicating the validity of the analysis (data not shown).

[000170] Method/Histopathology: Explanted tissues were stained by immunohistochemistry for expression of Neurofilament H to demonstrate axons (FIG. 15 A). Luxol Fast Blue staining was used to demonstrate myelination levels of the various regions of the explant (Fig. 15C).

[000171] Method/Electrophysiology: Nerve conduction studies (NCS) or neurography evaluations are non-invasive electrodiagnostic techniques used commonly for functional tests of the peripheral nervous system. Nerve injury or regeneration is evaluated by testing the ability of the nerve to conduct an electrical impulse. The technique involves recording electrical activity at a distance from the site where a propagating action potential is induced in a peripheral nerve.

The nerve is stimulated at one or more sites along its course, and the electrical response of the nerve is recorded using the same instrument. [000172] During ail recording sessions animals were lightly anesthetized by facility staff, per Biomere SOPs, and placed on a temperature-controlled warm pad. Animals were sedated with Ketamine (10 - 15 mg/kg) or Teiazol (5-10 mg/kg, IM) or Ketamine and Dexdomitor (~7.5 and 0.02 mg/kg mixture, IM. The anesthesia provided adequate sedation for handling while preserving peripheral evoked responses. Body temperature was recorded and included in Biomere records.

[000173] All recordings were performed at Biomere (Worcester, MA). All signal collections and post-collection data analyses were performed with a vendor-calibrated and certified clinical electromyography (EMG) machine (Natus Neurology System with Synergy software), per Predinical Electrophysiology Consulting SOPs. Subdermal platinum needle recording electrodes were used for recording (e.g., 0.703, 30-mm, 22G3, 1.25 in). ANatus pediatric stimulator was used for stimulation in order to maintain consistent distance between the cathode and the anode.

[000174] Signal measurement marks placed automatically by the software were manually verified post-collection. Onset latency was measured from the stimulus artifact to the initiation of the depolarization to the nearest 0.01 ms, amplitude was measured from baseline to the peak of the depolarization to the nearest 0.01 mU for sensory responses, and to the nearest 0.01 mV for motor responses. Group summaries were calculated with MSExcel. Differences in measures across groups were compared using Student's t-Test.

[000175] Nerve conduction was assessed for the radial motor and radial sensory branches. These measurements detected recovery of function at locations along the nerve. Fwvave responses were tested at the last timepoint. All recording procedures were adapted from routinely used neurological clinical protocols,

[000176] Locations for stimulation and recording w^?ere located relative to anatomical landmarks (e.g., elbow, spiral groove, axilla). An additional stimulation was elicited directly over the graft. Alternate locations along the nerve w^?ere sought where responses were not present, and the distance from the muscle recorded. Orthodromic compound muscle action potentials were elicited in the radial motor nerve and antidromic sensory nerve action potentials were elicited in the radial sensory_' nerve. Orthodromic and antidromic stimulations were relative to the physiological conduction in the respective nerves, per clinical conventions. [000177] For sensory branches, the recording electrode was positioned directly over the distal branches of the nerve and sensory nerye action potentials (SNAPs) were elicited. For radial motor branches the recording electrode was positioned over the belly of the extensor digitoruni communis muscle (EDC; innervated by the radial nerve) in a belly-tendon montage, and compound muscle action potentials (CMAPs) were elicited. The recording location was at. the junction of the upper third and middle third of the forearm.

[000178] At least two stimulation locations were used for accurate assessment of motor nerve conduction, in order to account for delays introduced by the neuromuscular junction. In this study the NCV was expected to vary across the length of the nerve with progressive recovery and therefore stimulation was performed with a pediatric stimulator at four locations proximal and distal from the graft (e.g., lateral from the ulna or biceps tendon in the antecubital fossa; at the spiral groove; across the graft; and at the axilla between the coracobrachialis and the long head of the biceps). The nerve conduction velocities w⁷ere averaged across the entire length of the nerve. Slightly different Left vs Right measurements are common.

[000179] The motor conduction velocity for each segment was calculated using the differences in onset latency and distance between each two points of stimulation along the radial nerve following supramaximal stimulation. The amplitude of the CMAP was determined at the peak of the response following supramaximal stimulation of the associated nerve. Segmental conduction velocities across the radial nerve were averaged.

[000180] For sensory stimulation, the stimulus strength was progressively increased until a response was evoked, and then increased further until a supramaximal response was elicited. Approximately 10 supramaximal stimuli w⁷ere averaged for each sensory nerve. Sensory recordings were performed directly over the radial sensory nerve as it passes over the extensor pollieis iongus tendon. Stimulation was performed over the radial side of the forearm, approximately 5 cm proximal to the recording site.

[000181] The distance from the recording site to the stimulation cathode was entered in the instrument during collections, for each site, and the conduction velocity was calculated by preset software protocols using the onset latency of the response and the distance (for sensor)-’ NCV) or the distance difference (for motor NCV calculations).

[000182] F-waves were elicited at the elbow⁷, distal from the graft. F-waves are late responses attributed to the antidromic activation of anterior horn motor neurons following peripheral nerve stimulation, which then results in orthodromic impulses returning along the involved motor axons (also known as ‘'backfiring of axons”). F -waves test motor conduction over long neuronal pathways including the proximal spinal segments and the nerve roots. The latency and amplitude of F-waves are known to vary and multiple stimulations (8-10) were performed to ensure a response. F-wave responses under 20 ms indicated the presence of motor conduction over long neuronal pathways.

[000183] The NCV measured in NCS studies is the velocity of the fastest fibers present in the nerve bundle tested. Decreased conduction velocity is assumed due to both axonotmesis (axonal loss) and neurapraxia (conduction block). Conduction velocity slowing alone, without conduction block, does not necessarily produce clinical weakness if sufficient motor units remain innervated. The presence of nerve conduction does not necessarily indicate fully functional muscle innervation. Uneven conduction within the nerve may indicate localized areas of demyelination, remyelination with immature myelin, loss of fibers, or connective tissue blockages.

[000184] Method/Functional Evaluation: A previously reported radial nerve injury model (Wang, D. et al. A simple model of radial nerve injury in the rhesus monkey to evaluate peripheral nerve repair. Neural Regeneration Research 9, 1041. ISSN: 1673-5374. (2014)) w¾s adapted to assess the functional recovery of xenogeneic and autologous nerve transplant recipients. Radial nerve injur)_'’ proximal to the el bow results in a loss of waist extension function, or “wrist drop,” loss of forearm muscle tonality, and digital extension due to motor denervation of the extensor carpi radial is longus and extensor carpi radialis brevis muscles (Injur)_'’ of Radial Nerve: Causes, Symptoms & Diagnosis Healthline. (2021); Gragossian, A. & Varacallo, M. in StatPearls (StatPearls Publishing, Treasure Island (FL), 2021)). Radial nerve functional assessments w¾re performed monthly for each recipient and included chair and cage-side observations of active and passive wrist angle flexion during the recipient’s retrieval of objects requiring wrist angle extension to obtain them. A series of wrist extension and gripping attempts by each recipient were video recorded, for each isolated arm, for each month. Observations were performed using food treats or mechanical stimulation to encourage wrist extension and gripping. This resulted in 16:13:33 hours of data, for 18 limbs of nine Rhesus Macaque transplant recipients. Over the entire study period, a combined 2,057 total events w¾re recorded. Results were analyzed by two independent investigators in a blinded manner with respect to the transplant type and location.

[000185] Qualitative regain of radial nerve functionality was monitored according to the following categorical scale: no observable impairment, mild impairment, moderate impairment, and severe impairment (FIG. 11).

[000186] Method/Immunogenicity: Total serum IgM and IgG (FIG. 9D) were measured using a commercial ELISA, and overall median and IQR were found in all nine animals included in the study. Binding of the xenoreactive antibody to pig ceils was measured as previously described i Mohan na, P. N., Young, R. C., Wiberg, M. & Terenghi, G. A composite poly- hydroxybutyrate-glial growth factor conduit for long nerve gap repairs. I Anat 203, 553-565. ISSN: 0021-8782 (Dec. 2003)). Cryopreserved genetically modified a-l,3-galactosyltransferase knockout (GalT-KO) porcine PBMCs were thawed, and cell concentration was determined using Coulter MD II (Coulter Corporation, Miami, FL). Cells were diluted to a concentration of 1.5 x 106 cells/mL in FACS buffer (IX Hanks" Balanced Salt Solution (BBSS) with calcium and magnesium, 0.1% BSA, and 0.1% sodium azide). Decomplementation of the serum samples was carried out by heat inactivation for 30 min at 56 °C and diluted at 1:2, 10, 100, 1,000, and 10,000 ratios using FACS buffer. 100 mΐ. of the ceils w'ere added into each well in 96-well u-bottom plate with 10 pL of the diluted serum samples and incubated for 30 min at 4 °C. Cells were washed one time using 200 pL FACS buffer. To prevent nonspecific binding, cells vrere incubated in 100 pL 10% goat serum for 10 min at room temperature, followed by one more additional washing. Cells w'ere stained with goat anti -human IgG PE and goat anti -human IgM FITC (Jackson ImmunoResearch Laboratories Inc., West Grove, PA) for 30 min at 4 °C. Cells were washed two times using FACS buffer and resuspended in 200 pL 0.5% PFA in MACS/ IX PBS buffer. Flow' cytometric analysis completed on Novocyte flow⁷ cytometer (ACEA Biosciences, Inc. San Diego, CA). Flow cytometry data was analyzed using NovoExpress 1.3.0 (ACEA Biosciences, Inc.).

[000187] Binding of IgM and IgG was assessed using relative mean fluorescence intensity (MFI): Relative Actual MFI value/MFI was obtained using a secondary' antibody in the absence of serum. IgG and IgM ELISA kits from Life Diagnostics were used for the quantification of total circulating IgG and IgM in Rhesus serum by following the manufacturer’s instructions. [000188] Binding of anti-porcine IgM and IgG was assessed using Median Fluorescence Intensity (MFI) and relative MFI was obtained as follows: Relative MFI = Actual MFI value / Limit of Blank (MFI obtained using secondary antibody only in the absence of serum).

[000189] Method/PCR: Twenty milligrams of the xenogeneic porcine tissue samples and 7 mg of autologous primate tissue samples were treated with the DNeasy Blood and Tissue Kit (Qiagen, Crawley, UK) as described by the manufacturer that included the RNase A-treatment step. The isolated DNA was quantified by UV spectrophotometry. Quantitative PCR amplification of 18S (Eurogentec, Seraing, Belgium) was carried out to assess the DNA homogeneity across samples. Serum samples were processed using the Viral RNA mini kit (Qiagen, Crawley, UK) as described by the manufacturer incorporating the DNA digestion step using DNase I to isolate viral RNA. Samples were then processed using the RNeasy MinElute Cleanup kit (Qiagen, Crawley, UK). All Serum samples were shown to have an IPC CT <32 progressed to PERV transcription analysis. PMBC samples were processed for DNA isolation using a modified version of the manufacturers ‘‘Whole Blood'’ protocol for the Centra Puregene Blood kit (Qiagen, Crawley, UK). The modified protocol involved homogenizing the PBMC samples prior to RNase A treatment, protein precipitation and, finally, isopropanol and 70% ethanol washes were added before DNA hydration. The DNA product was quantified using UV spectrophotometry and IBS amplification earned out to assess DNA homogeneity between samples while using 200 ng/reaction. PBMC samples were shown to have an 188 CT <27 progressed to PERV copy number and mixed chimerism analysis. At necropsy, tissue samples of the kidney, liver, lung, and spleen were harvested. RNA isolation was conducted on 35 mg of the tissue samples using the RNeasy mini kit (Qiagen, Crawley, UK) with homogenization using a Fast-Prep 24 (MP Biomedicals, Eschwege, Germany), The RNA product was quantified using UV spectrophotometry and IBS amplification w¾s carried out to assess DNA homogeneity between samples while using 200 ng/reaction. Tissue samples were shown to have an IBS CT <13 progressed to amplification, reverse transcription, and PERV copy number and mixed chimerism analysis.

[000190] Amplification was carried out using an Applied Biosystems Vii A 7 Real-Time PCR System with a polymerase activation step (10 min at 95 °C) and 40 amplification cycles of 15 sec at 95 °C, 30 sec at 53 °C, and 30 sec at 60 °C. All primate and porcine transplantation site samples shown to have an 18S CT <29 progressed to PERV copy number and mixed chimerism analysis. PERV genome copy number quantification and mixed chimerism was assessed by quantitative PCR (Q-PCR) using the QuantiTect virus kit (Qiagen, Crawley, UK), with identical cycling conditions described above. PERV was assessed using TaqMan primers specific to the PERV-pol gene as previously described (Scobie, L. et al. Long-Term IgG Response to Porcine NeuSGc Antigens without Transmission of PERV in Burn Patients Treated with Porcine Skin Xenografts. The Journal of Immunology 191, 2907-2915. ISSN: 0022-1767, 1550-6606). [000191] PERV quantification was carried out by comparison to standards of known PERV copy numbers. The limit of quantification (LQQ) for PERV using this assay is ten copies per reaction. Mixed chimerism was assessed using TaqMan primers for porcine centromeric DNA that were also used in the study described (Scobie, L. et a!. Long-Term IgG Response to Porcine NeuSGc Antigens without Transmission of PERV in Bum Patients Treated with Porcine Skin Xenografts. The Journal of Immunology 191, 2907-2915. ISSN: 0022-1767, 1550-6606.). Detection of porcine cells was quantified by comparison to standards of known porcine cell content. The LQQ for porcine cells using this assay is 0.026 cells per reaction,

[000192] Qualitative PCR for the reference gene RPL13A (ribosomal protein gene) w⁷as carried out to confirm that DNA was suitable for amplification. Fifty nanograms of DNA and 0.5 mM primers in a total volume of 25 id were cycled under the following conditions; 5 min at 94 °C, 50x (10 sec at 94 °C; 15 sec at 58 °C; 15 sec at 72 °C) 10 min at 72 °C. This PCR will detect both primate and porcine material.

[000193] Detection of primate-specific DNA was carried out using qualitative PCR as described previously (Tripathi, V. & Obermann, W. M. J. A Primate-specific Extra Domain in the Molecular Chaperone Hsp90. PLOS ONE 8. Publisher: Public Library of Science, e71856. ISSN: 1932-6203 (Aug. 9, 2013)) with modifications. Using the Taq PCR core kit (Qiagen, Surrey, UK) 75 ng of DNA and 0.2 pM primers in a total volume of 50 mΐ w¾s cycled under the following conditions; 94 °C for 5 min, 40x(3Q sec at 94 °C, 30 sec at 5 °C, and 60 sec at 72 °C), 10 min at 72 °C. PCR analysis was preferred for specificity wiiich is not seen by the use of antibodies to nerve components.

[000194] Method/Hematology and Clinical Chemistry: Serum chemistry blood samples were processed for serum or transferred to the Biomere Testing Facility laboratory and processed. Whole blood hematology samples were transferred to the Testing Facility laboratory without processing. Whole blood was analyzed on an IDEXX Procyte analyzer for erythrocyte count, hemoglobin, hematocrit, platelet count, leukocyte count, reticulocyte count, and mean corpuscular volume, hemoglobin and hemoglobin concentration. Serum samples were analyzed using an IDEXX Catalyst analyzer (Cbeml5, Lyte4, Trig, and AST slides for A/G ratio, alanine aminotransferase, albumin, alkaline phosphatase, aspartate aminotransferase, calcium, chloride, cholesterol, creatinine, gamma-glutamyl transferase, globulin (by calculation), glucose, inorganic phosphate, potassium, sodium, total bilirubin, total protein, triglycerides, and urea nitrogen. [000195] Clinical Outcomes: All ten subjects tolerated the surgical procedure resulting in the complete loss of radial nerve function bilaterally (FIGs. 6A-6C). Nineteen of the 20 surgical transplant procedures were successful. In one subject, histomorphologieal analysis at necropsy revealed a large neuroma proximal to the transplantation site as well as the lack of axonal continuity through and distal to the transplant indicating failure to maintain coaptation at the proximal anastomotic site. Thus, all functional, electrophysiol ogi cal, and morphological data for both of the transplant types from this subject were removed from nerve analyses, but clinical data were retained to assess immunological and toxicological outcomes.

[000196] Over the entire course of the study, no adverse events, negative veterinary' observations, or other deleterious systemic effects attributable to the xenotransplant were observed in any subject. At necropsy, there were no abnormal findings during inspection of internal organs and other tissues in any recipient, regardless of tacrolimus regimen. Hematology and chemistry analysis were within normal ranges (Chunasuwankul, R. et al. int Surg 87(2002)). Red blood cell (RBC), platelet (PLT) counts, hemoglobin (HGB), mean corpuscular volume (MCV), hematocrit (HCT), urea nitrogen (BUN), creatinine (CREA), and electrolyte levels were unremarkable for all subjects.

[000197] In Phase 1, during which all ten subjects received 0.15 mg/kg/day of oral tacrolimus for the first six months of the study, no serious adverse side effects, such as diarrhea, cachexia, or other effects were observed in any of the ten subjects. Postoperative trough tacrolimus blood levels for the first six months were targeted to between 20 to 30 ng/mL and varied between individual recipients (4.9 to 32.2 ng/mL).

[000198] In Phase 2, during which five subjects continued the regimen (Group 1), and five subjects ceased tacrolimus treatment (Group 2), gradual weight increase was observed in Group 2 recipients, and all survived without incident to the 12 -month end of study. [000199] However, subjects in Group 1 presented with progressing symptoms associated with tacrolimus toxicity, such as limited mobility in knee joints, muscle rigidity, stiffness, and atrophy, as well as significant weight loss. As a result of the tacro!imus-associated toxicity, at 8- months, the five subjects in Group 1 were euthanized.

[000200] Functional Evaluation: Following surgery, complete loss of functional wrist extension was observed bilaterally in all ten subjects for approximately three months regardless of nerve transplant type used. The distance from the proximal neurorrhaphy site to the site of innervation of the extensor carpi radialis longus and extensor carpi radial is brevis muscles measured 16.0cm ± 0.56 (Table 2). At a rate of axonal regeneration of Imm/day, functional recovery' was anticipated at. POD- 160.

[000201] By 4-months postoperative, six of ten xenogeneic transplants and all autologous began demonstrating varying degrees of functional recovery. By the end of the observation periods (8- and 12-months, respectively), all ten limbs repaired with the autologous nerve transplant demonstrated functional recovery values equal to baseline values, whereas seven limbs treated with the xenogeneic nerve transplant had recovered to preoperative levels. In the three nonresponders, two xenogeneic nerves were fresh, and one was cryopreserved.

[000202] In the 17 successful cases, the rate of recovery averaged across the subjects appeared to be equivalent between the two nerve types (FIG. 10B), while the magnitude of recovery' was greatest in limbs treated with autologous nerve transplants.

[000203] During Phase 1, the rate of functional recovery qualitatively appeared to be slower in the xenotranspl anted limbs (FIGs. 11-12). However, at the respective endpoints in Phase 2, there was no qualitative difference in the overall magnitude of functional recovery between limbs treated with a xenogeneic nerve transplant and the autologous control (FIG. 12). In addition, this recovery in phase 2 was regardless of whether the porcine nerve had been grafted fresh or frozen.

[000204] Electrophysiology: For all 18 limbs, preoperative median motor nerve conduction velocity was 64.26 m/s with an interquartile range (IQR) of 0.66 (FIG. 13 A), and median sensory' nerve conduction velocity was 53.72 m/s (IQR = 0.11) (FIG. 13B).

[000205] In Phase 1, at the first postoperative assessment (5-months), there was an overall reduction in median nerve conduction velocities to 36.50 m/s (IQR = 12.17) for autologous treated limbs, and 50.33 m/s (IQR = 21.16) for xenogeneic treated limbs. In sensory nerve, conduction velocity dropped to 25.00 m/s (IQR ^::: 13.50) in limbs with the autologous control, and 22.00 m/s (IQR = 4.50) with the xenogeneic transplant.

[000206] In Phase 2, at 8-months postoperative, median motor nerve conduction velocity increased to 56.33 m/s (IQR = 13.01) autologous, and 57.00 m/s (IQR = 11.83) xenogeneic, indicating partial remyelination of fast-conducting fibers had occurred (FIG. 14A). However, median sensory nerve conduction velocity did not return to preoperative baseline levels, only reaching 27.00 m/s (IQR = 12.5) autologous, and 27.00 m/s (IQR = 7.5) xenogeneic (FIG. 14B). [000207] At 12-months postoperative, the remaining four recipients in Group 2 demonstrated an increase in median motor nerve velocity' to 62.17 m/s (IQR = 8.76) and 63.34 m/s (IQR = 4.33) autologous and xenogeneic treated limbs, respectively (FIG. 14 A). Sensory nerve conduction velocity remained stable at 25.50 m/s (IQR = 4.00) for the autologous treatment group and 27.5 m/s (IQR = 3.00) in the xenogeneic experimental group (FIG. 14B). [000208] Median preoperative CMAP amplitudes for all 18 limbs w'as 20.03 mV (IQR = 4.54). At 5-months, a nearly complete loss of action potential was observed in all limbs: 1.93 mV (IQR = 1.41) autologous, and 2.05 mV (IQR ^::: 2,08) xenogeneic, levels at which nerves would be unable to reach threshold firing levels (FIG. 13C). At 8-months, CMAP amplitudes for the autologous nerve transplants had recovered to 9.20 mV (IQR ^:::: 4.27), compared to a CMAP of 7.30 mV (IQR of 6.13) xenogeneic (FIG. 14C). However, at 12-months, median CMAP amplitude magnitudes increased for both types of transplants, 15.44 mV (IQR = 4.64) for autologous, and 14.15 (IQR = 4.83) for xenogeneic.

[000209] Median preoperative CMAP duration values for all 18 limbs were 3.98 mV (IQR = 0.12) (FIG. 13D). At 5-months, median durations were measured at 3.14 mV (IQR = 3.27) for autologous treated limbs, and 5.12 mV (IQR = 2.66) for limbs treated with the xenogeneic nerve transplant. Over the course of the study, however, maximum recovery was observed at 8-months postoperative, 4.81 mV (IQR = 1.71) autologous; 4.62 mV (IQR = 1.99), xenogeneic (FIG.

14D). No major qualitative differences were observed in xenogeneic transplant vs autograft electrophysiology .

[000210] Imimmogenicity: Across both Phase 1 and Phase 2, white blood cell count (WBC) and individual component percentages remained within normal ranges (Koo et al; Chen et al) (FIGs. 9A-B). Neutrophil and lymphocyte percentages varied month-to-month, but absolute counts remained close to expected values (2.40 ± 6.18 K/pL and 2.46 ± 8.94 K/pL, respectively).

[000211] Total IgM levels were slightly elevated above preoperative levels at one or more postoperative time points in all recipients (FIG. 9C). The highest level of total IgM and IgG were observed 1 -month postoperative. Overall, changes detected in total serum IgM and IgG levels did not vary' more than 50% from baseline levels for each individual recipient in Groups 1 and 2 and remained stable over the course of the 12-month study.

[000212] Anti-porcine IgM and IgG levels showed an increase above preoperative levels following transplantation, followed by a gradual decrease to baseline values over time (FIG. 9D), The highest IgM fold increases were detected for all recipients at one week (0.25 months) and 3- months, and the highest IgG fold increases were detected for ail recipients at 1 and 3 -months. [000213] Histology: At necropsy, continuous resections of the nerve transplant including proximal and distal native nerve surgical beyond the neurorrhaphy site, were procured, and sectioned longitudinally via microtome to 5pm thickness and fixed in 10% NBF for histological analysis. Samples were stained with hematoxylin and eosin, Luxol Fast Blue, and NF200. Blinded evaluations were performed for the parameters in and the semi -quantitative scoring schema in Table 1.

[000214] At necropsy, neuromas of varying degree were observed at the proximal and distal anastomotic sites for both types of nerve transplants (FIGs. 16-17). Microscopic examination at these sites with H&E staining revealed fibrous tissue proliferation with variable inflammation, generally consisting of foreign body reaction around the sutures, as well as multidirectional proliferation of small diameter nerve branches consistent with neuroma formation. Mild fibrosis, with embedded nerve fibers and neurofibrils generally coursing longitudinally, was observed across the original defect site with fibrin deposits at the sites of anastomosis.

[000215] At the 8-month end point, the size of the nerve fibers across the defect site for all of the five subjects were comparable for both nerve transplants, ranging from 100 to 300pm, whereas when measured peri operatively, autologous nerve radius exceeded 300mhi. At the end of study for all ten subjects, xenogeneic axon diameter [2.50mih ± 0.40] was smaller than that of the autologous control [3.40mih ± 0.55], but neither fully recovered to the perioperative axonal diameter of the native radial nerve [4.00mih ± 0.00], (Table 3 and Table 4).

Table 3. Histopathology evaluated the listed parameters for the proximal and distal endogenous nerves and the nerve transplants.

Table 4. Histopathology Evaluation Axon Diameter

[000217] Data comparisons between autologous and xenogeneic nerve transplant sites, unless otherwise stated, are expressed as mean ± SD per group. Statistical comparisons were performed as one-way analysis of variance tests with the Student-Newman-Keuls multiple comparisons method. Statistical analyses were performed in Prism Graph Pad version 9.1.0 software (Prism, San Diego, CA USA), P values less than 0.05 were considered statistically significant.

[000218] Blinded histological analysis found no meaningful differences between nerve tissue excised from transplantation sites in limbs treated with either autologous or xenogeneic transplants. Nerve bundle diameter for perioperative explanted nerves not used for transplantation were >300 pm. For Group 1, the diameter of the regenerated nerve bundles across the defect site for all five recipients w¾s comparable for both types of nerve transplants, ranging from 100 to 300 pm. At the end of study for Group 2, xenogeneic nerve bundle diameters appeared smaller than those of the autologous control, with four autologous controls reaching preoperative diameters (FIGs. I5A-15B).

[000219] Overall, for all recipients and transplant types, full mye!ination in the nerve regions immediately proximal to the transplanted sites was observed, with discernible loss of myelin in regions distal to the transplants. For xenogeneic transplants, little to no myelination was observed in subjects from either group. Overall, autologous transplants appeared to result in regenerated nerves with a greater presence of myelination. (FIGs. 15C-15D). [000220] The degree of cell death (necrosis) and presence of fihrotic tissue (fibrosis) were comparable between tissue from autologous and xenogeneic transplant sites (FIGS. 15E-15F). Enlargement of the nerves was observed at the proximal and distal anastomotic sites for both types of transplants in all recipients (FIGs. 7A-7B). In the transplanted regions, there was mild fibrosis with embedded nerve fibers coursing mostly longitudinally along the long axis of the transplant. The extent of the fibrous tissue present w¾s consistent with the damage incurred as a result of the surgical procedure. Microscopic examination demonstrated foreign body reaction around the sutures, as well as multidirectional proliferation of small diameter nerve branches causing minor neuroma formation on all transplants, smaller in scale than that of the one surgical transplant failure.

[000221] There was a notable difference in the infiltration of inflammatory cells at the transplant sites between xeno-and-allo reconstructed limbs. At 8-months, the overall inflammation for the autologous treated limbs was observed to be minimal with scattered lymphocytes and macrophages (FIG. 7C), In contrast, the xenogeneic transplantation sites had a greater degree of inflammation consisting of lymphocytes and macrophages (FIG. 7D).

Similarly, by the 12-month time point, the xenogeneic treatment group showed more lymphocytes, macrophages, and inflammation than the autologous treatment group. Also, there were distinct tertiary lymphoid follicles scattered throughout the nerve sample (FIGs. 7E-F). [000222] Biodistribution of Porcine Tissue: Chinierism and PERV copy number and expression were analyzed to assess the presence of porcine cells by both conventional and Q- PCR. Samples analyzed included xenogeneic and autologous nerve tissues harvested at. 8- and 12-months postoperative, sera and PBMCs from the ten subjects obtained at various time points over the 12-month study, and spleen, kidney, liver, heart, and lung samples obtained at necropsy. Recipient PBMCs, sera, and tissues tested negative for PERV RNA and/or DNA amplification or microchirnerisrn, indicating that there was no evidence of microcbimerism or circulating porcine cells in any of the tissues/cells analyzed. Sera was also found negative for PERV RNA expression indicating that no active replication appeared to be taking place. All samples were positive for either the internal positive control (sera) or the control 18s housekeeping gene, indicating the validity of the analysis (FIG. 8).

[000223] The autologous nerve grafts lacked the presence of PERV or porcine cells. Surprisingly, the xenogeneic sites w^ere also negative for the presence of porcine cells by Q-PCR, indicating that there was no residual porcine tissue in the xenogeneic nerve tissue tested. Q-PCR for the porcine centromere was also negative (Fig. 8). However, the use of a primate-specific primer set using conventional PCR demonstrated the presence of primate cells in both autologous and xenogeneic transplants (FIG. 7G).

[000224] Discussion: Physical guidance and Sdrwann cell activity are vital components of nerve repair, and optimal axonal regeneration depends on growth in a conducive and complex biological environment. Thus, the therapeutic capacity of any nerve conduit, especially one sourced from a foreign species, is correlated to its ability to mirror the physiological conditions of the host environment.

[000225] Porcine nerves are well suited for this task, as many essentia! properties are conserved, closely resembling the neuroanatomy of humans. Characterization studies reveal length, number, pattern, and fascicular area to be comparable. This is highly favorable as endoneurial alignment and matched fascicular cross-sectional areas improve clinical outcomes. The architecture, composition, and distribution of essential components of the porcine ECM, such as collagen, laminin, fibronectin, and GAGs such as hyaluronic acid and chondroitin-4- sulfate are also highly similar, resulting in clinically advantageous structural integrity. Young’s modulus, and tensile strengths (Sunderland S; Burrell et al.). These characteristics allow^? for enhanced reliability of sutures to maintain stable coaptation under tension. Location and quantities of Sch wann cells distributed throughout the perineurium and endoneurium of porcine peripheral nerves closely mirror that of human nerves.

[000226] The regrown peripheral nerve in limbs treated with the xenogeneic transplant w⁷as capable of reinnervation of the extensor digitalis, allowing functional recovery and similar electrophysiological and morphological outcomes as the autologous control. Recovery of compound muscle action potentials and conduction velocities indicate adequately myelinated, fast-conducting fibers were restored, indicating a successful mixed-modal repair which would be expected to improve over time (Grinsell et al.). The neuro-regenerative capacity observed in this study demonstrates that the xenogeneic transplant was able to sufficiently replicate favorable regenerative conditions, including the presence and activity of Schwarm cells, and other repair mechanisms, despite its foreign origin.

[000227] The surgeries required to introduce and repair the 20 bilateral injuries required 10 surgical days in total, in addition to the surgical procurement of the porcine sciatic nerve. Over this time, uncertainty regarding the viability of the fresh nerve tissue over a two-week period w^?as an inherent challenge to an experimental design involving 10 subjects. The use of fresh nerve material procured from multiple source animals would introduce donor-to-donor variability and present an ethical conflict regarding the use and welfare of research animals. Cryopreservation is a well-characterized process that is recognized to preserve cell viability, basal lamina, and endoneurial structures, and its acceptable impact to tissue viability is supported in the literature. As a result, we chose to cryopreserve the materials intended for use in 5 subjects to provide the greatest achievable standardization given these limitations, as well as additional time necessary' to maintain quality and consistency with respect to surgical personnel, techniques, and conditions.

Tacrolimus is the most widely used immunosuppressive in nerve allotransplantation, and use of tacrolimus in this study w'as due to its reported positive impact on nerve regeneration, as well as mitigation of immunological rejection of the xenogeneic transplant. However, conversion of human clinical regimens for use in non-human primates was complicated by the variability of published protocols reported from other investigators. In consultation with veterinarians,

IACUC, and other investigators familiar with the use of tacrolimus and research involving nonhuman primates, the regimen used in this study was believed to be appropriate and scientifically justified. The presence of tertiary' lymphoid nodules at the conclusion of the study, located on the regenerated nerves in xenogeneic-treated limbs, as well as the post-operative presence of non- Gal antibodies as a result of a localized immune response to the GalT-KO porcine nerve transplant. The magnitude of the immune response; however, did not result in symptoms or signs related to graft rejection, attributed in part to the concomitant use of tacrolimus.

[000228] The data generated post the month 8 time point has been considered not relevant by the authors, due to the afore mention complications with the data that would have impacted the study’s statistical power. As a result, the qualitative histopathological analysis is hindered by the lack of quantitative, objective metrics, and could be improved in future studies with nerve stereoiogy and morphometry' for quantification and immunohistochemistry for specific antigens. [000229] Finally, the cellular components of the 4-cm porcine transplant w'ere found to be fully replaced and repopulated by the host cells at the rate of 1 mm/day, culminating in the complete, macrophage-mediated clearance of porcine cellular material and elimination of immunogenic porcine antigens. This w¾s supported by the lack of detectable porcine DNA using Q-PCR in both the xenogeneic and autologous nerve tissue at necropsy. In addition, assessment of blood and tissue from the recipients indicated no circulating porcine cells or replication of PERV elements. Both housekeeping Q-PCR and qualitative PCR assays detected DNA in the nerye samples, however, initially it was not possible to differentiate between primate and porcine as the control assays were not specific. Conventional PCR utilizing a primate specific gene did indeed show that primate ceils were detectable in both the autologous and xenogeneic transplants.

[000230] Previous testing of skin xenotransplants had demonstrated that porcine cells were detectable at the graft site, but this did not extend to the peripheral circulation (Holzer et al). Given the lack of detection here and the immunological responses shown, the data surprisingly indicate that the porcine transplant was fully replaced and repopulated by the host cells. These methods are highly sensitive and specific and are of more value than, for example, immunohistochemistry, which in addition to lack of sensitivity, can be complicated by the lack of suitable antibodies to distinguish ceil content. Indeed, use of PERV genes for detection increases the sensitivity of the molecular assay and may also be indicative of an inflammatory reaction in addition to testing for infection as is done for allotransplants. No significant immunological reaction w^?as seen in these animals again reflected in the lack of detection. [000231] Motor nerve conduction velocity showed partial recovery in ail subjects, with the limbs treated with the xenogeneic transplant exhibiting greater recovery'.

[000232] The field of allotransplantation has been a success of modern medicine, has been hindered by numerous shortcomings. The therapeutic success of such consumable xenogeneic materials has recently been demonstrated in human clinical trials of cryopreserved split-thickness skin xenotransplants for the treatment of severe burns. Critical foundational translational aspects of this program such as patient monitoring for zoonosis and immunogenicity, as well as regulatory requirements and ethical considerations related to animal transplant donors, can be directly applied to support a broader regenerative medicine platform.

[000233] In this study, peripheral nerve defects were successfully reconstructed with the use of genetically engineered, DPF porcine donor xenogeneic nerve transplants, without adverse event or impacts to safety attributed to the xenogeneic transplant. These data demonstrate that the transplantation of viable, xenogeneic nerve transplants derived from genetically engineered, DPF porcine donors, may be a promising source of viable donor nerves for transplantation across large- gap ( 4cm f segmental peripheral nerve injuries.

[000234] The embodiments of the invention described above are intended to be merely exemplary; numerous variations and modifications will be apparent to those skilled in the art. All such variations and modifications are intended to be within the scope of the present invention as defined in any appended claims.

Claims

1. A xenogeneic neural or nerve tissue from a non-wildtype, genetically reprogrammed nonhuman animal for xenotransplantation into a non-human primate or human subject in need of neural or nerve tissue replacement, reconstruction, and/or repair, wherein the xenogeneic neural or nerve tissue is biologically active and metabolically active, wherein the xenogeneic neural or nerve tissue is configured to be surgically grafted into the subject, optionally wherein the xenogeneic neural or nerve tissue comprises nerve fascicles, and optionally wherein the xenogeneic neural or nerve tissue has a length of > 4 cm.

2. The xenogeneic neural or nerve tissue of claim 1 , wherein the xenogeneic neural or nerve tissue is configured for xenophagy in the subject such that no xenogeneic cells are detectable in the subject within 1, 2, 3, 4, 6, 8, 10, or 12 months of surgical graft into the subject.

3. The xenogeneic neural or nerve tissue of claim 1, wherein the non-wildtype, genetically reprogrammed non-human animal comprises a genome that has been reprogrammed in a plurality of exon regions.

4. The xenogeneic neural or nerve tissue of claim 1, wherein the non-wildtype, genetically reprogrammed non-human animal comprises genetic modifications such that genes encoding alpha-1,3 galactosyltransfera.se, cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and/or pi,4-N-acetylgalactosaminyltransferase are altered such that the genetically reprogrammed non-wildtype, genetically reprogrammed non-human animal lacks functional expression of surface giycan epitopes encoded by said genes.

5. The xenogenei c neural or nerve ti ssue of claim 1 , wherein the xenogeneic neural or nerve ti ssue is a complete nerve.

6. The xenogeneic neural or nerve tissue of claim 1 , wherein the xenogeneic neural or nerve tissue is an axon, myelin, endoneurium, perineurium, epineurium, Schwann cells, a matrix-rich scaffold, and/or a fascicle.

7. The xenogeneic neural or neve tissue of claim 1, wherein the xenogeneic neural or nerve tissue is a cryopreserved tissue.

8. The xenogeneic neural or nerve tissue of claim 1 , wherein the xenogeneic neural or nerve tissue is a fresh tissue.

9. The xenogeneic neural or nerve tissue of claim L wherein the xenogeneic neural or nerve tissue has a length of at least 0.5, 1, 2, 3, 4, 5, 6, or more cm.

10. The xenogeneic neural or nerve tissue of claim 1, wherein the xenogeneic neural or nerve tissue is a motor nerve.

11 . The xenogenei c neural or nerve ti ssue of cl aim 1 , wherein the xenogeneic neural or nerve ti ssue is a sensory nerye.

12. The xenogeneic neural or nerve tissue of claim 1 , wherein the xenogeneic neural or nerve tissue is an afferent nerve, an efferent nerve, or a mixed nerve comprising both afferent and efferent axons.

13. The xenogeneic neural or nerve ti ssue of claim 1 , wherein the xenogeneic neural or nerve tissue is a mixed nerve.

14. The xenogeneic neural or nerve tissue of claim 1, wherein the xenogeneic neural or nerve tissue is a sciatic nerve, brachial plexus (radial nerve, median nerve, ulnar nerves), peroneal nerve (foot drop), femoral nerve, lateral femoral cutaneous nerve, spinal accessory nerve, or tibial nerve.

15. The xenogeneic neural or nerve tissue of claim 1 , wherein the xenogeneic neural or nerve tissue is of porcine origin and comprises reprogrammed nucleotides at SLA-MIC-2 gene and at exon regions encoding SLA-3, SLA-6, SLA-7, SLA-8, SLA-DQ, CTLA-4, PD-L1, EPCR, TBM, TFPI, wherein the reprogrammed nucleotides are from human exon regions of ortho!ogous human genes, and the xenogeneic neural or nerve tissue lacks functional expression of swine beta-2-microglobulin, SLA-1, SLA-2, and SLA-DR.

16. The xenogeneic neural or nerve tissue of claim 1, wherein the xenogeneic neural or nerve tissue is a peripheral nerve.

17. The xenogeneic neural or nerve tissue of any one of claims 1 - 16, wherein the xenogeneic neural or nerve tissue is free of at least the following zoonotic pathogens:

(i) Ascaris species, Cryptosporidium species, Echinococcus, Strongyloids sterocolis, and Toxoplasma gondii in fecal matter;

(!i) Leptospira species, Mycoplasma hyopneumoniae , porcine reproductive and respiratory syndrome virus (PRRSV), pseudorabies, transmissible gastroenteritis virus (TGE)/Porcine Respiratory Coronavirus, and Toxoplasma gondii by determining antibody titers;

(iii) Porcine Influenza;

(iv) the following bacterial pathogens as determined by bacterial culture: Bordetella hronchisceptica, Coagulase-positive staphylococci, Coagulase-negative staphylococci, Livestock-associated methicillin resistant Staphylococcus aureus (LA MRSA), Microphyton and Trichophyton spp.;

(v) Porcine cytomegalovirus; and

(vi) Brucella suis.

18. The xenogeneic neural or nerve tissue of any one of claims 1-17, wherein the xenogeneic neural or nerve tissue is configured to bridge a nerve gap having a length of 5 cm or more.

19. The xenogeneic neural or nerve tissue of any one of claims 1-18, wherein the xenogeneic neural or nerve tissue has a proximal diameter of 1 mm to 25 mm.

20. The xenogeneic neural or nerve tissue of any one of claims 1-19, wherein the xenogeneic neural or nerve tissue has a distal diameter of 1 mm to 25 mm.

21. The xenogeneic neural or nerve tissue of any one of claims 1-20, wherein about 0.1 to 20 percent of total number of living cells in the xenogeneic neural or nerve tissue are Schwann cells.

22. The xenogeneic neural or nerve tissue of any one of claims 1-21, wherein the xenogeneic neural or nerve tissue further comprises a neurotrophic growth factor configured to stimulate nerve regeneration.

23. The xenogeneic neural or nerve tissue of claim 22, wherein the neurotrophic growth factor comprises one or more of brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), neurotrophic factor (NGF), neutrophin-3 (NT-3), ciliary neurotrophic factor (CNTF), or leukemia inhibitor_}' factor (LIT).

24. The xenogeneic neural or nerve tissue of any one of claims 1 -23, wherein the xenogeneic neural or nerve tissue lacks functional expression of endogenous beta-2 microglobulin of the non-human animal .

25. The xenogeneic neural or nerve tissue of any one of claims 1-23, wherein the xenogeneic neural or nerve tissue expresses a human beta-2 microglobulin.

26. A composition comprising the xenogeneic neural or nerve tissue of any one of claims 1-25 in a cryopreservation medium.

27. A composition comprising the xenogeneic neural or nerve tissue of any one of claims 1-26 in a cell culture medium.

28. A method for the xenotransplantation of a xenogeneic neural or nerve product, into a nonhuman primate or human xenotransplant recipient comprising: obtaining the xenogeneic neural or nerve tissue of any one of claims 1-25; transplanting said xenogeneic neural or nerve tissue into a xenotransplant recipient; monitoring said recipient for at least one of: a) rejection of the xenogeneic neural or nerve tissue; b) increase in an immunogenic biomarker; and wherein upon said transplantation, said xenogeneic neural or nerve tissue exhibits a clinical benefit in the xenotransplant recipient, and wherein said xenogeneic neural or nerve tissue is configured for xenophagy in the recipient such that no xenogeneic cells are detectable in the recipient within 1, 2, 3, 4, 6, 8, 10, or 12 months of surgically graft into the recipient.

29. The method of claim 28, wherein said grafting repairs or facilitates the reconstruction of neuronal connections across up to large nerve gaps in the peripheral nervous system in said recipient.

30. The method of claim 28, comprising joining nerve gaps having diameters that differ by 10% or less using the donor xenogeneic neural or nerve tissue.

31. The method of any one of claims 28-30, wherein the diameter of the xenogeneic neural or nerve tissue is unequal to that of the nerve gaps.

32. The method of claim 30, comprising using a cable graft comprising multiple lengths of a smaller diameter donor xenogeneic neural or nerve tissue to approximate the diameter of the inj ured nerve and join the large nerve gaps in the peripheral nervous system in said recipient.

33. The method of any one or combination of claims 28-32, wherein the xenogeneic neural or nerve tissue is from expendable sensor)-’ nerves.

34. The method of any one or combination of claims 28-32, wherein the xenogeneic neural or nerve tissue is from sural and medial antebrachial nerve.

35. The method of any one or combination of claims 28-34, comprising surgically grafting the xenogeneic neural or nerve tissue in a reverse orientation to nerve conduit.

36. The method of any one or combination of claims 28-35, comprising treating a nerve injury.

37. The method of any one or combination of claims 28-36, further comprising administering one or more immunosuppressants.

38. The method of any one or combination of claims 28-37, further comprising administering tacrolimus.

39. The method of any one or combination of claims 28-38, wherein the transplanting step is by suturing.

40. The method of any one or combination of claims 28-39, wherein the transplanting step comprises use of a surgical adhesive.

41. The method of any one or combination of claims 28-40, wherein the transplanting step comprises use of a fibrin-glue.

42. The method of any one or combination of claims 28-41, comprising treating a nerve injury with an unequal diameter relative to the graft.

43. The method of any one or combination of claims 28-42, comprising surgically grafting the xenogeneic neural or nerve tissue at a tension such that the xenogeneic neural or nerve tissue harvested is less than 20%, 15%, 10%, or 5% longer than the recipient's nerve defect.

44. The method of any one or combination of claims 28-43, comprising surgically grafting the xenogeneic neural or nerve tissue to treat axonotmesis.

45. The method of any one or combination of claims 28-44, comprising surgically grafting the xenogeneic neural or nerve tissue to treat neurotmesis.

46. The method of any one or combination of claims 28-45, comprising combining the xenogeneic neural or nerve tissue with an autologous nerve graft before, during or after xenotransplanati on.

47. The method of any one or combination of claims 28-46, comprising combining the xenogeneic neural or nerve tissue with an allogeneic nerve graft before, during or after xenotransplanati on.

48. The method of any one or combination of claims 28-47, wherein the xenogeneic neural or nerve tissue is cable grafted.

49. The method of any one or combination of claims 28-48, comprising combining the xenogeneic neural or nerve tissue with a deceilularized nerve product.

50. The method of any one or combination of claims 28-49, wherein the immunogenic biomarker is one or more of antibodies, cytokines, chemokines, T cells, and B cells.

51. The method of any one or combination of claims 28-50, wherein the immunogenic biomarker is at least one of IgG, IgM, TNFa, a lymphocyte, brain-derived neurotrophic factor, surface- expressed axonal growth cone markers transient receptor potential channel 1 (TRPCl) and short transient receptor potential channel 3 (TrpC3), and neuronal damage markers cyclooxygenase 2 and prostaglandin E2 receptor 1, glial fibrillary acidic protein, expressed intracellularly and p75.

52. The method of any one or combination of claims 28-51, wherein said xenogeneic neural or nerve tissue exhibits a clinical benefit in the xenotransplant recipient comparable to an allogeneic neural or nerve product.

53. The method of any one or combination of claims 28-52, wherein said xenogeneic neural or nerve tissue exhibits a clinical benefit in the xenotransplant recipient comparable to an autologous neural or nerve product.

54. The method of any one or combination of claims 28-53, comprising surgically grafting the xenogeneic neural or nerve tissue such that peripheral nerve regeneration is equal to or greater than peripheral nerve regeneration effected by allograft nerve implantation.

55. The method of any one or combination of claims 28-54, comprising surgically grafting the xenogeneic neural or nerve tissue such that peripheral nerye regeneration is at least 90% of peripheral nerve regeneration effected by autograft nerve implantation.

56. The method of any one or combination of claims 28-55, comprising surgically grafting the xenogeneic neural or nerve tissue such that peripheral nerve regeneration persists after withdrawal of immunosuppressant.

57. The method of any one or combination of claims 28-56, comprising surgically grafting the xenogeneic neural or nerve tissue such that fast-conducting nerve fibers are recovered and remyelinated at 8 months after the xenotransplantation.

58. The method of any one or combination of claims 28-57, comprising surgically grafting the xenogeneic neural or nerve tissue such that motor conduction velocity of the xenotransplanted nerve product recovers to at least 90% of the recipient’s healthy motor conduction velocity at. 12 months after the xenotransplantation.

59. The method of any one or combination of claims 28-58, comprising surgically grafting the xenogeneic neural or nerve tissue such that motor conduction velocity of the xenotransplanted nerve product is at least 50 m/s.

60. The method of any one or combination of claims 28-59, comprising surgically grafting the xenogeneic neural or nerve tissue such that restoration of at least 60% of the recipient’s baseline wrist extension is observed within 4 months of the xenotransplantation.

61. The method of any one or combination of claims 28-60, comprising surgically grafting the xenogeneic neural or nerve tissue such that restoration of at least 80% of the recipient’s baseline wrist extension is observed within 6 months of the xenotransplantation.

62. The method of any one or combination of claims 28-61, comprising surgically grafting the xenogeneic neural or nerve tissue such that restoration of at least 90% of the recipient's baseline wrist extension is observed within 12 months of the xenotransplantation.

63. The method of any one or combination of claims 28-62, comprising surgically grafting the xenogeneic neural or nerve tissue such that, the compound muscle action potential (CMAP) amplitude of the xenograft is at least 90% of the CMAP amplitude of autograft.

64. The method of any one or combination of claims 28-63, comprising surgically grafting the xenogeneic neural or nerve tissue such that the CMAP amplitude of the xenograft continues to increase after withdrawal of immunosuppressant.

65. The method of any one or combination of claims 28-64, comprising surgically grafting the xenogeneic neural or nerve tissue such that the xenotransplantation of a nerve product exhibit restoration of CM AP amplitude of at least 50% of that of the recipient’s baseline peripheral nerve.

66. The method of any one or combination of claims 28-65, comprising surgically grafting the xenogeneic neural or nerve tissue such that radial motor nerve conduction velocity (NCV) of the xenograft is at least 90% that of the radial motor N VC of autograft.

67. The method of any one or combination of claims 28-66, comprising surgically grafting the xenogeneic neural or nerve tissue such that radial motor NCV of the xenograft continues to increase after withdrawal of the immunosuppressants.

68. The method of any one or combination of claims 28-67, comprising surgically grafting the xenogeneic neural or nerve tissue such that radial motor NCV of the xenograft, post, transplantation is at least 90% of a baseline peripheral nerye.

69. The method of any one or combination of claims 28-68, comprising surgically grafting the xenogeneic neural or nerve tissue such that sensory⁷ nerve conduction velocity (NCV) of the xenograft is at least 90% that of the sensory NVC of the recipient’s baseline sensory NCV.

70. The method of any one or combination of claims 28-69, comprising surgically grafting the xenogeneic neural or nerve tissue such that the recipient’s peripheral blood mononuclear cells (PBMCs) are negative for porcine endogenous retrovirus (PERV) RNA and/or DNA amplification or microchimerism at 8 months post transplantation.

71. The method of any one or combination of claims 28-70, comprising surgically grafting the xenogeneic neural or nerve tissue such that the recipient’s serum is negative for porcine endogenous retrovirus (PERV) KNA and/or DNA amplification or microchimerism at 8 months post transplantation. , The method of any one or combination of claims 28-71, comprising surgically grafting the xenogeneic neural or nerve tissue such that the recipient’s tissue is negative for porcine endogenous retrovirus (PERV) RNA and/or DNA amplification or microchimerism at 8 months post transplantation.