Large‐gap peripheral nerve repair using xenogeneic transplants in rhesus macaques

Surgical intervention is required to successfully treat severe, large‐gap (≥4 cm) peripheral nerve injuries. However, all existing treatments have shortcomings and an alternative to the use of autologous nerves is needed. Human and porcine nerves are physiologically similar, with comparable dimensions and architecture, presence and distribution of Schwann cells, and conserved features of the extracellular matrix (ECM). We report the repair of fully transected radial nerves in 10 Rhesus Macaques using viable, whole sciatic nerve from genetically engineered (GalT‐KO), designated pathogen free (DPF) porcine donors. This resulted in the regeneration of the transected nerve, and importantly, recovery of wrist extension function, distal muscle reinnervation, and recovery of nerve conduction velocities and compound muscle action potentials similar to autologous controls. We also demonstrate the absence of immune rejection, systemic porcine cell migration, and detectable residual porcine material. Our preliminary findings support the safety and efficacy of viable porcine nerve transplants, suggest the interchangeable therapeutic use of cross‐species cells, and highlight the broader clinical potential of xenotransplantation.

nerve transplants, suggest the interchangeable therapeutic use of cross-species cells, and highlight the broader clinical potential of xenotransplantation.

K E Y W O R D S peripheral nerve, tacrolimus, Xenotransplantation INTRODUCTION
Severe trauma to the extremities frequently results in neurotmesis, the complete transection of peripheral nerves, a devastating injury. 1,2 It is estimated that 20 million Americans suffer from peripheral nerve injury (PNI), resulting in nearly 50 000 surgeries annually. 1 Treatment of injuries ≥4 cm, termed large-gap PNIs, are especially challenging as direct co-aptation is only possible for smaller defects. 3,4 In such cases, a nerve conduit (NC) is needed. The use of autologous nerves, such as the sural, is considered the standard of care despite complications such as donor site morbidity, chronic pain, paresthesia, insufficient length, or improperly matched fascicular areas and patterns. 5,6 Alternatives such as allogeneic nerve transplants or synthetic, non-biological conduits exist, 7,8 but all current options have numerous shortcomings and outcomes are suboptimal. 2 Therefore, a reliable, high-quality, and widely available alternative is highly desirable in the repair of large-gap PNIs. 2 The goal of surgical repair with NCs is to facilitate a complex, natural repair process, thereby maximizing the potential for the reinnervation of distal targets. Within 24 h of nerve trauma, an irreversible cascade of apoptosis known as Wallerian degeneration occurs, characterized by the dissolution of axonal cell membrane and cytoskeleton, release of axoplasm, retraction of the proximal and distal nerve stumps, and chromatolysis, the disruption of neurotransmitter production necessary for synaptic activity and axonal growth. 9 Schwann cells and macrophages phagocytose myelin and axon debris and release neurotrophic growth factors, such as GDNF, NT-3, and NGF, creating a microenvironment favorable for axonal repair. 10 Despite degradative protease activity, basal laminae are spared, leaving channels formed from residual endoneurial structures to direct an axonal growth cone emerging from Nodes of Ranvier at the proximal site towards a downstream synaptic target. Components of the conserved extracellular matrix (ECM), such as transmembrane cell adhesion molecules, laminin, fibronectin, and glycosaminoglycans (GAGs), provide stimulation of neuronal activity, Schwann cell migration, 11 and modulation of neurite extensions resulting in regeneration at a rate of 1-2 mm/day. 2,12 In the repair of large-gap PNIs, many conduits are unsuitable 2,13,14 due to mechanistic limitations. An unguided growth cone will result in a disorderly axonal mass forming a neuroma, a clinically painful outcome. 15 Optimal nerve conduits should contain a matrix-rich ECM scaffold, Schwann cells, neurotrophic growth factors, and a fascicular area comparable to or greater than that of the injured native nerve. Material properties such as plasticity, durability, and tensile strength should be sufficient to resist mechanical injury. 5,10,16 In addi-tion, research indicates that return of perfusion is critical, as diffusion of oxygen, nutrients, and cytokines relies on a network of longitudinally arranged blood vessels that courses throughout the nerve. 17 Therefore, vasculature that can be co-opted to restore perfusion would be advantageous. 18 Lastly, manufacturing ability, storage, and clinical acceptability are other critical considerations.
Viable xenogeneic nerve transplants offer the potential for a biological nerve conduit comprised of mixed-modal nerves, which can facilitate nerve recovery in large-gap PNIs and also support efferent and afferent conduction through the conduit without the additional morbidity and paresthesia from self-harvest and limitations of clinical availability.
Previously, the use of wild-type xenografts was explored, yielding mixed results. Evans et al. 19 reviewed all published research on xenograft nerve repair from 1880 to 1991, spanning more than 40 studies and hundreds of human and non-human subjects in which nerve sources were predominantly dogs, rabbits, and rodents. However, general optimism for xenografts diminished after research and experience demonstrated inferior outcomes when compared to autografts, 14,20 as well as undesirable immunological responses. 19,[21][22][23] The adverse immunological responses are better understood in the case of human recipients, primarily mediated by preformed antibodies against Galactose-α-1,3-galactose (α-Gal), an oligosaccharide expressed on all non-primate mammalian cells. 24 In some instances, xenografts were decellularized to diminish the rejection phenomenon as well as the possibility of zoonosis, but this resulted in the loss of essential cell populations in the process. 2 Surprisingly, few of these studies investigated the potential of porcine nerves, given the greater physical and genetic similarities between Sus Scrofa and Homo Sapiens. Recently interest in the use of porcine donors has gained momentum, but limited research exists in this area. The similarity of critical physiological characteristics between pig and human nerves, including size, length, architecture, and extracellular matrix composition, 1,25-28 would suggest the potential for regenerative capacity.
Genetic engineering of porcine donors as well as mitigation strategies and possible treatments for zoonosis, have made clinical xenotransplantation a more achievable goal. 21,24,[29][30][31][32][33] Thus, we hypothesized that instead of traditional xenografts, viable xenogeneic nerve transplants derived from specialized, Designated Pathogen Free (DPF), GalT-KO porcine donors could offer an alternative solution for repair of large-gap (≥4 cm) PNIs.
Here, in a two-phase, 12-month pilot study, we report successful axonal regeneration, distal muscle reinnervation, and recovery of conduction velocity following surgical repair of fully transected radial nerves in 10 Rhesus Macaque recipients via the use of xenogeneic nerve transplants.

Animals
This study's surgical procedures, protocols, and guidelines for animal

Cryopreservation
Following procurement, nerve xenotransplants were prepared via standardized institutional protocol where the nerve was packaged in cryovials (Simport, T310-1A, Beloeil, QC) and cryoprotective media

Surgical procedures
The porcine sciatic nerve was selected as the source of the xenogeneic transplant due to its superstructural similarity to human and primate nerves. 1 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. 35 The porcine donor was euthanized and prepared for surgery as previously described. 34 To isolate the sciatic nerve prior to harvesting, a linear incision was made midway between the sacrum and the ischium and extended ventrally along the posterior aspect of the femur, longitudinally dissecting the gluteus medius, gluteus maximus, piriformis, and biceps femoris muscles, to the proximal tibiofibular joint. The sciatic nerve was visualized and harvested by radial transections distal to the nerve origin and proximal to the bifurcation into the tibial and common peroneal nerves. This process was repeated on the bilateral side.
Two porcine sciatic nerves were harvested from one donor per protocol, and stored at −80 • C for a period of 7-8 days, after which they were thawed as previously described. 36 This cryopreserved nerve was used as the source of the donor nerve to repair the radial nerve defects in the remaining five Rhesus Macaque recipients 7-8 days later.
Cryopreservation of the sciatic nerve was necessary to limit variability of surgical personnel, techniques, and conditions, as the number of surgeries and availability of surgical team required more than one series of surgical operations.
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.
Bilateral, 4 cm complete transections of radial nerves were surgically introduced in a total of ten Rhesus Macaque recipients. Recipients, under anesthesia, 37 were positioned in lateral recumbency with the shoulder at 90 • flexion, full internal rotation, and neutral abduction.
The subcutaneous tissue and deep fascia were 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. 38 The intramuscular plane between the long and lateral head of the triceps was developed approximately This process was performed bilaterally per each of the ten recipients; both xenogeneic and autologous nerves 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. In the contralateral arm, excised Rhesus Macaque radial nerve segments were rotated 180 • and reimplanted as a surgical control (Figure 1a). 2 The 10 recipients were randomly, evenly divided between two surgical series, one week apart (Table S1). 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.

Immunosuppression
Intramuscular injection of tacrolimus, at a dosage of 0.15 mg/kg/day, began 10 days before surgery and was continued until 8-months for Group 1 (N = 5) and 6-months for subjects in Group 2 (N = 5) as previously described. 39 Trough levels were maintained between 20 and 30 ng/ml. Dosing for subjects experiencing levels above or below the range was adjusted to bring the trough level within the target range.

Pathology
At the designated necropsy time point, the animals were sedated  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. 40 PERV quantification was carried out by comparison to standards of known PERV copy numbers. The limit of quantification (LOQ) 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. 40 Detection of porcine cells was quantified by comparison to standards of known porcine cell content. The LOQ for porcine cells using this assay is 0.026 cells per reaction.
Qualitative PCR for the reference gene RPL13A (ribosomal protein gene) was carried out to confirm that DNA was suitable for amplifica- Detection of primate-specific DNA was carried out using qualitative PCR as described previously with modifications. 41 In brief, the primers

Electrophysiology
Nerve conduction studies (NCS) 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.

Nerve conduction velocity and functional evaluation
The Nerve Conduction Velocity (NCV) measurement 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

Functional assessment
A previously reported radial nerve injury model was adapted to assess the functional recovery of xenogeneic and autologous nerve transplant recipients. 42 Radial nerve injury proximal to the elbow results in a loss of wrist extension function, or "wrist drop," loss of forearm muscle tonality, and digital extension due to motor denervation of the extensor carpi radialis longus and extensor carpi radialis brevis muscles. 43,44 Wrist extension testing and evaluation were performed monthly. Subjects were offered a food treat outside of the of the cage in a manner to encourage them to reach out to grab it with a wrist angle extension required. This is done with one hand then the other. The test was video-  Binding of the xenoreactive antibody to pig cells was measured as previously described. 45 Cryopreserved genetically modified α-

Statistical analysis
The study design includes several acknowledged limitations that introduced variability between subjects and the non-normality of these data. As a result, a detailed statistical analysis is not appropriate for these findings.

Hematology and clinical chemistry
Blood samples were obtained monthly and processed for serum or transferred to the Biomere Testing Facility laboratory and processed.

Clinical outcome
All 10 subjects tolerated the surgical procedure resulting in the complete loss of radial nerve function bilaterally (Figures 1a-c). Nineteen In Phase 2 (post 6 months), 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.
However, subjects in Group 1 presented with progressing symptoms associated with tacrolimus toxicity, 49 such as limited mobility in knee joints, muscle rigidity, stiffness, and atrophy, as well as significant weight loss. As a result of the tacrolimus-associated toxicity, at 8-months, the five subjects in Group 1 were euthanized.

Functional evaluation
Radial nerve functional assessments were performed monthly for each The distance from the recording site to the stimulation cathode was entered in the instrument during collections, for each site, and the con-   (Table S2).

Hematology and clinical chemistry
Across both Phase 1 and Phase 2, white blood cell count (WBC) and individual component percentages remained within normal ranges 47,48 ( Figure 4a, b). Neutrophil and lymphocyte percentages varied monthto-month, but absolute counts remained close to expected values.
RBCs and platelets were in the normal range for all subjects throughout the study period (data not shown). creatinine (CREA), and electrolyte levels were stable at expected baseline levels for the duration of the study ( Figure S1). Glucose (GLU) levels were above expected values and were elevated for all months except zero and twelve. We expect this was due to the recipients' increased sugar consumption during radial nerve functional evaluations. These results were reviewed by the study veterinarian.

Pathology
Spleen, liver, heart, kidney, and lung sections obtained at necropsy were stained with H&E and assessed microscopically. All organs were determined to be consistent with normal primate organs.

Immunogenicity
Total IgM levels were slightly elevated above preoperative levels at one or more postoperative time points in all recipients (Figure 4c). 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.
Anti-porcine IgM and IgG levels showed an increase above preoperative levels following transplantation, followed by a gradual decrease ( Figure 4d). The IgM increased to its highest level for all recipients at 1 month and remained elevated for 6-months, returning to baseline after that timepoint. Anti-porcine IgG increased to a peak at 1 and 3 months, gradually decreasing but remained significantly elevated, 5.4 fold above baseline throughout 12 months. A comparison rMFIs of the anti-porcine IgG for fresh versus frozen at 1 month and 6 months shows a higher rMFI for subjects receiving the frozen nerve but the difference was not significant, p = .29 and p = .37, respectively (Table S3).
The specificity of the anti-porcine IgG has not been addressed at this time, although others have detected high levels of anti-non-αGal in burn patients after being treated with pig-skin dressings. A nonhuman sialic acid Neu5Gc was identified as one of the target antigens. 40 Since the porcine nerve structure was in vivo for up to a year, it is possible a number of additional epitopes could also be targets.

Histology
Blinded histological analysis found no meaningful differences between nerve tissue excised from transplantation sites in limbs treated with either autologous or xenogeneic transplants.
The pathologist scored the nerve bundle diameters and perioperative explanted autologous nerves not used for transplantation were positive controls with scores 4, >300 μm. For Group 1, the diameter of the regenerated nerve bundles across the defect site for all five recipients was comparable for both types of nerve transplants with scores of 2 and 3100 to 300 μm. At the end of study for Group 2 subjects, xenogeneic nerve bundle diameters scored more 2s,100-200 μm, and appeared smaller than those of the autologous control, with four autologous controls with scores of 4, >300μm and reaching preoperative diameters (Figure 5a, b).

Biodistribution of porcine tissue in autografts and xenografts
Chimerism 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 10 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 microchimerism, indicating that  Figure S2).
there was no evidence of microchimerism 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 ( Table 2).
As expected, the autologous nerve grafts lacked the presence of PERV or porcine cells. Surprisingly, the xenogeneic sites were also negative for the presence of porcine cells by Q-PCR, suggesting there was no residual porcine tissue in the xenogeneic nerve tissue tested.
Q-PCR for porcine centromeric DNA was also negative ( Table 2). However, the use of a primate-specific primer set using conventional PCR demonstrated the presence of primate cells in both autologous and xenogeneic transplants (Figures 6g and S2).

DISCUSSION
Physical guidance and Schwann 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. involving non-human primates, the regimen used in this study was believed to be appropriate and scientifically justified. 49 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. 80 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. 49 The data generated post the month 8time point has been considered not relevant by the authors, due to the afore mentioned 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 stereology and morphometry for quantification and immunohistochemistry for specific antigens.
Finally, we expected the cellular components of the 4-cm porcine transplant to be fully replaced and repopulated by the host cells at the rate of 1 mm/day, 19,73,81 culminating in the complete, macrophagemediated 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 PERV elements. Both housekeeping Q-PCR and qualitative PCR assays detected DNA in the nerve 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 cells were detectable in both the autologous and xenogeneic transplants.
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. 34 Given the lack of detection here and the immunological responses shown, this supports the hypothesis that the porcine transplant is 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 cell content. Indeed, recently, it has been suggested that the 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 allo-transplants. 82

CONCLUSION
The field of allo-transplantation has been a success of modern medicine, has been hindered by numerous shortcomings. 1