Porcine Kidney Xenotransplantation in Patients With End-Stage Kidney Disease

The mismatch between organ supply and demand results in the deaths of thousands of Americans each year. Our research group aims to solve this unmitigated health care crisis by translating advances in xenotransplantation to humans and expanding organ supply in a sustainable fashion using genetically modified pigs as a source of organs. We propose here a phase I clinical trial of porcine kidney xenotransplantation into 20 people with end-stage kidney disease. Source donor animals are pigs with 10 gene edits (10-GE) which attenuate immunologic harm to the kidney xenograft. 10-GE pigs are housed in a designated pathogen-free facility within 30 minutes of the transplantation center. Xenotransplantation procedures follow conventional practices currently employed in allotransplantation and comply with multiple regulatory standards to ensure ethical treatment of research subjects and source animals. Recruitment and xenotransplantation will occur over 5 years with study follow-up extending 1 year after xenotransplantation. Primary outcome variables surround patient safety, such as patient survival and the rate of zoonotic disease transmission. Secondary outcome variables include commonly used metrics of graft survival and function.

Twenty patients with ESKD listed for kidney allotransplantation at UAB will be enrolled to receive either one or two porcine kidney xenotransplants from a 10-GE pig donor, contingent on the pig's overall size at the time of procurement.

Recruitment and xenotransplantation will occur over a five-year time period. Patients will undergo follow-up of one year post-xenotransplant with study extension if graft survival exceeds one year.

Participants will undergo prospective crossmatching with a 10-GE pig to determine histocompatibility prior to xenotransplant. After performance of a negative crossmatch, procurement of the donor pig will occur in a surgical suite at the designated pathogen-free facility near the UAB campus where the donor herd is maintained.

Porcine kidney(s) will be transported under sterile and hypothermic conditions to the main UAB hospital. The porcine kidney(s) will be transplanted into the research subject in standard surgical fashion within the abdomen and immunosuppression will be administered. The induction immunosuppression regimen utilized will mirror that used in human-to-human allotransplantation; this regimen represents current standard-of-care. After transplantation, kidney health will be assessed biochemically, histologically, and radiographically. Subjects will be monitored for potential zoonotic disease transmission and blood-based chimerism as well as thrombocytopenia or indicators of consumptive coagulopathy, development of anti-human leukocyte antigen antibody/alloantibody sensitization.

FOLLOW-UP PHASE

Xenotransplant recipients are expected to remain in the hospital under close clinical surveillance by the study team and associates for approximately 1-3 weeks, pending kidney recovery and surveillance for potential complications.

Post-transplant Medications. The post-transplant immunosuppression and maintenance regimen consists of conventional agents that are routinely used in kidney allotransplantation. Although immunosuppression practices in allotransplantation can vary widely from center to center, the approach below utilizes agents and doses employed by UAB's Incompatible Kidney Transplant Program. As the ideal immunosuppression regimen for xenotransplantation has not yet been established, Investigator and team have adopted strategies used in incompatible kidney allotransplantation based on the assumption that significant antigenic variation remains between the species and antibody responses of unknown specificity below the level of detection of the crossmatch may impact later function or graft longevity.

Maintenance immunosuppression will be administered as below:

Note: Induction immunosuppression will be given at the time of xenotransplantation and will include ATG 1.5mg/kg, solumedrol 500mg, and eculizumab 1200mg. The remaining doses of ATG will be given to a total of 6mg/kg to complete induction. The ATG dosing schedule will vary depending on degree of hemolysis and/or lineage depletion, as is routinely done in allotransplantation.

Tacrolimus in divided doses daily, to a target level of 8-12 ng/mL Mycophenolate Mofetil (1000mg twice daily as a starting dose and tapered as needed per UAB incompatible kidney allotransplantation protocol) Daily prednisone, tapered from induction to a target dose of 10 mg per day. Eculizumab, 1200 mg every two weeks Investigator and study team have opted to include eculizumab as a maintenance agent to avert or attenuate complement-mediated injury of the graft. Although the 10-GE pig expresses a number of human transgenes designed to regulate complement biology, the finding of TMA in the brain-dead human recipient (\*Porrett et al, AJT 2022) suggests that additional blockade of complement may be beneficial. Given that eculizumab increases the risk of infection with encapsulated bacteria, research subjects will be required to complete the FDA REMS and be vaccinated accordingly. Ciprofloxacin 500 mg twice daily (adjusted for GFR) for 6 weeks after transplantation in individuals vaccinated with meningococcal vaccine as above. Subjects will be monitored and counseled about side effects of prolonged ciprofloxacin use, such as tendonitis and tendon rupture. When eculizumab is discontinued (such as after graft removal), ciprofloxacin will be discontinued 6 weeks after the last eculizumab dose.

Thrombosis Prophylaxis: Recipients may be maintained on a heparin infusion and/or receive aspirin as clinically indicated. Subcutaneous heparin will be administered for DVT prophylaxis. Early ambulation will be encouraged.

Antimicrobial Prophylaxis: Surgical wound prophylaxis will be discontinued within 24h of exiting the operating room. Prophylaxis against opportunistic infections will be continued after transplant.

For PCP prophylaxis: Sulfamethoxazole (or Dapsone if allergic to Sulfa). This will be continued for 12 months.

For viral/CMV/HSV prophylaxis:

Valganciclovir to prevent CMV reactivation if recipient is CMV positive. This will be renally dosed and continued for 6 months post-transplant.

If recipient is CMV negative, valacylovir will be selected for HSV prophylaxis and continued for 6 months post-transplant.

Note: Although the transplantation of CMV+ donors to CMV- recipients occurs often in human allotransplantation, this situation is unlikely to occur in the setting of this trial as porcine donors in the DPF are CMV negative. Should a porcine donor test positive for CMV, prophylaxis in the recipient would be altered accordingly (i.e., valganciclovir or ganciclovir would be provided in consultation with Transplant Infectious Disease specialists)

For Candidal prophylaxis (i.e. oral thrush): Nystatin swish/swallow

Follow-up Visits and Procedures

Frequency:

Xenotransplant recipients will be closely monitored for the duration of their participation in the study. After discharge from the hospital, the study team anticipates seeing these patients in the outpatient clinic according to the table below, with additional clinic visits as needed. Recipients will have 24h access to the study team and will be extensively educated about clinical triggers (i.e., signs and symptoms of infection, myocardial infarction, etc.) to seek care. Each visit will consist of routine care for kidney transplant patients, including an assessment of medication compliance, collection of vital signs, history, a review of systems, and physical examination including examination of the surgical wound (for the first 6-8 weeks after transplantation

Post-transplant time period Clinic visit schedule 0-1 months Twice Weekly 2-3 months Weekly 3-6 months Twice Monthly 6-12 months (or study endpoint) Monthly

Protocol biopsies will occur 24h after transplant, 3 months, 6 months, and at 12 months assuming graft survival. Biopsies may be performed either percutaneously or may require an open surgical approach. Percutaneous biopsies will be performed under ultrasound guidance. Biopsy schedule may be amended for cause. Biopsies will be submitted for routine pathologic analysis (H\&E, immunofluorescence microscopy, etc.), as well as research studies. Biopsy findings will be scored using the Banff criteria.

Imaging Studies:

Ultrasound: Kidney xenograft perfusion will be monitored with ultrasound. Grafts will be studied with ultrasound daily for the first week then weekly thereafter while recipients are in the hospital. Monthly ultrasounds will be performed in the outpatient setting for the first year (or for the duration of the graft if less than one year). Schedule may be amended for cause.

Nuclear medicine scanning: Kidney perfusion scans will be performed monthly to acquire additional information about xenograft excretion and perfusion.

Laboratory Assessments:

Most of the laboratory tests below will be performed daily for the first 2 weeks after transplantation and then gradually transition to weekly and/or monthly schedules pending clinical stability of the recipient. Testing schedule may be escalated for cause. Tests will be performed on either blood or urine and are largely non-invasive or minimally invasive (such as phlebotomy). This testing schedule will largely replicate the routine monitoring of transplant recipients.

Complete blood count with differential Complete metabolic panel (including LFTs) Coagulation panel (including PT/PTT/INR) Tacrolimus level Mycophenolate level Urinalysis Urine culture 24h urine collection (Creatinine, Protein, Albumin, Urea, Sodium, Potassium, Protein Electrophoresis and Immunofixation etc.) Daily on postoperative day 1,2,3 Weekly for two weeks Monthly for first year Random urine protein Random urine creatinine Vit D 25 and Vit D 1,25 level Intact PTH Hemoglobin A1C Lipid Profile Lymphocyte profiling (e.g. CD4, CD8,CD19, CD20) Note: blood will also be analyzed in the research laboratory where higher dimensional immune profiling can be performed using multiparameter flow cytometry, single-cell RNA-sequencing, etc.) Iron, TIBC, Ferritin Samples for blood chimerism studies Immunologic monitoring Single antigen bead screening to assess for changes in anti-HLA antibody titers which may have been provoked by cross-reactivity with SLA.

Retrospective crossmatch Pathogen monitoring CMV PCR will be performed monthly for the first transplant year. Serum and urine will be tested monthly for BK virus reactivation for one year. Monitoring of donor-derived infection

Eligibility Criteria:
Inclusion Criteria:

* Ages 18-75 years
* Body mass index (BMI) 18-40 kg/m2
* Waitlisted for kidney allotransplantation at UAB
* Dialysis dependent
* Proficient in the English language
* Presence of risk factors for

  * high wait list mortality, AND/OR
  * anticipated or actual prolonged wait time on the wait list, AND/OR
  * inability to access a suitable organ offer.
  * Note: Examples of risk factors include high cPRA, frequent incompatible crossmatches leading to prolonged wait times, listing at an advanced age, and impending loss of dialysis access, recurrent disease in previous transplants.
* Crossmatch compatible with porcine donor
* SARS-CoV-2 vaccination in accordance with most recent CDC guidance
* Willingness to obtain other standard-of-care vaccinations for kidney transplant recipients (MMR, HBV, Herpes Zoster, etc.) and for patients receiving eculizumab (Menactra® and Bexsero®)
* Reside within a 60-minute radius of the UAB hospital (by ground transport)

Exclusion Criteria:

* Age \<18y or ≥ 76y
* BMI ≤ 18 or ≥ 41 kg/m2
* Current pregnancy
* Presence of severe comorbid disease, including but not limited to uncontrolled HTN or DM (hemoglobin A1C (HgA1C) \>10%), advanced cardiovascular disease, absent surgical targets for implantation, etc.
* Presence of hypercoagulable disorder
* Inability to accept a blood transfusion
* Intolerance of immunosuppression
* History of medical non-compliance
* Presence of untreated psychiatric disease
* Significant psychosocial vulnerability and/or poor social support
* Current use/abuse of illicit drugs and/or abuse of alcohol
* History of psychiatric hospitalization
* Inability to provide informed consent
* Pre-emptive transplant
* Inability to comply with study protocols and procedures