Ex Vivo Perfusion of Gene-Edited Porcine Livers in Patients With Severe Hepatic Failure

This is a single-center, prospective, open-label clinical trial designed to enroll one patient with severe hepatic failure to evaluate the safety and efficacy of extracorporeal perfusion using a gene-edited pig liver as a bridge-to-transplant therapy.

Background and Rationale:

Severe hepatic failure carries extremely high short-term mortality, and the scarcity of human donor livers limits treatment options. Xenotransplantation using genetically modified pigs represents a promising solution to organ shortage. This study builds on preclinical success in pig-to-non-human primate liver xenotransplantation and recent subclinical trials in brain-dead human recipients. By employing an extracorporeal (ex vivo) perfusion approach, the study minimizes surgical trauma to the patient while allowing assessment of xenogeneic liver function and compatibility.

Study Population:

The study will recruit one adult patient (age 18-70 years) with severe hepatic failure meeting the diagnostic criteria of the "Guidelines for Diagnosis and Treatment of Hepatic Failure (2024 Edition)." Eligible participants must have grade II or higher hepatic encephalopathy and a predicted poor prognosis with high short-term mortality (28-day mortality ≥15%). A third-party independent committee of three experts will assess natural mortality expectations to select appropriate candidates. Key exclusion criteria include immune dysfunction (e.g., HIV), prior allogeneic transplantation, or inability to tolerate surgical trauma.

Intervention:

The intervention involves connecting the patient to an extracorporeal perfusion circuit containing a liver from a six-gene-edited Bama miniature pig (provided by Sichuan Zhongke Aoge Biotechnology Co., Ltd.). The pig liver is procured using standard techniques with hypothermic perfusion (Schüssner's solution and hypertonic citrate-purine solution) and transported under strict cold storage conditions (1-6°C).

Surgical Procedure:

Under local anesthesia, vascular access is established via one internal jugular vein and one femoral vein. The patient is connected to the perfusion platform, and the preserved gene-edited pig liver is integrated into the extracorporeal circuit.

Immunosuppression and Anti-inflammatory Regimen:

Participants will receive a multi-drug immunosuppressive protocol including:

Rituximab (375 mg/m²) pre-operatively Rabbit anti-human thymocyte immunoglobulin (ATG, 1-1.5 mg/kg/day for 2-3 days) Tacrolimus (target trough 15-25 ng/mL) Mycophenolate mofetil (1 g twice daily) Etanercept (anti-TNF-α, 25 mg twice weekly) Methylprednisolone (high-dose tapering regimen) Eculizumab (900 mg, 24 hours pre-perfusion) for complement inhibition

Anticoagulation and Supportive Care:

Heparin sodium will be administered to maintain APTT at 1.5-2.5 times normal and ACT at 200-300 seconds. Ganciclovir will be provided for cytomegalovirus prophylaxis. Standard supportive care for liver failure, including anti-infection therapy, hepatoprotective measures, and nutritional support, will be provided according to clinical guidelines.

Primary Objectives:

To observe changes in vital signs, physiological parameters, biochemical indicators, coagulation function, inflammatory responses, and immune-related markers in patients with severe hepatic failure following extracorporeal perfusion of gene-edited pig liver To evaluate whether the procedure can significantly improve liver function and serve as an effective bridge-to-transplant therapy

Secondary Objectives:

To evaluate the survival and functional viability of the pig liver through monitoring of bile secretion (volume and color), blood flow in portal vein/hepatic artery/hepatic vein, and histopathological examination of biopsy specimens every other day To explore perioperative management strategies and optimize the treatment protocol for extracorporeal liver perfusion To assess biosafety through daily monitoring of porcine endogenous retrovirus (PERV) and porcine cytomegalovirus to evaluate cross-species transmission risks

Outcome Measures:

Primary: Improvement in liver function parameters (bilirubin, INR, hepatic encephalopathy grade), coagulation function, and inflammatory markers; successful bridging to human liver transplantation or clinical recovery Secondary: Graft survival time, bile production quality, vascular patency on ultrasound, histological evidence of rejection (ISHLT grading), incidence of adverse events, and viral safety parameters

Study Duration and Follow-up:

The study period is from October 2025 to December 2026. Extracorporeal perfusion will continue until one of the following occurs: (1) successful bridging to allogeneic liver transplantation, (2) clinical recovery allowing discontinuation of support, (3) graft failure, (4) participant death, or (5) maximum duration of 14 days. If the initial graft fails, a second gene-edited pig liver may be used for continued perfusion.

Safety Monitoring:

Comprehensive safety monitoring includes hourly vital signs, daily laboratory assessments (CBC, liver/renal function, coagulation, tacrolimus levels), daily abdominal ultrasound, and rigorous adverse event documentation. An independent ethics committee oversees the study, and participants (or family members) may withdraw consent at any time prior to or during the procedure.

Statistical Analysis:

As a single-participant study, data will be analyzed descriptively. Continuous variables will be expressed as mean ± standard deviation, with changes from baseline documented longitudinally.

Inclusion criteria

• Age and Gender: Adults aged 18-70 years, any gender.
• Diagnosis of Severe Hepatic Failure: Patients meeting diagnostic criteria from the "Guidelines for Diagnosis and Treatment of Hepatic Failure (2024 Edition)" including:

Acute liver failure: Acute onset without underlying liver disease history, developing grade II or higher hepatic encephalopathy within 4 weeks Subacute liver failure: Acute onset without prior liver disease, clinical manifestations of liver failure appearing within 4-24 weeks Acute-on-chronic liver failure: Acute deterioration of liver function on the basis of chronic liver disease (with or without cirrhosis), presenting as liver and/or extrahepatic organ failure with high short-term mortality (28-day mortality ≥15%) Chronic liver failure: Progressive liver function decline on the basis of cirrhosis resulting in chronic hepatic decompensation (recurrent ascites and/or hepatic encephalopathy) All patients must have: Grade II or higher hepatic encephalopathy, confirmed by a third-party independent expert panel of three members assessing poor treatment response and extremely high short-term mortality risk.

• Surgical Tolerance: Other organs besides the liver must be able to tolerate the trauma of extracorporeal liver perfusion surgery.
• Informed Consent: Parents, spouses, and adult children (immediate family members) must be fully informed and voluntarily sign the informed consent form, with ability to complete the trial as required by the protocol.

Exclusion criteria

• Immune Dysfunction: Individuals with HIV infection, diagnosed systemic immune diseases, or those undergoing immunosuppressive therapy affecting systemic immune function.
• Prior Transplantation: Previous recipients of allogeneic tissue or organ transplantation.
• Religious/Ethnic Restrictions: Individuals who cannot accept swine-derived materials due to religious beliefs, ethnic considerations, or other reasons.
• Recent Trial Participation: Participation in other clinical studies within 3 months prior to enrollment.
• Surgical Contraindications: Pathological changes at the planned operative site rendering surgery inoperable, or presence of critical organ failure or systemic severe infections precluding surgical intervention.
• Investigator Discretion: Patients deemed unsuitable for participation by the investigator for other reasons.