Genetic Engineering of the Kidney to Permanently Silence MHC Transcripts During ex vivo Organ Perfusion.

Yuliia Yuzefovych,Constanca Figueiredo,Dirk Wedekind,Jessica Schmitz,Emilio Valdivia,Tamina Rother,Jan Hinrich Bräsen,Rainer Blasczyk,Faikah Gueler,Franziska Hack,Cyril Moers,Song Rong,Nadine Wenzel

doi:10.3389/fimmu.2020.00265

Abstract

Organ gene therapy represents a promising tool to correct diseases or improve graft survival after transplantation. Polymorphic variation of the major histocompatibility complex (MHC) antigens remains a major obstacle to long-term graft survival after transplantation. Previously, we demonstrated that MHC-silenced cells are protected against allogeneic immune responses. We also showed the feasibility to silence MHC in the lung. Here, we aimed at the genetic engineering of the kidney toward permanent silencing of MHC antigens in a rat model. We constructed a sub-normothermic ex vivo perfusion system to deliver lentiviral vectors encoding shRNAs targeting β2-microglobulin and the class II transactivator to the kidney. In addition, the vector contained the sequence for a secreted nanoluciferase. After kidney transplantation (ktx), we detected bioluminescence in the plasma and urine of recipients of an engineered kidney during the 6 weeks of post-transplant monitoring, indicating a stable transgene expression. Remarkably, transcript levels of β2-microglobulin and the class II transactivator were decreased by 70% in kidneys expressing specific shRNAs. Kidney genetic modification did not cause additional cell death compared to control kidneys after machine perfusion. Nevertheless, cytokine secretion signatures were altered during perfusion with lentiviral vectors as revealed by an increase in the secretion of IL-10, MIP-1α, MIP-2, IP-10, and EGF and a decrease in the levels of IL-12, IL-17, MCP-1, and IFN-γ. Biodistribution assays indicate that the localization of the vector was restricted to the graft. This study shows the potential to generate immunologically invisible kidneys showing great promise to support graft survival after transplantation and may contribute to reduce the burden of immunosuppression.

Highlights

Kidney transplantation remains the best treatment for end stage renal diseases
In Germany there are currently 7,526 patients waiting on a kidney transplantation with waiting times exceeding 10 years (DSO 2018) [1]
The pRRL.PPT.eFS.pre lentiviral vector plasmid encoding for the sequence of a secreted form of luciferase from Oplophorus gracilirostris (NanoLuc, NanoLuc Luciferase (NL)) as a reporter gene was used for cloning of an RNAi cassette [18]

Summary

Introduction

Kidney transplantation remains the best treatment for end stage renal diseases. the limited availability of organ donors contribute to increased waiting times and high morbidity and mortality on the waiting list. Allograft loss after kidney transplantation is the result of a tight and synergistic interplay between innate and adaptive immune responses This involves complex molecular mechanisms based on T and B-cell activation, autophagy, apoptosis, and inflammatory responses [12]. In the interest of a precise regulation, we used RNA interference to downregulate MHC class I and II transcript in a rat kidney transplant model as a strategy to reduce the immunogenicity of the allograft. This could offer many new opportunities in transplant settings and contribute to gender and diversity equality

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