Improved immune-compatibility and fetal kidney complementation in gene edited sheep

Abstract

There is a global shortage of organs for transplantation, with the highest demand for kidneys. Livestock have long been regarded as a potential source of donor organs to alleviate this burden. While investigations have been focusing on pigs, sheep also have similar physiology and anatomy to humans, potentially being a culturally more widely accepted alternative donor species. There are two approaches to produce transplantable kidneys in livestock: i) sourcing human immune-compatible organs from animals lacking highly immunogenic xenoantigens or ii) growing human organs in animal hosts after genetically disabling livestock organogenesis. In this thesis, both approaches were addressed by using Cas9-mediated genome editing in ovine fetal fibroblasts. Random nucleotide insertions or deletions were introduced to both alleles of CMAH and GGTA, responsible for producing the xenoantigens Neu5Gc and α-Gal, respectively, and the kidney developmental gene SALL1. Double (CMAH/GGTA) and triple (CMAH/GGTA/SALL1) knockout clonal strains were screened for frameshift mutations before use in somatic cell transfer cloning to produce edited animals. Five female CMAH-/- GGTA-/- double knockout ewes were produced and confirmed negative for Neu5Gc and α-Gal. Two different editing strategies were performed for SALL1. First, targeting after the fourth zinc finger domain produced variable fetal kidney phenotypes, ranging from agenesis/severe hypoplasia to mild hypoplasia. Second, removing all zinc finger domains resulted in a single fetus with complete kidney agenesis. For the kidney complementation approach, rescue of the anephric phenotype was examined by generating intraspecific chimaeras between triple knockout hosts and mCherry fluorescent donor cells. Fetuses were generated with varying levels of chimaerism. High contribution of mCherry donor cells resulted in the most successful rescue of kidney development, with organ size resembling that of wild-type females. Low level of mCherry contribution produced fetuses with kidney hypoplasia similar to the original SALL1 knockout fetus, with donor cells restricted to meso- and metanephros in chimaeras with lowest contribution. This study combined the two approaches to kidney generation in livestock and demonstrated sheep as a viable alternative to pigs for xenotransplantation research.