Towards a new, glyco-humanized rabbit line.

Humans lack Neu5Gc and αGal moieties on glycosylated proteins due to mutations in the enzymes
CMAH and GGTA1, respectively. For a variety of reasons, rabbits produce exquisitely effective
antibodies that are suitable for use as human therapeutics. However, as they display these Neu5Gc
and aGal epitopes, a strong humoral response is elicited upon injection in humans, which reduces
clinical efficacy and shortens half-life. In this context and in accordance with the scope of our
University, FEDER-backed EPI-SHIELD initiative, we aimed at inactivating CMAH and GGTA1, relying
on the CRISPR/Cas9 technology. Six-month old, superovulated does were sacrificed and their
zygotes harvested ~14 hours after mating. A mix of 2 ribonucleoproteins, each made of Cas9 and a
specific guide-RNA, was injected in the pronucleus, and injected embryos were either cultured for 5
days (up to the blastocyst stage) or transferred in the oviducts of a synchronized, pseudopregnant
doe. High proportions of five-day old blastocysts displayed indels at expected locations, with success
rates of ~43 (3/7) and ~71% (5/7) for GGTA1 and CMAH, respectively. After transfer, a litter of 4
kits was born. Among the 4 kits, the CMAH of one is expected to be inactivated, due to insertion of
a single G generating a premature stop codon.