Establishment And Characterization Of Wiskott-Aldrich Syndrome Rabbit Model

Wiskott-Aldrich syndrome(WAS)is an X-linked recessive inherited immune disorder resulted from loss-of-function mutations in the WAS gene.The WAS protein(WASP)is an actin nucleation promoting factor specifically expressed in the hematopoietic system.WASP defection can cause abnormalities in hematopoietic cells and immune cells.At present,the pathogenic mechanism of WAS is still unclear.And the only effective treatment is hematopoietic stem cell transplantation for classic WAS patients whose median survival is less than 15 years without treatment.Mouse is the only available mammalian model used for investigation of WAS.However,these mice do not faithfully recapitulate WAS clinical phenotypes,including mild thrombocytopenia,no sign of bleeding,no infections and enjoying normal lifespan,which limits its application for WAS clinical research.Therefore,a more suitable animal simulating human WAS accurately is a necessity for exploring the mechanism and developing new diagnosis and treatment approaches of WAS disease.Rabbits were widely used as reliable models of many human diseases.The physiology,anatomy,and genetic background of rabbits are more similar to those of humans than those of mice.Therefore,our intention is to generate a gene editing rabbit model that can authentically recapitulate human WAS.In this study,two sgRNAs targeting the exons 2 and 7 of WAS gene were designed.Ten rabbits were obtained by microinjecting the two sgRNAs and Cas9 mRNA into the cytoplasm of the rabbit prokaryotic stage fertilized eggs.9 out of the 10 rabbits carried the expected mutation.The gene editing efficiency was as high as 90%.Through further breeding,we obtained 33 offspring,of which 17 were heterozygous and 8 were homozygous with mutations including indel or large fragment deletion.Western blotting results showed that WASP was completely eliminated in the WAS KO rabbits(WAS-/or WAS-/-)and decreased by half in the WAS+/-rabbits.Peripheral blood analysis exhibited that the platelet and lymphocyte numbers of the WAS KO rabbits substantially decreased.Further analysis revealed that the peripheral T cells of WAS KO rabbits significantly decreased.Especially,considerable reduction of CD8+T cells also were found in the peripheral blood.The substantial reduction of lymphocytes,particularly T cells,would make WAS KO rabbits susceptible to infection of microbial pathogens.Correspondently,we found that most WAS KO rabbits developed severe infections,indicating that the immunity of WASKO rabbits was indeed impaired.Consistent with the severe reduction of platelets in WAS KO rabbits,subcutaneous hemorrhage and massive hyperemia in inward organs including lung,stomach and kidney were observed in WAS KO rabbits,indicating that WAS KO rabbits have a bleeding tendency.In addition,the growth and survival of these rabbits were evaluated.The KO rabbits had a lighter body weight and a shortened lifespan compared with that of the wild type rabbits.In this study,we first established a WAS rabbit model by CRISPR/Cas9.These rabbits completely lack WASP,and mimic the typical symptoms of WAS patients including severe thrombocytopenia,bleeding tendency,peripheral lymphopenia,severe infection and greatly reduced lifespan.This novel rabbit model will be an important tool for further study of WAS mechanism and clinical treatment.