Development Of A Cynomolgus Monkey Model Of Beta-Thalassemia Based On HBB Knockout

Beta-thalassemia is a kind of hereditary hemolytic hemoglobin disease caused by mutation of HBB,which leads to the decrease or total inability of the synthesis of beta-globin peptide chains used to synthesize hemoglobin.It can imbalance the alpha-globin peptide chains and beta-globin peptide chains ratio in red blood cells,and accelerate the rate of erythrocyte lysis.At present,very limited patients can get effective treatment by transplanting matched hematopoietic stem cell.Most patients can only slow down the diseases development process through regular blood transfusion therapy,but it can increase the iron level in the blood and have serious complications,including endocrine glands and major organ damage.There is urgent need of an ideal beta-thalassemia disease animal model which can be used for vivo tests to investigate the physiological and pathological mechanism of beta-thalassemia diseases development and potential new drugs clinical trials.CRISPR/Cas9 technology is a widely used gene editing technology.It is simple,high efficient,and low cost technology.It plays an important role in constructing gene mutation animal models.In this study,CRISPR/Cas9 gene editing technology was used to target and knockout the HBB gene coding region in the cynomolgus monkey.It can cause abnormal expression of HBB gene and defective production of beta globin peptide chain to construct a cynomolgus monkey model of beta-thalassemia disease.The experimental design,content and results of this paper are as follows:1.Design and construct the vector.Three sg RNAs targeting the second exon of the HBB gene of cynomolgus monkey were designed and transcribed in vitro to prepare the vector.2.Verify the efficiency of three sg RNAs on cynomolgus monkey embryos and cultivate HBB knockout cynomolgus monkeys.The prepared vector was microinjected into embryos of the cynomolgus monkey,and blastocysts were obtained after 7 days of culture in vitro.T7E1 digestion and sequencing of 10 blastocysts showed that 80%(8/10)of the embryos were targeted for cleavage by Cas9 nuclease.Then,a newborn monkey was obtained by embryo transplantation,named the KO monkey.And its blood and ear tissues were collected for sequencing.It was found that a 217 bp deletion occurred in the same site of the second exon of the HBB gene.3.Phenotypic analysis of the KO monkey.(1)From birth to the age of 8 months,hematological tests showed that the levels of hemoglobin,red blood count and hematocrit gradually decreased with development,and were about 50%lower than those in the control group.(2)Blood smears showed that it had appeared the typical erythrocyte morphology of beta-thalassemia,such as target shape,acanthocyte,water drop-shaped and fractured.(3)Hemoglobin electrophoresis combined with q PCR showed that the production of beta globin was defective.And the content of beta globin in the control group was about 10~5times higher than that in the KO monkey.(4)Morphological analysis showed that its skin and ears were pale and bloodless,and the fur was yellowish and messy.(5)Growth curve showed that the growth and development of this KO monkey was slower,especially at the age of 8 months.The weight of this KO monkey was reduced by about 30%compared with the control group.(6)The results of B-mode ultrasonography of spleen and liver showed that the density of spleen tissue increased.And the thickness of spleen tissue increased by2.6 mm,1.43 times higher than that in the control group.The length of spleen tissue increased by 6.45 mm and 1.23 times higher than that in the control group.The maximum oblique diameter of the right lobe of the liver increased by 4.56 mm,1.18 times higher than that in the control group.Those all indicated that the enlargement of the liver and spleen,accompanying by fibrous hyperplasia of the spleen.(7)Analysis of behavioral activities found that the activity ability of the KO monkey was weak.The average time of inactive behavior was about 3.57 times higher than the control group,and the average time of exploration behavior in the control group was about 1.63 times that of the KO monkey.With the increase of age,this KO monkey began to have special behavioral activities such as supining,lying prone,suddenly falling down.Based on the above experimental results,we successfully developed a cynomolgus monkey as beta-thalassemia disease model,which provides an opportunity to investigate the mechanism of beta-thalassemia diseases development and run animal model based clinical trials.