CRISPR/Cas9-mediated Anti-HIV-1 New Strategy And Molecular Mechanisms Of Drug Resistance Of Acute Myeloid Leukemia

Precise genetic modification can be achieved at the cellular level and contributes to disease prevention and treatment.To achieve this purpose--the researchers could take advantage of genetic engineering technologies including knocking out disease-related genes,correcting the mutated genes or inserting a protective gene.Nowadays,highly active antiretroviral therapy(HAART)is the most effective treatment for AIDS.However,even though the treatment can control disease progression and prolong the life of patients,it cannot eradicate HIV virus which has infected host cells successfully and is latent in virus reservoir.Furthermore,it is of high-cost and long-term drug treatments can lead drug resistance.There is no effective vaccine targeting HIV.Therefore,it is urgent to develop new therapy for AIDS.The famous Berlin patient rekindled interests in gene therapy for AIDS.More and more researchers want to achieve the similar effect by using genome engineering technologies.Researchers isolate cells from patients and confer them resistant to HIV infection after modifications in vitro and infuse modified cells back to patients.CRISPR/Cas9 system is based on small guide RNA(sgRNA)which directs Cas9 nuclease to make DNA double strand breaks causing disruption of gene expression.It is effective and reliable to precisely modify genes in living cells.In this study we aim to specifically knock out the co-receptor CXCR4 which mediates the entry of HIV-1.We designed ten sgRNAs and five out of ten showed efficiency in CXCR4 gene editing while two of them could ablate CXCR4 with a high efficacy.We acquired loss-of function mutations of CXCR4 effectively in Ghost cell line,Jurkat T cells and human and Rhesus primary CD4+ T cells through CRISPR/Cas9 system.After modifications,the cells were resistant to HIV-1 infection.We did not find off-target effect in the study.Our study provided a theoretical basis for the gene therapy of AIDS,which indicated that CXCR4 may be potentially used as a target in the gene therapy of AIDS.Due to its high efficiency in genome editing and the ability of targeting multiple genes or multiple sites in one gene,CRISPR/Cas9 system is applicable in genome-wide screen.RNA interference can only knock down the expression of genes transiently with high off-target efficiency.It is limited to transcribed genes.Combined with sgRNA library,CRISPR/Cas9 system is more sensitive and effective and it can target almost of all the DNA sequences.The gain of function mutations in FMS-like tyrosine kinase 3(FLT3)is the most common active mutagenesis in acute myeloid leukemia(AML)which is considered as a poor prognosis.Quizartinib(AC220)is the second generation small-molecule inhibitor of FLT3 which can inhibit FLT3 potently with a high kinome selectivity.It is now in clinical II trial and can be used to treat relapsed and refractory AML patients.Our study applied the second generation of human CRISPR/Cas9 library to perform an unbiased genome-scale screen in AML MV4-11 cells for genes critical for AC220 drug resistance.In our findings,loss of function of some genes may lead to drug resistance to FLT3 inhibitor AC220.Compared with wild-type cells,knockout cells showed enhanced tolerance to AC220.Next,we chose SPRY3 which is an intracellular inhibitor of FGF signaling and GSK3,a canonical Wnt signaling antagonist to validate our screen.When we knocked out SPRY3 or GSK3 respectively in AML cell line or primary cells from patients,the knock-out cells were highly resistant to AC220.The IC values of SPRY3 and GSK3 knockout MV4-11 cells were approximately 8 nM and 6 nM respectively,while the IC value of wild type cells was only 2 nM.We also analyzed some patient samples and find that the expression of SPRY3 and GSK3 are lower in patients who are not sensitive to AC220 than those who are sensitive.Our research provides new insights for drug screen and determines the mechanism of drug resistance.On this basis,our study also provides the technical and theoretical support for further improving clinical AML treatment and may help futher development of drugs against AML.