Improve The Inclusion Of Exon7 Of SMN2 Gene In Spinal Muscular Atrophy Based On Adenine Base Editing System

Background and ObjectiveSpinal muscular atrophy(SMA)is an autosomal recessive genetic disease with progressive myasthenia and muscular atrophy caused by characteristic involvement of motor neurons in the anterior horn of the spinal cord.It is the most common fatal neurogenetic disease in infants and young children.The pathogenic gene of SMA is the survival motor neuron(SMN)gene 1,which encodes a full-length SMN protein involved in the splicing of precursor messenger RNA(pre-m RNA),axon growth and synergistic anti-apoptotic functions of Bcl-2 protein family.There are highly homologous SMN1 and SMN2 genes in the human genome,and a key base difference in exon7 c.840C>T causes the pre-m RNA alternative splicing of these two genes to be different,resulting in SMN2 gene mainly producing exon 7 deletion proteins,SMN?7.The majority of SMA patients had homozygous deletion of SMN1 gene,while the highly homologous SMN2 gene could not compensate for the loss of SMN1gene function because it could not produce sufficient full-length SMN protein.Therefore,increasing the expression of full-length SMN protein is the main strategy for the treatment of this disease,SMN2 gene is a potential target for the treatment of SMA.The antisense oligonucleotide treatment of SMA,Nusinersen,requires repeated intrathecal injection and is expensive,so it is urgent to explore new therapeutic methods.The successful application of the third-generation CRISPR/Cas9 gene editing technology in mammalian cells has shed light on gene therapy research for SMA.The classic CRISPR/Cas9 system has two enzyme active domains-Ruv C digestion activity and HNH digestion activity.The two active domains can respectively cut DNA double strands to form DNA double strand breaks(DSBs).The10th amino acid aspartic of Cas9 protein is mutated to alanine(D10A)to inactivate the Ruv C enzyme cleavage domain of the protein(ie Cas9 nicked,Cas9n),and the only remaining HNH enzyme cleavage activity cut the single-stranded DNA complementary to single guide RNA(sg RNA)to produce a single-stranded DNA nick Adenosine deaminase fusion expression with Cas9nicked(D10A)can realize the conversion of adenine to guanine,which was named Adenine Base Editing system(ABE).Under the guidance of sg RNA,the ABE editing system only cuts the single strand of DNA and uses adenosine deaminase to realize the substitution of A base to G base,which has been widely favored in gene therapy for human single nucleotide mutation diseases.Team early to set upthe source of SMA-I patients'i PSC cultivation and directional differentiation to get motor neuron system,at the same time has the SMN^-/-,SMN2^2TG/0and SMA-?7 two mice model.In this study,directed mutations(A-G)of SMN2 minigene were used to rapidly screen the effective sites,which can improve Exon7 inclusion.Adenine base editing system was used to screen the endogenous effective editing sites in HEK293T cells,SMA-I derived mouse embryonic stem cells,and SMA-I-derived induced pluripotent stem cell models and to identify the editing tools and sg RNA.The m RNA level of SMN-FL,the expression of SMN protein,motor neuron differenciation,the anti-apoptotic ability of SMN protein and the improvement of sn RNP assembly capacity in vitro were tested in SMA-i PSCs and splicing correction(SC)-SMA-i PSCs.The anterior horn organs of spinal cord were preliminarily differentiated by SMA-i PSC and SC-SMA-i PSC.Methods1.pSMN1,pSMN2 and pSMN2-minigene-Mut(A to G)were constructed and overexpressed in HEK293T,and the improvement of exon 7 inclusion SMN-FL m RNA level by A to G directed mutation was detected by RT-PCR and q PCR.2.sg RNA was designed for exonic splicing silencer of SMN2,four adenine base editing tools after editing the endogenous SMN2 in three cell types,comprehensively considering the editing efficiency,whether the editing site changes the amino acid sequence,and RNA off-target,and finally mini ABEmax is determined to combine sg RNA6 to carry out follow-upexperiments.3.Using mini ABEmax combined with sg RNA6 to edit SMA-i PSC to obtain SC-SMA-i PSC monoclonal cell line.Detection SMN-FL and SMN protein expression and anti-apoptotic capability of SMN protein;verify the effectiveness of mini ABEmax on post-mitotic motor neuron precursors and editing effects of cortical neurons in SMA-?7 mice.4.In order to ensure that the amino acid sequence of SMN protein was not changed after editing,the effective editing sites were further screened in the 3'UTR region of SMN2 gene,and the m RNA expression level of SMN-FL,the expression level of SMN protein and the improvement of sn RNP assembly ability in vitro were verified in the repaired SC-SMA monoclonal cell line,and the spinal cord anterior horn organ model derived from SMA and SC-SMA differentiation was preliminarily established.Results1.In the pSMN2-minigene-Mut directed mutation screening,13 sites on Exon7that can significantly increase the inclusion of exon7 and the level of SMN-FL m RNA were obtained,and there is one effective editing site on the 3'splicing site of E8.2.For the exonic splicing silencer of SMN2,four adenine base editing tools were used to edit endogenous SMN2 in three cell types.Comprehensively consider the editing efficiency,whether the editing site changes the amino acid sequence,RNA off-target,and other factors,mini ABEmax was finally determined to carry out follow-upexperiments,in combination with sg RNA6.3.mini ABEmax combined with sg RNA6 was used to obtain SC-SMA^{A36G A38G}and SC-SMA^A36Gmonoclonal cell lines.The m RNA level of SMN-FL,the protein expression of SMN and the anti-apoptosis ability of SMN protein of the corrected monoclonal cell lines were significantly higher than that of SMA,which were basically equal to or even higher than that of WT.mini ABEmax combined with sg RNA6 can effectively edit post-mitotic SMA motor neuron precursor cells and SMA-?7 mouse cortical neurons in vitro.4.An effective editing site was obtained in the 3'UTR region of SMN2 gene,and the SC-SMA-E8^A-10Gmonoclonal cell line was obtained by using mini ABEmax combined with sg RNA35.The amino acid sequence of SMN protein was not affected by editing,and the m RNA level of SMN-FL and the expression of SMN protein were higher than that of SMA,which were basically equal to or even higher than that of WT.The sn RNP assembly ability of SC-SMA was basically the same as WT.We preliminarily established a new protocol to differentiate the spinal cord anterior horn organs from SMA and SC-SMA,and completed the characterization of some cells.ConclusionsUse the optimized version of adenine single base editing system(mini ABEmax)to edit the endogenous SMN2 gene to obtain multiple SMA-strains through single or multiple base editing in exon7-ESSB and 3'splicing site of exon8.These splicing corrected i PSC strains show increase the expression of SMN-FL transcript and SMN protein.Through in vitro differentiated motor neuron anti-apoptosis experiments and in vitro sn RNP assembly experiments,it is confirmed that the increased expression of SMN protein has normal function,and spinal anterior horn organoid model is preliminary established.Comprehensive comparison of various factors,the E8^A-10Gsite is more preferred,which can be used as a therapeutic target for subsequent adult mouse gene therapy experiments.