| Background and ObjectiveSpinal muscular atrophy(SMA)is a lethal neurodegenerative disease characterized by progressive myasthenia and amyotrophy.The majority of SMA patients are caused by homozygous deletion of Survival Motor Neuron 1(SMN1).In humans,a highly homologous gene SMN2 can partially compensate for the deletion of SMN1.However,some different bases make SMN2 produce more truncated transcript without exon7,and then produce truncated SMN protein,which is malfunctional and easily degradable.More importantly,all SMA survivors exist at least 1 copy SMN2.Therefore,to increase the full length transcript ratio produced by SMN2 to completely compensate for the deletion of SMN1 is an important strategy of SMA gene therapy.Base editing was derived from CRISPR/Cas9 systems.By fusion express mutant Cas9 and deaminase,Base editor can directed mutant one or more bases under the guide of sg RNA without DNA double strands breaks.Previous study show that mutant SMN2 exon 7 by base editor can increase the SMN2 full length transcript of neuron and make a proof of the principle that base editor can apply in the SMA gene therapy.However,bystander effect can lead to missense mutation,which is hard to avoid when editing exon,and make it difficult to evaluate the safety of in vivo gene therapy.Thus,the previous strategy needs to be optimized.In this study,we try to mutant the splicing juctions of SMN2 intron7 by high-fidelity base editors in HEK-293 T cells and SMA patient induced pluripotent stem cells,which may lead to the retention of intron7 and subsequently avoid the exclusion of exon7 in these cells.Then,we will evaluate if this new strategy can increase the expression of exon7-including transcripts and full length SMN protein.Methods1.We designed and constructed 9 adenine base editor and cytosine base editor plasmids targeting SMN2 intron7 splicing sites and transfected HEK-293 T cells by liposome.Then,fluorescent protein positive cells were purified by flow cytometry and evaluated editing efficiency by Sanger sequence and next generation sequence.SMN2 transcripts were detected by reverse transcription(RT)-PCR.To confirm transcripts alteration of specific mutations,we transfected HEK-293 T by mutative SMN2 minigene,and detected miniSMN2 transcripts by RT-PCR.2.The pluripotency of SMA patient derived induced pluripotent stem cells(SMA-i PSCs)were detected by immunofluorescent staining.High efficient base editor plasmid were transfected into SMA-i PSCs by electroporation and several edited cell monoclonal cell strains were established and then genotyped by Sanger sequence and next generation sequence.The SMN2 transcripts alteration of these cell strains were detected and quantify by RT-PCR and q PCR.The SMN protein expression were semi-quantify by western-blot.Result1.SMN2 intron7 splicing junction acceptor-2A was mutant into G by ABE with 70% efficiency and produced a long transcript with intron7 retention in HEK-293 T cells.2.SMN2 exon7 G52 and G53 were mutant into A by CBE which can increase the SMN2 full length transcript in HEK-293 T cells.Mutant minigene also confirmed that G52 A and G53 A mutations can increase mini-SMN2 full length transcript.3.Edited i PSCs monoclonal cell strains with different copy numbers of A-2G mutation were established by ABE.Similarly,these edited cell strains produced a long transcript with intron7 retention.The exon7-included transcript(i.e.,add up long transcript with intron7-retentive and full length transcript)of all edited cell strains were higher than unedited SMA-i PSCs.The SMN protein of most edited cell strains was higher than SMAi PSCs.Conclusions1.A-2G mutation in SMN2 intron7 splicing junction acceptor can produce intron7-retentive transcript.This study gives proof of the principle of using base editors to induce intron7-retention for SMA gene therapy.2.G52A and G53A mutations in SMN2 exon7 can significantly increase the quantity of full length transcript which are candidate loci for SMA gene therapy using base editors. |
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