FBN1 Gene Mutations Lead To Poor Prognosis Of Aortic Dissection And Its Gene Editing Repair Studies

BackgroundDistal aneurysmal expansion of false lumen of aortic dissection(DAEAD)is a manifestation of aortic dissection progression.After long-term follow-up,one out of every three Stanford aortic dissection patients developed DAEAD after endovascular isolation,and the vast majority of patients required re-treatment during long-term follow-up.However,once it progresses to DAEAD,due to the high complexity of its anatomy,DAEAD with extensive involvement of visceral branch arteries has high risks whether open surgery or endovascular treatment is used.Currently,there is increasing evidence that aortic disease is associated with genetic mutations.Addressing genetically related aortic disease from a gene therapy perspective faces two problems.On the one hand,there is a lack of clear and effective delivery vectors for gene repair tools to reach aortic target sites.On the other hand,how to perform gene editing for genetically related aortic diseases is also an urgent problem to be studied.Therefore,this study intends to explore the correlation between DAEAD and gene mutation from the perspective of genetic research;to explore suitable AAV serotypes as delivery vehicles for gene therapy;and to explore whether the sequence repair of primary aortic smooth muscle cells in patients with Marfan syndrome with FBN1 point mutation can be achieved at the genetic level.Objective1.To explore the clinical and genetic correlation of aneurysmal dilation distal to the false lumen of aortic dissection.2.To explore the adeno-associated virus(AAV)serotypes that can best infect the aortic root and thoracic aorta,and the infection situation of AAV9 with the highest infection efficiency in different segments of the aorta was compared and analyzed.3.Through CRISPR/Cas9 technology,the repair of primary aortic smooth muscle cells from patients with Marfan syndrome with FBN1 point mutation is realized.Methods1.Retrospectively analyzed the patients with aneurysm-like dilation distal to the false lumen of aortic dissection and normal postoperatively diagnosed by imaging in our center from April 2013 to October 2020.Disease gene analysis and comparison with healthy control alleles from healthy people in the database.2.Using molecular cloning technology,after targeted design into the nucleus and carrying a plasmid expressing red fluorescence,the viruses of different AAV serotypes(AAV1,AAV2,AAV4,AAV6 and AAV9)were packaged,and the C57 mice were injected into the tail vein.The infection efficiency at aortic root and thoracic aorta was compared from immunofluorescence and real-time quantitative PCR.In addition,the infection efficiency of AAV9 in different segments of the aorta was compared and analyzed.3.Using CRISPR/Cas9 technology,after screening suitable sgRNAs,repair the primary aortic smooth muscle cells of patients with Marfan syndrome with FBN1 point mutation,and verify the sequence before and after repair.Results1.The long and short diameters of the false lumen in the celiac trunk plane,the left renal artery plane and the right renal artery plane in the DAEAD group were longer than those in the non-DAEAD group,and the short diameter of the false lumen in the largest infrarenal plane was longer than that in the non-DAEAD group,and the differences were statistically significant(P < 0.05).Genetic analysis showed that the pathogenic variant rate was 58.3%(7/12)in the DAEAD group,and no pathogenic variant was found in the non-DAEAD group.At the same time,3 variants of unknown significance(VUS)were found in the DAEAD group;the VUS risk variable analysis was further extended to important pathways related to aortic aneurysm and dissection.more than the healthy control group,the difference was statistically significant(P<0.05).2.A Plasmid expressed in the nucleus and expressed red fluorescence was successfully constructed.The infection efficiency at the aortic root and thoracic aorta was counted and found that there were significant differences between AAV1,AAV4,and AAV9,except that AAV2 and AAV6 had no infection(P<0.05).Among the different serotypes(AAV1,AAV2,AAV4,AAV6,and AAV9),AAV9 was the best infecting aortic root and thoracic aorta,followed by AAV4,and AAV1 had almost no infection.Neither AAV2 nor AAV6 were infected.In the infection of AAV9 in different segments of the aorta,the infection efficiency of each segment was not the same,and the difference was statistically significant(P<0.05).Except for the infection between the aortic arch and the thoracic aorta,the abdominal aorta and the brachiocephalic trunk,there was no significant difference(P>0.05),and the differences between the other segments were statistically significant(P<0.05).The infection efficiency of AAV9 in different segments of mouse aorta was not found in renal artery and iliac artery.The infection efficiency of other segments was: aortic root was the best,followed by aortic arch and thoracic aorta,abdominal aorta and brachiocephalus Dry infections are minimal.This suggests that AAV9 is differentially responsible for the infection of different segments of the mouse aorta.3.In this part of the experiment,the primary smooth muscle cells were cultured after the tissue was obtained from the aorta of the Marfan patient(c.3959G>T)with early onset and heterozygous single base mutation.By means of sanger sequencing,combined with the mutation sites,related sgRNAs were designed,and after screening of three sgRNAs,the best sgRNA was selected.Gene editing of primary aortic smooth muscle cells using CRISPR/Cas9 technology.Sanger sequencing verified that the sequence of FBN1 was successfully repaired by single-base heterozygous mutation before and after repair,and the normal changes were restored.Conclusions1.This study suggests that some known causative genes of aortic disease are not only associated with the pathogenesis of aortic dissection,but also with poor prognosis of its long-term progression.The occurrence and development of DAEAD is genetically related,and it may be a highly penetrant pathogenic variant in a monogenic disorder or a combination of multiple genes.2.The results of animal experiments showed that AAV9 had the best infection efficiency at the aortic root and thoracic aorta of the five serotypes AAV1,AAV2,AAV4,AAV6 and AAV9,followed by AAV4.However,AAV9 has the best infection efficiency in the aortic root in different segments of the aorta(aortic root,aortic arch,thoracic aorta,abdominal aorta,brachiocephalic trunk,renal artery and iliac artery).The arterial root and thoracic aorta have obvious advantages over other serotypes.3.Gene editing of primary aortic smooth muscle cells using CRISPR/Cas9 technology.Sanger sequencing verifies that the sequence of FBN1 is successfully repaired by single-base heterozygous mutation before and after repair and demonstrated the possibility of successful repair of gene editing in primary aortic smooth muscle cell lines with FBN1 mutation in patients with Marfan syndrome.