PGEA-based Gene Vectors Mediated CRISPR/Cas9 Delivery In Vivo For Cardiovascular Applications

CRISPR/Cas9 has been widely used as an emerging gene editing tool for gene editing in vivo and in vitro,Including correction of disease-causing genes,verification of functional genes,etc.However,due to the large size of CRISPR/Cas9,effective and safe delivery poses the major obstacle for their further development toward human applications.Viral vectors are often limited by their immunogenicity and packaging capacity.Non-viral vectors are gaining more and more attention due to their flexibility,but are also restricted by lower transfection efficiency and higher toxicity.Although various vectors were reported for CRISPR/Cas9 delivery in tumor diseases,their use in cardiovascular diseases is minimal.Pullulan or cholesterol-terminated PGEA vectors showed great potential for gene delivery.In view of the above problems,this paper based on the gene carrier of PGEA to achieve an effective CRISPR/Cas9 system delivery for cardiovascular disease.The proprotein convertase subtilisin/kexin9(Pcsk9)is mainly expressed in the liver and small intestine,and is an important target for the treatment and prevention of coronary heart disease(CHD).There are currently clinically available monoclonal antibodies of Pcsk9 for the treatment of CHD.But their price is very expensive,and multiple injections are needed to achieve therapeutic effects.Using CRISPR/Cas9 to edit Pcsk9 and reducing the side effects of systemic administration will provide a strategy for the treatment of coronary heart disease.Based on this we constructed a pullulan modified PGEA(Pul-PGEA)as a delivery vehicle for the CRISPR/Cas9 system.Pullulan has the potential for liver targeting and can reduce side effects of systemic administration.Pul-PGEA exhibits excellent CRISPR/Cas9 transfection performance and gene editing ability both in vivo and in vitro.Pul-PGEA/pCas9-sgPcsk9 nanoparticles could significantly reduce plasma Pcsk9 level and plasma total cholesterol levels.At the same time,Pul-PGEA also showed great potential for delivery of short-chain mimic-cy3 RNA and long-chain Cas9 mRNA.Mutations in Fbn1 are often associated with the development of aneurysms.It is very urgent for diagnosis and sequent gene therapy to determine whether these mutated variants are pathogenic or benign.Due to the complete endothelial barrier,effective delivery of CRISPR/Cas9 system to vasculatures remains a challenge for in vivo gene editing of genetic vascular diseases especially in aorta.In third chapter,a cholesterol-modified CHO-PGEA with abundant hydroxyl groups can deliver a plasmid-based pCas9-sgFbn1 system for gene editing of Fbnl was reported.This is the first report that a polycation-mediated CRISPR/Cas9 system for gene editing in aorta of adult mice.CHO-PGEA/pCas9-sgFbn1 nanosystems could effectively contribute to the knockout of exon 10 in Fbn1 in vascular smooth muscle cells in vitro,which led to the change of the phosphorylation of Smad2/3 and the increased expression of two downstream signal of Fbn1.For in vivo application,the aortic enrichment of CHO-PGEA/pCas9-sgFbn1 was achieved by administrating a pressor dose of angiotensin ?(Ang ?),resulting in efficient gene editing.Thus,the combination of CHO-PGEA/pCas9-sgFbn1 nanosystems with the Ang ?infusion could provide the possibility for in vivo gene editing in aorta.