Gene Editing Of Duchenne Muscular Dystrophy Through Novel Biological Nanoparticles

Duchenne muscular dystrophy(DMD)is an X-linked recessive genetic disease with high mortality rate.The dystrophy gene mutation results in the loss of dystrophin protein expression,which disrupts the stability of the muscular membrane structure.DMD disease cause huge physical and mental pains as well as financial burdens to both patients themselves and their families.Up to date,existing therapies for this disease mainly focus on delaying the development of the disease and cannot cure the disease fundamentally.Therefore,it is particularly urgent to develop effective gene therapy methods to cure this gene-deficient disease.The best treatment strategy for genetic diseases is gene editing or genome editing that is correcting the wrong genes and restoring normal gene expressions.Currently,DMD gene editing therapy are also faced with the same problem,that is the effectiveness and safety of many experimental therapies need to be improved at the same time.The delivery system of gene editing vectors is the key factor to the function of gene drugs.Therefore,based on the previous studies from our lab,the third-generation gene editing tool CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats/associated protein 9)was selected as a gene editing therapy tool,and a lentivirus-like particle(LVLP)-based Cas9 ribonucleoprotein(RNP)delivery system was innovatively designed in this study,hoping to achieve an efficient,rapid and safe gene editing of the genome.This Cas9/sgRNA RNP packaging and delivery system is attempted to be used to restore the dystrophin reading frame by a single sgRNA-guided gene editing cleavage method,and in this way,the host’s tissue cells can produce truncated but partially functional dystrophin to achieve the purpose of treating DMD.The topic of this study mainly includes two parts:Part one:Construction of an efficiency gene editing system for Cas9/sgRNA ribonucleoprotein delivery based on lentiviral capsid-based bionanoparticlesFirst of all,a plasmid co-expressing Cas9 protein and aptamer-modified sgRNA was constructed,and the ribonucleoprotein formed by Cas9 and sgRNA was packaged into LVLP by using the specific interaction of aptamer MS2 and aptamer MS2 coat protein(MCP).To find out the appropriate positions for aptamer replacement,we transfected plasmid and transduced packaged lentiviral particles into the constructed IL2RG-HBB-GFP reporter cell line.It was found that there are 3 positions suitable for aptamer replacement in sgRNA,namely Tetraloop,ST2 loop and 3’end.Among them,aptamer MS2 replacing sgRNA Tetraloop can obtain the best gene editing activity(about 8.1%GFP positive cell rate)of Cas9/sgRNA RNP.On this basis,aptamers and aptamer-binding proteins were selected from the four aptamer and aptamer-binding protein groups of MS2/MCP,PP7/PCP,Box B/λN22 and com/COM,and the com/COM selected as the best packaging sgRNA aptamer and aptamer-binding protein group(producing about 11%GFP-positive cell rate).The Cas9 protein expression in Cas9/sgRNA RNP was confirmed by Western blotting.In order to verify that the gene editing activity from Cas9/sgRNA RNP,a plasmid co-expressing Cas9 protein and aptamer-modified sgRNA was split into two plasmids expressing only Cas9 protein or only sgRNA.Plasmid transfection and lentivirus-like particle transduction experiments were repeated,and the experimental results showed that co-transfection of two plasmids into the IL2RG-HBB-GFP reporter cell line yielded a GFP-positive cell rate of approximately 17%which is similar to the previous experiment(16%),and the two plasmids were packaged separately into LVLP and co-transformed in the IL2RG-HBB-GFP reporter cell line,almost no gene editing activity(1%)were detected.This result indicates that Cas9 protein can protect sgRNA during the process of transducing cells.Furthermore,the effect of aptamers on LVLP packaging was confirmed by using real-time quantitative PCR(rt-qPCR).The results showed that although the presence of aptamers reduced the packaging efficiency of LVLP by about 11%,there was no significant difference in the overall packaging number of lentiviral-like particles.The packaging efficiency of com~+HBB sgRNA1 into LVLP is 50 times higher than com~-HBB sgRNA1 in the presence of Cas9 protein.Combining Western blotting with real-time quantitative PCR results,we found that the aptamer com-modified sgRNA was essential to package Cas9/sgRNA RNP into the core capsid of LVLP,and the complex formed by aptamer com-modified sgRNA and Cas9 protein could protect sgRNA during LVLP transduce cells.Since the purpose of purifying Cas9/sgRNA RNP is to concentrate virus particles with a diameter of less than200 nm,many membrane structures(such as exosomes)also exist in the concentrated virus.Meanwhile,under transmission electron microscopy,the size of common lentiviral particles is basically the same as that of LVLPs with or without sgRNA,and there are a considerable number of exosomes.After removing the membrane structure of the concentrated virus with STE solution containing 0.5%Triton X-100 and 10%sucrose,it was found that aptamer-modified sgRNA can increase the anti-wash LVLP concentration by about 5.8 times.In addition,we found that a single LVLP we produced contains about 600 RNPs which normalized to the commercial Cas9 protein standard purchased from the company by Western blotting.Based on the next-generation sequencing results of g DNA from LVLP-infected samples,RNP LVLP showed high gene editing efficiency(35.5%on-target gene editing efficiency)and extremely low off-target effects(off-target rate 0.9%).Additionally,compared with other viral vectors,the amount of RNP we produced is more than 10 times lower compared with biochemically purified RNP.Finally,the Cas9/sgRNA RNP LVLP transduced in this study showed the gene editing ability 11 hours after transduction by using the Incu Cyte S3 monitoring system.In summary,the Cas9/sgRNA RNP delivery system innovatively designed in this study can achieve efficient,fast and safe gene editing.Part two:A study of using novel bionanoparticles gene editing for Duchenne muscular dystrophyAfter successfully developed the highly efficient,fast and safe Cas9/sgRNA RNP LVLP delivery system,we would like to verify the strategy working efficiency in our humanized mouse model of hDMDdel52/mdx(the deletion exon 52 is integrated into mouse chromosome Gene 5 of human DMD patients).This project attempts to treat DMD by use gene editing technology to restore the dystrophin reading frame in vitro and in vivo experiments.Firstly,the CRISPOR system was used to predict all available single-cut sgRNA target sites near exon 52 of DMD.Combined with the DMD53-GFP reporter gene detection system we designed and constructed,these sgRNA target sites were tested for gene editing activity.Subsequently,next-generation sequencing was used to further analyze the percentage of Indel restore dystrophin reading frames by gene editing,from which three sgRNAs,Sa Cas9-DMD53-g2(19.5%GFP-positive cell rate),Sp Cas9-DMD53-g1(21%GFP-positive cell rate)and Sp Cas9-DMD53-g2(21.4%GFP-positive cell rate),were selected for LVLP packaging experiments.Then,LVLP transduction experiments of three kinds of sgRNA were carried out in myoblasts.The results of next-generation sequencing showed that the gene editing trend of Cas9/sgRNA RNP prepared by different sgRNA in myoblasts was consistent with that found by GFP reporter gene detection system.Among them,Sa Cas9-DMD53-g2 produced 0.11%indel rate(PAM sequence polymorphisms,can’t guide Sa Cas9 cut target sequence),Sp Cas9-DMD53-g1 produced 19.1±1.1%indel rate and Sp Cas9-DMD53-g2 produced 31.8±2.5%Indel rate.Applying our new Cas9/sgRNA RNP LVLP delivery system for gene editing can generate more than 50%Indel rate in host cells,and more than 50%of them are Indel types that can reconstruct the dystrophin reading frame(insertion 3n+2 bp).At the same time,we used double-cut sgRNA as control and intuitively detected by agarose gel electrophoresis,the high concentration of Cas9/sgRNA RNP showed extremely high editing efficiency which is close to 100%in HEK293T cells,however,only 20%in myoblasts.In addition,the analysis results show that in addition to targeting their respective regions,some of our selected sgRNAs also target exon splicing enhancer sequences,which will cause exon 53skipping.In the negative control(myoblasts treated with RNP not targeting DMD exon 53),54.3%of the cDNA showed exon 53 skipping,while in the myoblasts treated with Sa-gRNA1 RNP,Sp-gRNA1 RNP and Sp-gRNA2 RNP,58.5%,56.4%and 45.3%respectively skipping rate were observed.This indicates that we have not detected any significant increase in exon 53 skipping in exon 53 sgRNA-targeted myoblasts.Therefore,we cannot use the same sgRNA to simultaneously cause the reconstruction of dystrophin reading frames and exon 53 skipping to increase single-cut sgRNA to restore dystrophin expression.Next,RNA was extracted from muscle progenitor cells(which extracted from mouse muscle)and differentiated mouse myoblasts,and cDNA accordingly was reverse-transcribed for next-generation sequencing of the DMD53 region.The results showed that the longer of in vitro culture time(up to 14 days),the higher the expression of spontaneous dystrophin in cells.The same results were found in in vivo experiments in hDMDdel52/mdx mice.Four weeks after Sp Cas9-DMD53-g2 RNP was injected into hDMDdel52/mdx mice,dystrophin staining of posterior thigh muscles’cryo-sections showed that hDMDdel52/mdx mice restored some dystrophin expression.In a word,in the process of exploring a fast,safe and effective DMD gene editing therapy,we first designed and constructed a new Cas9 gene editing delivery system.The delivery system takes lentivirus like particles as the carrier and uses the specific interaction of aptamer and aptamer binding protein to package Cas9/sgRNA RNP into lentivirus-like particles.To some extent,this delivery system innovates the delivery mode of Cas9 protein in gene editing,which not only improves the efficiency of gene editing,but also increases the biosafety of gene editing without reducing the infectivity.In addition,using this new and efficient delivery system,gene editing of DMD was attempted to restore dystrophin expression in vitro and in vivo experiments,and restored dystrophin expression to different degrees.