Inhibition Of HBV Replication Using AAV-delivered CRISPR-Cas9 In Vivo

Hepatitis B is an infectious disease caused by Hepatitis B virus(HBV).According to the report of World Health Organization(WHO),about 2.4 billion people are chronically infected with Hepatitis B,with 0.68 million died of complications,including cirrhosis and hepatocellular carcinoma.Despite the achievement of Hepatitis B vaccines,curing HBV infection is still a knot.It is believed now that the only way of curing HBV infection is the thoroughly removal of cccDNA,yet current therapies failed to achieve such goal.The recent CRISPR(Clustered regularly interspaced short palindromic repeats)-Cas9 has drawn much attention in DNA editing because it is more simple,convenient and efficient compared to previous gene editing technology.CRISPR-Cas9 has now brought light to treatment of virus infection,genetic disease and chromosomal defect,these diseases were difficult to be cured in the past.Up to now,researchers including our group have successfully using CRISPR-Cas9 system to suppress HBV expression with high efficiency in cells and HBV hydrodynamics mouse models.These studies showed the great value of CRISPR-Cas9 in eliminating HBV infection.However current ways of using CRISPR-Cas9 in HBV trans-genetic mice,a mouse model of HBV which is more similar to HBV infected patients,didn't come to good results because of the lack of an efficient and safe gene transfer vector.The popular kind of Cas9 is SpCas9,short for Cas9 from Streptococcus pyogenes.Though SpCas9 is suitable for lentivirus and adenovirus,gene transfer vectors are not ideal either for high immunogenicity or for integration into the host's genome.Recently emerged recombinant Adeno-associated virus(r AAV)is widely used in research of nerve system,ophthalmology,liver,cardiac and carcinoma and in gene therapy because it is safe and efficient.But the size of SpCas9(7kp)is too large for AAV to load,use of AAV and CRISPR-Cas9 is delayed.Feng Zhang and his colleagues from Massachusetts Institute of Technology(MIT)published their lasted research on nature in May,2015.Their article revealed a new kind of Cas9,SaCas9(Staphylococcus aureus Cas9),and its perfect combination with AAV.According to their result,the efficiency of Sa Cas9 is equal to SpCas9,which provides hope for using CRISPR-Cas9 to inhibit HBV DNA in vivo.The purpose of this study is to use rAAV8 mediated CRISPR-SaCas9 system to inhibit the expression of HBV in HBV trans-genetic mouse.Through continuous monitoring to measure the efficiency,duration,inhibitory effect,potential off-target effects and damage of animals.1.Screen for efficient HBV targets that suits CRISPR-SaCas9Our group had successfully inhibiting expression and replication of HBV in cell models and hydrodynamics mouse models.But,different from the PAM sequence of CRISPR-SpCas9,which is NGG,PAM of CRISPR-SaCas9 is NNGRRT.Thus we need to screen for new targets for SaCas9 under the circumstance of lacking target predicting tools.(1)A new method of design targets on HBVThe mouse model we used in this study has a 1.28 copy of HBV full sequence integrated into mouse chromosome.But to obtain targets suitable for different genotypes of HBV genome,we compared the genome of 26 different sources and genotypes of HBV,then use Vector NTI Advance 11.5.1 software to search for area with the PAM of NNGRRT,eventually we got 21 target sequences.(2)Screen for an efficient target of HBVWe constructed 21 CRISPR-Sa Cas9 expression vectors from the targets we selected and co-transfected these expression vectors with HBV expression vector into 293 T and HuH-7 cells separately to test their efficiency in inhibiting HBV antigen and HBV DNA by corresponding detection methods.Experimental results showed all targets could inhibit the expression of HBV on different levels and do no harm to cells.Of which,Sa4 on Pre C region of HBV shows the highest efficiency,Sa4 could suppress HBsAg and HBeAg in cell supernatant on the same time.The shearing action of Sa4 on HBV is versified by T7 EI experiment.2.Using rAAV8::CRISPR-SaCas9 to inhibit expression of HBV in HBV transgenetic mouse efficiently(1)Package of SaCas9 into recombinant adeno-associated virus(AAV)Different serotypes of AAV have different tissue specificity.According to recent reports,rAAV8 has the highest liver specificity and barely infects other organs.To reduce bad influence of other organs in mice,we use recombinant adeno-associated virus(rAAV8)as vectors to transfer CRISPR-SaCas9.Based on our highly efficient gRNA-Sa4,we packed rAAV8:Sa4 in HEK293 cell line with the help of pAAV-RC and pHelper plasmid.Viruses were purified using chloroform.According to the result of dot hybridization,the drop of the virus we packed reached 1012 v.g./mL.(2)Appraisal of HBV trans-genetic mouseThis mouse model we used has a 1.28 length of HBV full sequence integrated into mouse chromosome,with HBsAg level of 4000-6000 IU/m L,HBeAg reaches 800-1000 S/CO and HBV DNA of 108 v.g./mL in serum.HBc Ag could also be detected in the liver.This HBV trans-genetic mouse model we use is ideal.(3)Assessment of the effect of low dosage CRISPR-SaCas9Based on Randomly grouping,different dose of rAAV8::CRISPR-SaCas9 virus was injected through tail vains into 4-6 weeks male HBV trans-genetic mice.Continue monitoring revealed low dose of virus in vivo(5×1010v.g.per mouse)failed to inhibit HBV replication,no difference was discovered between experimental and control group.(4)High dosage of CRISPR-SaCas9 inhibit replication and expression of HBV in vivoHigh dose of virus(2×1011v.g.per mouse)exists and successfully surprised the expression of HBsAg,HBeAg and HBV DNA in serum for 38 days long,HBsAg and HBeAg in serum decreased 62.96 ± 7.59% and 65.18 ± 3.08% separately in experimental group,while the drop of HBV DNA in serum is 92.82 ± 3.67%.Analysis of the liver immune fluorescence slice showed 76.88 ± 18.39% decrease of HBcAg,the difference of experimental and control group is visually obvious.Liver HE staining of the control group mice has obvious inflammatory infiltrates and hepatomegaly,while experimental mice did not have such pathological changes.In the same time,we noticed rAAV8:Sa4 existed 38 days in HBV trans-genetic mice with no harm done to animal organs.No off-target effects were detected and no harm to mice was found.No experiment animal was found abnormal or dispirited.The effect of rAAV8:Sa4 on HBV genome is verified by T7 EI experiments and deep seqeuncing.In conclusion,we have selected a highly efficient target of CRISPR-SaCas9 targeted HBV C region,and first present a highly efficient study on inhibiting expression and replication of HBV DNA using CRISPR-Cas9 in HBV trand-genetic mice.The result of our study will developing application of CRISPR-Cas9 in anti-virus infection.Our result would benefit and provide theory and technical support for the use of CRISPR-Cas9 in curing hepatitis B.