Transcriptional Regulation And Gastric Cancer Oncolytic Virus Engineering Of LAT Gene Of Herpes Simplex Viruses

Herpes Simplex Viruses(HSVs)are common human Herpes pathogens,including Herpes Simplex Virus type Ⅰ(HSV-1)and Herpes Simplex Virus type Ⅱ(HSV-2).After Gendicine,the first oncolytic virus in the world,was approved for clinical usage in China in 2003,T-VEC(Talimogene laherparepvec)was approved by the US food and drug administration(FDA)in 2015 for the treatment of clinically advanced melanoma.Understanding the biological process of HSV is the basis for the development of HSV-based oncolytic virus.HSV can remain latent in neurons for years or decades after infection and can be activated in response to stress.LAT(latency-assistant transcript)gene is the key to regulate the latency and activation of HSV.However,the transcriptional regulatory sequences and transcriptional regulatory factors of LAT gene are rarely reported.This study intends to analyze the transcriptional regulatory sequence and transcriptional regulatory factors of LAT,construct the oHSV with the deletion of LAT,and explore the anticancer effect of oHSV in gastric cancer cells,so as to provide experimental basis for the development of new anticancer oncolytic virus drugs.Objective:To find transcriptional regulatory sequences and factors of herpes simplex virus LAT gene,and construct oHSV with LAT deletion,to provide experimental basis for the application of oncolytic herpes simplex virus type I in the treatment of gastric cancer.Methods:1.Sequence alignment determined the transcriptional regulation region sequence of the LAT gene of the new strain HSV-1-LXMW,2.Bioinformatics analysis of transcriptional regulatory sequences of LAT genes.3.The LAT guide RNA sequence was designed for LAT gene modification using CRISPR/Cas9 technology.4.Recombinant plasmids for LAT gene engineering were constructed.Results:In this study,the sequence of LAT gene transcription regulatory region of the new strain HSV-1-LXMW was determined.Through phylogenetic evolution analysis,it was found that HSV-1-LXMW was 99%similar to HSV-1 strain 17 in the UK.Using weight matrix matching software program,I found 10 LAT transcriptional regulatory sequence and nine transcription factors in 6 strains of HSV:AP-1,C-REL,Comp1,E2F,Hairy,HFH-3,Kr,TCF11/MAFG,v-Myb.By literature analysis I found the transcription factors v-Myb and AP-1 have the function of regulating the LAT transcription,especially AP-1 transcription factor has the function of regulating HSV latent infection and maintaining latent infection.Most importantly,seven other novel HSV LATs transcription factors and their corresponding transcriptional regulatory sequences were predicted.Further verification by multi-sequence comparative analysis showed that the transcription regulatory sequence of LAT gene of HSV contained 11 conserved regions.In HSV-1 or HSV-2 strains,LAT transcription regulatory sequences are conserved in Hairy,HFH-3,v-Myb,Comp1,C-REL and TCF11/MAfG binding sites,thus supporting the possibility that LAT transcription regulatory sequences may have biological function.Meanwhile,experimental data indicate that HSV-1 and HSV-2 have no common transcription regulatory sequences.These findings add to the understanding of the biology of HSV and transcriptional regulation of oncolytic HSVs.Based on the LAT sequence analysis of HSV-1-LXMW,gRNA of LAT sequence was designed and successfully cloned into PX458-LAT plasmid,which laid a foundation for the subsequent construction of recombinant oHSV and the treatment of gastric cancer.Conclusions:1.The LAT gene transcription regulatory region sequence of the newly isolated Chinese strain HSV-1-LXMW in our laboratory was determined,and it was found that its homology with HSV-1 strain 17 in the UK was as high as 99%.2.By analyzing the sequences of 6 HSV strains including HSV-1-LXMW,10 transcription regulatory sequences of HSV LAT gene and 9 LAT transcription regulatory factors(AP-1,C-REL,Comp1,E2F,Hairy,HFH-3,Kr,TCF11/MAFG and v-Myb)were first found.Among them,function of transcription regulators v-Myb and AP-1 have been verified in HSV literature,supporting their function in regulating and maintaining latent infection of HSV.The other seven are newly discovered HSV LAT transcription regulators.This finding has important implications for our understanding of the biology of HSV and the treatment of oncolytic HSV.3.The LAT gene sequence of the newly isolated Chinese strain HSV-1-LXMW in our laboratory was determined,the unique oncolytic HSV with the deletion of LAT was designed,and the gRNA plasmid for engineering oHSV with the deletion of LAT gene by CRISPR/Cas9 technology was constructed,which laid a foundation for the subsequent construction of oHSV-deltaLAT and its therapeutic effect study on gastric cancer.