Herpes simplex virus type 1 (HSV-1) is one of the well-known gene therapy virus vectors. Scientists have conducted in-depth research on the pathogenesis, clinical diagnosis and treatment of infection. As a highly safe therapeutic viral vector, the first oncolytic herpes virus (T-VEC) was approved for marketing by the FDA in 2015. Recently, another study has shown that the combination of oncolytic herpes virus and immune monitoring point drugs (PD1/PD-L1) can significantly improve the therapeutic effect of tumors. Therefore, the clinical application of oncolytic herpes virus is further broadened. One of the major issues that need to be solved in this industry is to figure out how to prepare, optimize and transform HSV-1 virus better and more efficiently.
A recent research paper published on PNAS systematically describes a method of constructing the oncolytic herpesvirus through fragment synthesis and multi-segment simultaneous optimization and transformation. The significant advantages of this method are: perfect fusion of gene synthesis technology and yeast recombination technology, rapid and complex modification and preparation of HSV-1 virus.
The KOS sequence in the HSV-1 genus—KOS-37 BAC (KOS genome clone in the form of BAC plasmid) was selected. The entire gene component was divided into 11 fragments, with an average of 14 kb per fragment. Each fragment has an overlapping area of 80 base pairs to prepare for the yeast assembly later. The 7th fragment was modified with BAC and YCp (yeast centromere sequence plasmid) so that it could replicate in both. The assembled HSV-1 genome is labeled KOSYA (KOS yeast assembled).
Through the combination of TAR cloning and cre-loxp technology, 11 fragments are separated and collected, each fragment is linked to the TAR cloning vector, and a PmeI cleavage site is introduced on the vector (this site will not cut the HSV-1 genome fragment). After PCR verification, it was transferred to E.coli for cultivation. And the positive plasmid was sequenced and verified after extracting the plasmid and verifying with PCR and enzyme digestion method.
Enzyme digestion of the genomic fragment-positive plasmid obtained above results in 11 independent fragments, which are then co-transfected into yeast. Under the effect of the yeast recombination system, according to the overlap between the previously designed fragments, the fragments were sequentially assembled to the corresponding positions. Sequencing proved that there was no non-HSV-1 sequence and PmeI site at the link. Transfer the plasmid into E. coli to extract the plasmid and transfer it into the cell to obtain the complete virus.
After getting the virus, the scientists carried out a cell infection, and the cells showed typical signs of disease. Crystal violet staining also confirmed this conclusion. Subsequently, the researchers tested the growth viability of the virus and found that compared with wild-type KOS, the growth viability of KOSYA was inhibited. This may be due to the presence of the YCp / BAC sequence, which shows that the virus synthesized in this way has the same growth properties as wild-type KOS.
At present, the oncolytic virus is generally administered by intratumoral injection. Although this method can exclude the body`s anti-HSV-1 immune response, it has brought difficulties to the treatment of many solid tumors and can only be found Solid tumors are treated; by comparison, intravenous injection can solve this problem, and can suppress primary and metastatic lesions. But the biggest obstacle is to overcome the body`s antiviral immune response to reach the effective concentration of treatment. Therefore, it is necessary to find a new carrier to transport the oncolytic virus to the tumor tissue to play a therapeutic role. Recent studies have shown that after packaging oncolytic viruses with certain cells such as antigen-specific T cells, macrophages, dendritic cells, etc., they can be prevented from being cleared by the body`s immune system and can be targeted for transport to tumors tissue, which in turn can infect and kill tumor cells. This method is a new technology with great prospects, and is expected to become an important means of tumor biotherapy.
When recombinant HSV-1 is infected with cells latent in wild HSV-1, the two may undergo homologous recombination and produce toxic viruses carrying biologically active genes. Although no re-activation of wild viruses in animal experiments has produced significant harm, the harm to humans is not yet clear. Moreover, this is not only harmful to the patients receiving treatment, but also the possibility of being in close contact with patients.
In summary, HSV-1 can target and kill tumor cells through a variety of mechanisms. A large number of preclinical and clinical trial results show that HSV-1 has good safety and anti-tumor efficacy in the treatment of various malignant tumors. However, most HSV-1 drugs are still in preclinical or clinical trials, and their safety and effectiveness need to be further verified. In addition, a lot of research work needs to be done on the administration method to eliminate the body`s antiviral immune response and improve tumor targeting. With the continuous progress of research in various fields, the existing problems will definitely be solved, and HSV-1 will also become one of the main methods of cancer treatment.
