Gene editing technology

Compared with viral vectors that can only add genes, gene editing technology can achieve the effects of adding genes, eliminating genes, correcting genes, and editing various other genomes. Gene editing can be performed either in vitro in cells, or gene editing tools can be delivered to the body for in situ genome editing. To edit the target gene, the first step is to perform double-strand breaks mediated by nucleus, triggering DNA recombination and repair in the cell. Non-homologous end link (NHEJ) recombination can effectively insert, delete or inactivate a gene. The homology-directed repair (HDR) can be used to create the correct corresponding sequence in the presence of the correct DNA template to achieve the purpose of repairing genes. Early gene editing methods relied on specific zinc finger nucleases (ZFNs) or large-scale nucleases (meganucleases). For each target DNA region to be edited, a specific sequence must be redesigned in terms of operability and ease of use. In 2009, the transcription activator-like effector nuclease technology (TALENs) developed based on a protein called TALEs bacteria can effectively cut any target sequence of DNA, allowing gene editing technology to advance, but this technology still needs design specific nucleases for each DNA target sequence, so there are certain limitations.

By 2012, the researchers discovered a bacterial defense mechanism, including regular cluster interval short palindrome repeats (CRISPR) and related endonuclease (Cas) CRISPR/Cas9 systems in the presence of guide RNA (gRNA). It can easily and accurately cut DNA, which has led to the CRISPR/Cas9 gene editing technology being born, and is quickly applied to various eukaryotic animal cells, greatly simplifying the workload of gene editing and reducing costs. Through the CRISPR/Cas9 system, genes can be added, knocked out, turned on or turned off, and any mammalian cell genes can be replaced and modified to improve the function of defective genes or curb disease-causing genes, even by knocking out cell surface proteins. The purpose of viruses invading human cells, or to reduce immune rejection for organ transplant patients by editing immune cells, etc., are promising in many disease treatment fields. Researchers are currently improving the off-target effect and safety of the technology so that it can bring the gospel to a wider range of patients.

Compared with viral vectors, gene editing technology can edit genomes more accurately, overcoming the problem of random insertion of viral vectors, thereby avoiding possible side effects such as activation of proto-oncogenes and inactivation of tumor suppressor genes due to random integration, and, through genes introduced by gene editing can be controlled by the endogenous promoter of the target tissue, making gene expression more physiologically relevant. However, it is still difficult to deliver all the components required for gene editing into the target tissue cells. If only NHEJ editing is required, then non-integrating viral vectors can be used to achieve the goal, but if HDR editing with DNA templates is required, the current technology still needs to be optimized to achieve acceptable results in some specific cells.

Cell engineering

In recent years, immunotherapy that has gained a reputation in the field of cancer research is one of CAR-T gene therapy based on engineered T cells. CAR in CAR-T refers to the engineered chimeric antigen receptor, which consists of an antigen binding domain from an immunoglobulin molecule or T cell receptor and mediates the intracellular signaling domain fusion, thereby activate and enhance the function and persistence of T cells. CAR-T treatment first requires the isolation of T cells from the patient's body, and the delivery of engineered CARs to T cells in vitro to produce antigen-specific T cells, allowing them to recognize and kill cancer cells. Injected into the patient's body to achieve the effect of eliminating cancer cells. In theory, CAR-T can achieve a once and for all healing effect. Within one year in 2017, the US FDA approved two CAR-T therapies for the treatment of refractory acute lymphocytic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL). Current research in this area aims to speed up the preparation of individualized CAR-T for patients, expand the application of CAR-T to myeloma and solid tumors, and reduce the risk and side effects of treatment.

CAR-T therapy can target more than cancer. Compared with antigen receptors that are physiologically present, CARs can be designed to recognize proteins, carbohydrate glycolipids, HLA-peptide complexes, etc., so in the future there is potential to treat other diseases based on T cells, such as autoimmunity Sexually transmitted diseases and AIDS, etc.

Summary

Gene therapy can be regarded as the most complex and diverse drug therapy ever. Starting from a research-based academic environment, through the cooperation with biotechnology companies and pharmaceutical companies into industrialized drug development approach. In recent years, advances in basic theory of gene editing technology, the mature results of multiple clinical trials, and regulatory approvals have shown that gene therapy has entered an era of both safety and effectiveness. However, there are still many challenges in this field, including handling the genotoxicity of integrated gene carriers, reducing off-target effects of gene editing, and improving the efficiency of gene delivery and editing to bring it to a level of clinical benefit. Various results show that the potential of gene therapy is enormous, and it is hoped that in the near future, we will solve some persistent diseases that the medical profession has been helpless to bring lasting benefits to human health.