Current gene therapy strategy is mainly based on five types, of which, gene replacement and gene repair are typically the most ideal methods. Current strategies for gene therapy are mainly based on the following:

(1) Immune regulation: The gene of antibody, antigen, or cytokine is introduced into the patient's body to change the immune state and achieve the purpose of prevention and treatment of diseases. For example, the gene of interleukin (IL) can be introduced into the body of tumor patients to improve the level of 1L to activate the anti-tumor activity of the immune system and to achieve the purpose of tumor treatment.

(2) Gene silencing (aka Gene Inactivation): Using antisense RNA, ribozyme, or nuclear phthalate to inhibit the expression of some oncogenes, inhibit the proliferation of tumor cells, and induce the differentiation of tumor cells. This strategy can also block the expression of drug resistance genes in tumor cells and improve the efficacy of chemotherapy.

(3) Gene replacement (Supplemental): The defective gene still exists in this treatment method. It is the main method of gene therapy currently used in clinical studies. The target gene is transferred into the diseased cells or other cells, and the expressed products can strengthen or correct the function of the defective cells.

(4) Gene replacement (Complete): The normal gene is used to replace the pathogenic gene in situ, so that the DNA in the cell is completely restored to normal.

(5) Gene repair: Directly repair the abnormal part of the gene.

Gene Augmentation

Gene augmentation (enhancement) therapy is used to treat diseases caused by loss-of-function mutations, which prevent the gene from producing a functional product. This gene therapy technique introduces DNA containing a functional version of the lost gene into the cell and aims to produce a functioning product at sufficient levels to replace the protein that was originally missing. At present, gene augmentation is mainly used to delivering of a new, functional protein-coding gene, increasing the expression of growth factors and cytokines, and genes related to autophagy activation (to accelerate the elimination of abnormal proteins).

Gene augmentation therapy is the most common treatment option for spinal muscular atrophy (SMA). SMA is caused by a deficiency of a motor neuron protein called SMN1, so the basic concept of gene therapy treatment is to insert the normal SMN1 gene into the diseased cell. Importantly, vector AAV9 can deliver cDNA of SMN1 gene to the cells. The first gene therapy treatment for SMA was approved by the Food and Drug Administration (FDA) in 2019. Despite being an effective option to minimize the progression of SMA and improve survival, this treatment is costly.

Gene Silencing

In cases where the addition of a functional gene does not resolve the disease phenotype, gene silencing therapy may be used to shut down (silence) the expression of an abnormal gene. For diseases caused by dominantly inherited disorders, just one abnormal allele can manifest the disease phenotype and related dysfunctionality of cells or organs. A common example is the constitutive expression of oncogene mutations in tumor cells, which requires gene therapy to inhibit the function and expression of pathogenic genes. RNA interference (RNAi) therapy has been applied in research across many polyglutamine (PolyQ)-related diseases, including Huntington’s disease (HD) and spinocerebellar ataxias (SCAs), with the aim to reduce the expression of toxic proteins. Although single-stranded ASOs can mediate gene silencing – small/short interfering RNA (siRNA), short hairpin RNA (shRNA), and microRNA (miRNA) therapies typically provide stronger inhibitory function and durability.

Gene Suicide

Suicide gene therapy is also known as virus-directed enzyme prodrug therapy (VDEPT); its principle is to transfer suicide genes into tumor cells in a targeted manner, using viruses as vectors. The specific enzymes encoded by this gene can metabolize the drug precursors which are not highly toxic to cells into toxic products in tumor cells, thereby preventing replication and causing these cells die. For example, thymidine kinase gene/ganciclovir system (TK/GCV) and cytidine deaminase/5-fluorocytosine system are based on this principle and are some of the most cited in lung cancer research.【1】

Gene Editing

The use of gene editing technology in gene therapy applications has been closely tied to the development of Targeted Gene Editing-Pro technology, which has made gene editing in organisms much easier and more economical. Importantly, Targeted Gene Editing-Pro gene editing technology has become widely used in gene therapy and served as a breakthrough approach to many previous restrictions, such as the limitations by disease type (recessive or dominant disease), gene length, and in vitro or in vivo experimental model development. Those experimental limitations could be solved by gene editing (Targeted Gene Editing-Pro) technology. Below are four key gene editing strategies for gene therapy:

(1) Gene Disruption: Directly inhibiting the expression of pathogenic genes, which is used for treating dominantly inherited diseases;

(2) NHEJ Gene Correction: Correcting the faulty gene via single base editing or direct knockouts (KOs);

(3) HDR Gene Correction: Using homologous fragment repair strategy, which depends on the improvement of homologous recombination efficiency;

(4) HDR Gene Addition: Introducing the normal gene by homologous recombination at the safe site, similar to gene augmentation.

At present, gene editing is the most popular and promising technology in gene therapy. This technique utilizes gene-editing tools – such as transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and Targeted Gene Editing - are used to insert repair genes into cell DNA. The advantage of this technology is that it not only avoids the risk of random gene insertion but also ensures the continuity of therapeutic effect through repair or insertion.

In the next article, we will present a case study in gene therapy that leverages the gene-modified animal model and viral vectors and discuss the progress of gene therapy applications.

Research Resources on Gene Therapy

With the growing understanding of disease mechanisms and effective therapeutic targets as well as the development of gene-editing technology, large-scale clinical application of gene editing therapy have become possible, especially for those diseases that are difficult to be cured by traditional therapy - such as rare diseases, genetic diseases, and more.

Herein, we 've collected and analyzed some resources related to gene therapy and the application of gene-editing animal models in this field - including background and current development of gene therapy, success and failure case studies, the prospect of gene editing and animal models in the future development of gene therapy.

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