On November 8, 2017, Nature published a paper: Regeneration of the entire human epidermis using transgenic stem cells. Through retroviral vector gene therapy, a 7-year-old patient with junctional bullous epidermolysis (JEB) had a total 80% of the skin was rebuilt, and the skin function was completely normal.
On November 2, 2017, NEJM published a paper: Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy, gene therapy using adeno-associated virus type 9 (AAV9) as a vector successfully extended the life of 15 children with type 1 spinal muscular atrophy (SAM1).
On August 30, 2017, Novartis’ Kymriah CAR-T therapy using lentivirus as a carrier for B-cell acute lymphoblastic leukemia was approved by the FDA and became the first gene therapy approved by the FDA.
The above three cases of gene therapy use three different gene therapy vectors, namely retrovirus (RV), adeno-associated virus (AAV), and lentivirus (LV).
Gene therapy vectors are divided into two categories: viral vectors (mainly including lentivirus, adenovirus, retrovirus, adeno-associated virus, etc.), and non-viral vectors (mainly including naked DNA, liposomes, nanocarriers, etc.)
Before talking about gene therapy vectors, let’s talk about two concepts in gene therapy: in vivo and ex vivo.
In vivo: direct transfer of living body, injecting the carrier with genetic material directly into the laboratory animal or human body. Applicable carriers: adeno-associated virus, adenovirus, non-viral vector, etc.
Ex vivo: Transfer in vivo, take out the cells of the experimental subject, culture and introduce recombinant genes in vitro, and then retransmit these genetically modified cells back into the experimental animals. Applicable carriers: lentivirus, adenovirus, retrovirus, etc.
Next, we introduce the following commonly used gene therapy vectors.
- Viral vectors in gene therapy
Retrovirus (RV): A single-stranded RNA virus that can efficiently infect many types of cells, and can randomly insert and stably integrate foreign genes into the host cell genome for continuous expression. Among them, the γ-retroviral vector was first modified and widely used in gene therapy, and has achieved a lot of great success.
Disadvantages: (1) Cannot infect non-dividing cells; (2) Transcription termination ability is relatively weak, which may cause transcription through reading; (3) May produce replication-capable viruses; (4) May cause insertion mutations, such as those 10 patients with X-linked severe combined immunodeficiency disease (X-SCID) treated with retroviral vectors, 4 of them developed leukemia due to vector integration near the proto-oncogene LMO2, etc., and activation of downstream gene expression.
The insertion site of the retrovirus is random, but it is more inclined to insert the first intron of the human gene and the transcription start site. Since then, people have begun to re-examine the risks posed by the use of gene therapy vectors. After using retrovirus to treat JEB, the research team identified insertion sites through whole-gene sequencing and found more than 27,000 insertion sites, but all focus on non-coding sequences, without destroying known tumor suppressor genes.
Lentivirus (LV): Gene therapy vector developed on the basis of HIV-1 (human immunodeficiency virus type I). It belongs to retrovirus and can effectively infect dividing and non-dividing cells. Randomly insert and stably integrate into the host cell genome for continuous expression. A series of clinical research results are very satisfactory, and have broad application prospects.
Because lentiviruses are also retroviruses, there is also a risk of gene mutation due to random insertion, but a series of clinical studies have confirmed that lentiviral vectors are safer and more applicable than retroviruses.
Adenovirus (AdV): Non-enveloped linear double-stranded DNA virus. The adenovirus vector has a wide range of host cells. It effectively infects dividing cells and non-dividing cells. It does not integrate with the host cell genome, so there is no risk of insertion mutation.
Disadvantages: (1) Lack of specific targeting and low infection efficiency in some cells lacking their corresponding receptors; (2) The expression time of the target gene is short and may require repeated treatment; (3) Has strong immunogenicity, such as Jesse Gelsinger died in the clinical treatment of adenovirus (AdV) led by Professor Jim Wilson due to a strong immune response. Since then, Professor Wilson has found a more suitable and safer viral vector for gene therapy: adenovirus Related viruses (AAV).
Adeno-associated virus (AAV): One of the simplest single-stranded DNA-defective viruses found so far. It requires an auxiliary virus (usually an adenovirus) to replicate. Because of its good safety, wide host cell range, low immunogenicity, and long time to express foreign genes in vivo, it is regarded as the most promising gene therapy vector, which has been obtained in gene therapy and vaccine research worldwide widely used.
In addition to the several commonly used viral vectors mentioned above, there are also vaccinia virus vectors, pox virus vectors, herpes simplex virus vectors, etc. in clinical gene therapy, which will not be introduced one by one because they are used less.
- Non-viral vectors in gene therapy
In clinical gene therapy, viral vectors have potential safety issues and the capacity of viral vectors is limited. These shortcomings have promoted the development of non-viral vector systems. Non-viral vectors have the advantages of low cost, simple preparation, large-scale production, high safety, and unlimited length of foreign genes. The simplest non-viral vector is naked DNA, which can be directly injected into specific tissues, especially muscles, and can achieve a higher level of gene expression. Naked DNA has a wide range of applications in clinical gene therapy.
Although non-viral vectors have many advantages, they also have obvious disadvantages: (1) the transfection efficiency is not ideal; (2) the expression time of foreign genes after transduction into host cells is short, (3) non-specific targeting is high, etc. Clinical treatment also needs to do more in-depth research and improvement.
In 2017, a series of successes in the field of gene therapy announced the arrival of the era of gene therapy. The ultimate goal is to cure cancer and genetic diseases safely and effectively. This previously impossible task is being achieved gradually through gene therapy.