(2) Adeno-associated virus: non-integrated virus vectors have a lot to offer.

Adeno-associated virus is a type of single-stranded linear DNA-deficient virus with a genomic DNA of less than 5Kb, no envelope, and a bare icosahedral particle. Adeno-associated viruses (AAV) cannot be replicated independently. Only in the presence of helper viruses, such as adenovirus, herpes simplex virus, etc., AAV can replicate, infect and lyse host cells, otherwise only lysogenic latent infections can be established (infected host cells and latent, but not lyse host cells).

Compared with the first- and second-generation of retroviruses, the advantages of adeno-associated viruses are mainly reflected in the two aspects:

Genomic non-integration: Adeno-associated viruses, like lentiviruses, can infect both dividing and non-dividing cells, but the adeno-associated virus genome carrying the gene of interest enters the host cell and is not inserted into the genome of the host cell and is stably present in the host cell (some types of wild-type adeno-associated virus genomes are inserted into the host genome) in the form of free DNA, and synthesize normal proteins for a long time to achieve treatment of the disease. Due to the "non-integration of the genome", the adeno-associated virus vector does not cause insertional mutations in the host cell genome, thereby improving the safety of gene therapy.
Tissue targeting: at present, scientists have discovered more than a dozen of adeno-associated virus subtypes. Different subtypes have different affinities for different tissue cells, thus partially solving the problem of viral vector tissue cell targeting. For example, the most commonly used serum type 2 adeno-associated virus (AAV2) has high affinity for skeletal muscle cells, neurons, smooth muscle cells, and hepatocytes.
In addition to the common problems of viral vector virus toxicity, immunogenicity, and expression level of the target gene, the adeno-associated virus also has problems such as small target gene fragments and easy dilution of the target gene.

The portable gene fragment of interest is small: the capacity of the retrovirus carrying the gene of interest is usually around 8 Kb, while the adeno-associated virus is only 4.5 Kb.

The target gene is easily diluted: the adeno-associated virus genome is present in the host cell as free DNA and is not inserted into the host genome. Therefore, when the host cell undergoes division and proliferation, the "compensation gene" introduced by the viral vector does not follow the DNA replication of host. When the host cell divides into two daughter cells, the number of genes of interest is also diluted and eventually lost. Therefore, the adeno-associated virus has an obvious advantage in infecting long-term non-dividing cells or cells with fewer divisions.

Non-viral vector: another way for genetic modification
In any case, the virus vector will have potential risks. Therefore, in recent years, scientists have tried to use the non-viral vector to transport the target gene to the patient's cells, which can be roughly divided into physical methods and chemical methods. Wherein, the physical methods are represented by microinjection, gene gun, electrical transduction, etc., and chemical methods are represented by liposome methods, nanoparticles, and the like.

Compared with viral vectors, non-viral vectors have the advantage of low cytotoxicity and weak immunogenicity; at the same time, the production process of non-viral vectors is more standardized than viral vectors, so mass production is much easier. However, the major drawback of non-viral vectors is the low transfection efficiency, which is to say, it is still difficult to use the non-viral vector to introduce the target gene into the cell to be as efficient as the viral vector, and unlike the viral vector, the applicable cell type is limited. At present, the non-viral vector technology is still being optimized. After the technology is further matured, its application range is expected to be expanded.