The viroid delivery technology between viral and non-viral vectors

The viroid delivery technology between viral and non-viral vectors. Since the earliest gene editing tools came out, gene editing technology has a history of nearly 30 years. Especially since 2012, with the emergence of CRISPR, gene editing technology has gradually matured. Unfortunately, relative to the rapid evolution of gene editing tools themselves, the development of their delivery technology is extremely slow and difficult. And delivery is as important to gene editing therapy as rockets are to moon landing. Due to the lagging development of delivery technology, the clinical application of in vivo gene editing therapy is difficult, and the entire field is looking forward to a breakthrough in delivery technology.

Nobel laureate Jennifer Doudna systematically discussed the prospects and challenges of gene editing therapy in a review written to Nature in early 2020. While looking forward to the bright application prospects of CRISPR, this year’s Nobel laureate made a lament that “Delivery remains perhaps the biggest bottleneck to somatic-cell genome editing” (detailed) See BioArt report: “Collector’s Edition” Nature Review | Opportunities and Challenges of Gene Editing Technology in Clinical Application).

In recent years, the development of gene editing tools itself has been dazzling. Various Cas9 variants, base editing tools and their variants, Prime Editor, etc. have emerged one after another. However, the clinical application of these tools must return to delivery, and the delivery tools available so far are still decades-old’old three’: AAV, lentivirus, and nanomaterials. Although these delivery vehicles are widely used in basic research, they are not suitable for direct clinical use.

The clinical application of gene editing has double standards of safety and effectiveness. On the one hand, viral vectors will cause uncertainty in safety due to long-term expression of gene editing enzymes; on the other hand, nanomaterials face efficiency challenges. In the first half of 2020, Editas, a well-known gene editing therapy technology company with Zhang Feng as the founder, implemented the first clinical study of human gene editing therapy (Nature Biotechnology volume 38, page382(2020)).

However, because the study uses AAV as a vector, CRISPR will coexist with patients for a long time, with certain risks. The ideal gene editing delivery tool needs to be both instantaneous and efficient to ensure the safety and effectiveness of the treatment.

On January 4, 2021, Yujia Cai’s team from the Institute of System Biomedicine of Shanghai Jiaotong University published an article titled: Lentiviral delivery of co-packaged Cas9 mRNA and a Vegfa-targeting guide RNA prevents wet in Nature Biomedical Engineering. The research paper of age-related macular degeneration in mice invented a virus-like particle (VLP) delivery technology between viral vectors and non-viral vectors. VLP can deliver CRISPR/Cas9 mRNA to realize safe and efficient gene editing in vivo.

Lentiviral vectors can efficiently infect almost all cells, while the non-viral component mRNA has transient characteristics. Cai Yujia’s team used the principle of mRNA stem-loop structure and the specific recognition of phage capsid protein, and through virus engineering technology, the advantages of the two were perfectly combined to create a new delivery technology VLP-mRNA. When Cas9 mRNA is delivered by VLP-mRNA, the existence time of Cas9 is only 72 hours. Studies have found that compared with viral systems that express Cas9 for a long time, VLP-mRNA can significantly reduce or even completely avoid off-target effects. In addition, VLP-mRNA can deliver the entire CRISPR element (Cas9 and gRNA), which overcomes the small carrying capacity of AAV vectors, and can even deliver larger base editing tools.

Researchers also use VLP-mRNA technology for the treatment of ophthalmic diseases. Age-related macular degeneration (AMD) is a degenerative fundus disease. The patient manifests as decreased central vision, deformed vision, and dark spots in the peripheral or central visual field, which have a great impact on the quality of life of the elderly. According to statistics, more than 40% of the elderly over 70 in Western developed countries suffer from AMD.

With the increasing number of elderly people in many countries, the incidence of macular degeneration is also increasing. In addition, diabetic patients may also suffer from diabetes-related macular degeneration, with an overall incidence of about 10%. Currently, the treatment for macular degeneration is VEGF monoclonal antibody. However, antibodies need to be administered repeatedly; in addition, antibody spillover can cause serious side effects.

Using a laser-induced mouse macular degeneration model, the research team found that CRISPR is specifically distributed in retinal pigment epithelial cells (RPE) through subretinal injection, and RPE cells are the main source of VEGF in the eye. VLP delivery of CRISPR achieved 44% knockout of vegfa gene and reduced the area of ​​new blood vessels by 63%.

Second-generation sequencing showed that VLP-mRNA did not induce off-target effects. For the previously reported large fragment deletion caused by AAV delivery of CRISPR, the research team used third-generation sequencing and could only find a signal that was barely above the background. It is worth mentioning that VLP mRNA did not cause an immune response whether in vitro or in the eye. These experimental results strongly support the clinical application potential of CRISPR in gene therapy of macular degeneration.

In general, VLP-mRNA is a universal and transient CRISPR delivery tool with the advantages of high efficiency and safety. This technology will help CRISPR in vivo gene editing therapy to go to the clinic, bringing new hope to patients with hereditary, acquired and infectious diseases that are drugless or refractory to drugs. It is worth mentioning that this technology is an original gene therapy vector developed completely independently by China, reflecting China’s technological progress in the field of gene therapy.

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