AAV Vector Optimization Strategy: Enhancing Infection Efficiency And Targeting

AAV has broad application prospects as a vector. In addition to being used for the treatment of genetic diseases, it can be used for the treatment of tumors and can also be used as a vector to construct vaccines. AAV also has certain applications in influenza prevention. AAV-mediated antibody expression can protect elderly and immunodeficient mice from influenza virus damage.

In order to achieve lower and safer vector doses, it is necessary to improve AAV transduction efficiency, escape neutralizing antibodies, and improve cell/tissue specificity. Modifying AAV itself, such as modifying the capsid protein, can not only improve the transduction efficiency and targeting of AAV, but also reduce humoral immunity and cellular immunity, thereby reducing the immunogenicity of recombinant viral proteins. Different serotypes of AAV can also be selected based on different tissue affinities and use specific promoters for a certain organ tissue. This article will review the research progress on enhancing AAV vector transfection efficiency and its targeting.

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Enhance The Infection Efficiency of AAV on The Host

➤ Currently, there are more than 10 different serotypes of AAV that can be used to construct viral vectors, but the transfection efficiency of rAAV vectors of different serotypes into different tissues and cells varies greatly. Therefore, only by selecting the appropriate serotype can the gene of interest be specifically expressed in target tissues and cells.

For example, in a study in 2021, researchers quantitatively analyzed the expression characteristics of AAV1, 2, 5, 6, 8, and 9 in different brain regions and cells of mice through experiments. They injected different serotypes of AAV into different brain regions, and 21 days later, they compared the expression efficiency of different serotypes in the striatum, hippocampus and auditory cortex by detecting green fluorescent protein (GFP) expression. Among the analyzed brain regions, rAAV5, 8, and 9 had similar expression efficiencies in different brain regions and showed good expression levels, while rAAV2 had the lowest expression in all brain regions.

➤ By modifying and mutating the capsid of AAV, AAV mutants with high infection efficiency can be screened, which is beneficial to the delivery of target genes by AAV vectors.

In 2019, researchers such as Bertin carried out molecular engineering design of the capsid of AAV2. By glycosylation modification of the capsid, transgene expression increased by 1.3 to 2.5 times in three cell lines (HeLa, Huh7 and ARPE-19). In the hemophilia B mouse model, hepatic gene transfer of AAV with modified glycosylation sites caused a 2-fold increase in the level of human coagulation factor (F) IX, while its T/B cell immune response did not change. This study showed that intravitreal gene transfer increased green fluorescent protein expression approximately 2 to 4 times and enhanced penetration throughout the retina. It shows that gene delivery can be enhanced by glycosylating the capsid protein and has good translation potential in gene therapy for liver and eye.

Suppresses Immune Responses Associated with AAV

Although AAV has low immunogenicity, it can still cause an immune response in the human body. 40% to 80% of adults have been infected with AAV, so it may cause immune rejection. The presence of neutralizing antibodies against AAV in humans poses a major challenge that limits clinical trials of gene therapy using recombinant AAV vectors. Mutations or modifications to the AAV capsid can effectively avoid the effects of neutralizing antibodies.

➤ Engineered AAV

Designs can be developed through capsid modification, surface conjugation, and encapsulation to address the limitations of native AAVs. Chemically modified capsids showed reduced interaction with neutralizing antibodies, revealing that chemical coupling of AAV may reduce vector immune responses. The introduction of click chemistry technology can modify the capsid after the virus particles are assembled. Oligonucleotides can be site-specifically coupled to the AAV capsid surface through click chemical amino acid methods, thereby effectively protecting AAV particles from phagocytosis by neutralizing antibodies.

➤ Reduce AAV immune response through exosome encapsulation

The researchers used standard AAV8 and exosome-related AAV8 vectors (exo-AAV8) to systemically administer mice, and found that both methods could effectively infect a large number of immune cell populations, including CD4+ T cells, CD8 +T cells, B cells, macrophages and dendritic cells. Using Exo-AAV as a liver gene delivery system prevents humoral immunity to the capsid while reducing the therapeutic vector dose. Exo-AAV vectors improve the safety and efficacy of liver-directed gene transfer.

Improve AAV targeting

To more efficiently transduce therapeutic genes and target specific tissues or cells, AAV vectors need to be modified to improve targeting.

➤ Directed evolution of AAV to screen out highly targeted AAV capsid structures

Researchers established the BRAVE (barcoded rational AAV vector evolution) method to optimize AAV. With the BRAVE method, each viral particle has a peptide of known function on the AAV capsid surface and a unique molecular barcode in the packaged genome. RNA expression barcode sequencing from a single generation screen can simultaneously map putative binding sequences for hundreds of proteins, allowing large-scale selection of engineered AAV capsid structures in a single screen in vivo.

Using the BRAVE method and clustering based on Hidden Markov Model (HMM) statistics, researchers demonstrate 25 finely characterized synthetic capsid variants engineered to target human dopamine neurons in vivo, and transported along connected pathways in the brain, allowing unprecedented therapeutic accuracy.

➤ AAV promoters are two key factors that determine the dynamics of transgene expression

The promoter element of a viral vector determines the specificity of viral expression. For targeted research or gene therapy, researchers need to continuously develop promoters suitable for specific cells or tissues and continuously optimize them to achieve targeting to specific tissues. Specific promoters enable the expression of target genes in specific tissues and cell types. Some modified specific promoters can be used, such as specific promoters fused with enhancer sequences. These specific promoters often ensure tissue specificity while also possessing high priming activity. At the same time, try to use a shorter promoter to expand the loading capacity of the target gene.

To circumvent the inherent weaknesses of tissue-specific promoters, many different promoter optimization strategies have been devised. For example, adding enhancer elements upstream of a tissue-specific core promoter can increase the expression level of the target gene while maintaining its tissue specificity.

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Conclusion

Current applied research on AAV includes protein replacement therapy, gene editing, gene silencing, etc. With the development of AAV-related research, AAV has gradually overcome many challenges in gene therapy, such as transgene maintenance, safety, host immune response and target diseases, and is increasingly able to meet people's expectations. It is believed that in the near future, more and more highly safe and effective AAV drugs will be clinically launched to serve more people in need.

Reference

Mary B, et al. Molecular engineering of adeno-associated virus capsid improves its therapeutic gene transfer in murine models of hemophilia and retinal degeneration.Molecular pharmaceutics, 2019, 16(11): 4738-4750.