Multiple Genes Delivery

In the current, gene therapy using adeno-associated viral (AAV) vectors represents one of the most promising techniques for the treatment of many polygenetic diseases. To be noticed, the main obstacle precludes widespread AAV applications is their limited packaging capacity, which restricts their usage to the treatment of disorders caused by multiple genes mutations. As a well-recognized expert in the gene therapy area, Creative Biolabs has made considerable efforts to investigate and optimize strategies to increase the transfer capacity of AAV vectors. Our new developed viral vector design services highlight the advancements and overcome the limitations to expand the applicability of AAV to certain diseases due to mutations in multiple genes.

Overview

AAV has been identified as a naturally replication-defective parvovirus in the past decades. Because of its non-pathogenicity, low immunogenicity, and high efficiency in gene delivery, recombinant AAV (rAAV) is engineered by replacing all viral coding fragments with a gene expression cassette of interest. To date, this vehicle has become one of the most valuable and widely used gene transfer systems. As known, it is originally performed in gene replacement therapy to treat several monogenic diseases. More recently, AAV has been expanded to new applications, including polygenic disorders, gene and RNAi editing, further spreading the applicability of AAV vectors. As a tool with great potential for gene replacement, AAV has gained remarkable progress in early clinical trials.

Strategies for Multiple Genes Delivery at Creative Biolabs

Table 1. Summary of multiple gene delivery by two or three AAV vectors. (Patel, 2019)

Despite the small packaging capacity and single cargo modality of AAV particles, there're various transgene delivery strategies have been investigated to achieve the transport of multiple genes at the same time. Based on the size of expression cassettes, multiple transgenes can be delivered by one, two, or three AAV vectors, and express the complete proteins after reconstitution.

At Creative Biolabs, our specialists pay attention to advanced AAV engineering of vector genome and offer robust AAV-mediated delivery services by co-delivery of two or three AAV vectors to transfer multiple transgene expression cassettes. Our strategies include but not limited to the below platforms.

• Single AAV Vector System

  If the size of two genes is within the packaging limit of 5 kb, the coding sequence of two transgenes can be engineered into a single AAV. The expression of these transgenes could be regulated by a sole promoter in the same expression cassette, or by two independent promoters in two different expression cassettes.

• Co-delivery of Two AAV Vectors System

  If the size of both genes is over the packaging limit of 5 kb, we can take advantage of two AAV vectors that carry two different intact gene expression cassettes. This requires that multiple AAV vectors containing two distinct expression cassettes enter the same target cell and modulate the expression of these two transgenes. Co-delivery of two AAV systems has been applied to package and express two therapeutic genes in many disease models.

• Co-delivery of Three AAV Vectors System

  Some common disorders, like Parkinson's disease, may benefit from co-expression of three different therapeutic proteins. Therefore, we have developed simultaneous delivery of three AAV vectors carrying three desired transgene expression cassettes leading to the expression of these different proteins in specific tissues.

Advantages

• AAV-mediated delivery of multiple transgenes with high precise and efficacy
• Numerous AAV vector systems can be provided for particular cases
• Professional teams with hundreds of cases experience

The number of clinical practices in which AAV vectors were used for in vivo gene transfer therapy has steadily elevated. Owing to the excellent safety profile and high efficiency of transduction for target cells, AAV technology has been implemented in the medical for a variety of conditions, including inherited blindness, coagulation disorders, and neurodegenerative diseases. With our continued efforts on AAV cloning and developing, Creative Biolabs has launched numerous viral vector service strategies to accomplish the multiple transgenes delivery by modifying and fine-tuning AAV vectors, creating enhanced AAV tools for treating more diseases. For more information, please feel free to contact us.

Reference

1. Patel, A.; et al. (2019). Design of AAV vectors for delivery of large or multiple transgenes. Methods Mol Biol. 19-33.