Large Gene Delivery

The adeno-associated virus (AAV)-mediated gene therapy has evolved from laboratory to the clinic, and currently is a valuable therapeutic choice for certain genetic diseases. Nevertheless, the small packaging capacity of AAV vectors precludes the delivery of a number of genes that exceed the fixed size and the use of large physiological regulatory elements. As a famous expert in the field of gene therapy, Creative Biolabs has launched a series of advanced platforms to delivery large gene expression cassettes, including split AAV gene delivery and oversized AAV gene delivery system. We have obtained some success in animal experiments and always devote to the development of AAV-mediated transport progress of large gene elements for global clients.

Overview

Typically, AAV usually measures 20-25 nm in diameter, and a standard-sized AAV vector has a complete gene expression cassette, containing a promoter, transgenic sequence, polyadenylation (polyA) signal, as well as other cis-transcriptional signals. This gene expression cassette is respectively flanked by an inverted terminal repeat (ITR) at both ends. The AAV particle can easily enter target cells and efficiently transfer the gene expression cassette into the cell nuclei to induce transgene expression. Despite its increasing popularity, the AAV vector has a significant challenge of relatively small packaging capacity and can't effectively encode sequences more than 5 kb of DNA.

Strategies for Large Gene Delivery at Creative Biolabs

The DNA cargo capacity of AAV vectors obviously prevents this technique from treating genetic diseases with mutations of large genes. At Creative Biolabs, AAV engineering focusing on the vector genome has been pursued and we have useful viral design strategies to overcome the size limit of AAV genomes. One strategy is to split large transgenes into two or three parts, forming dual or triple AAV vectors that delivery large gene expression cassettes. Another strategy is to harness factors that facilitate the expression of a large transgene from a single AAV vector. Based on these theories, our services include but not limited to:

Design of Split AAV Vector Systems

To deliver a large gene expression cassette, this large expression cassette is separated into two or three parts, carried by two or three individual AAV vectors. After transfer dual or triple AAV to target cells simultaneously, the whole large gene expression cassette is reconstituted within the host cells and the complete protein is expressed. Here, we provide three different split AAV vectors with distinct reconstitution formats, including trans-splicing, overlapping, and hybrid AAV systems.

• Trans-splicing AAV Vectors - Taking use of inherent AAV concatamerization by ITR-mediated intermolecular recombination. One AAV vector contains the promoter, 5' part of the coding sequence, and a splicing donor signal, while the other contains a splicing acceptor signal, the remaining coding sequence, and a polyA signal.

Figure 1. Schematic representation of the trans-splicing dual AAV approach for large gene reconstitution. (Trapani, 2019)

• Overlapping AAV Vectors - Exploiting homologous recombination within the coding sequence to rebuild a large gene expression cassette. One AAV vector carries the promoter and 5' part of the coding sequence, while the other carries 3' part of the coding sequence and a polyA signal. The coding sequence in the above two AAV vectors shows a homologous region.

Figure 2. Schematic representation of the overlapping dual AAV approach for large gene reconstitution. (Trapani, 2013)

• Hybrid AAV Vectors - Combining the mechanisms of both trans-splicing and overlapping AAV vectors, which are supposed to have higher efficacy in transferring large gene cassettes.

Figure 3. Schematic representation of the hybrid dual AAV approach for large gene reconstitution. (Trapani, 2019)

• Triple AAV Vectors - Developed for gene delivery of several giant genes, like 10.1 kb CDH23 gene, 11 kb dystrophin coding sequence, and 12.5 kb ALMS1 gene, when dual AAV vectors can only expand their packaging capacity up to 10 kb.

Design of Oversized AAV Gene Transfer Systems

Besides split AAV vectors, we also provide other strategies to express the large expression cassette through a single AAV vector. These oversized AAV vectors have been known to produce detectable expression of large transgenes.

• Fragmented Oversized AAV Gene Delivery - When AAV comprising mixed DNA fragments of oversized AAV genomes are delivered into desired cells, homologous recombination of fragmented genomes can restore the whole expression cassette and mediate the expression of complete large proteins.
• Other Strategies - There're additional strategies that have been established to transfer an oversized AAV genome, including capsid manipulation, improvement of virus production, optimization of transcriptional elements, and administration of proteasome inhibitors.

Advantages

• Coding sequence exceeds 5 kb, even oversized gene of over 10 kb
• Multiple AAV package strategies can be chosen to bypass size limitation (split AAV gene delivery and oversized AAV gene delivery)
• Years of experience in viral vector design and challenging projects

With increasing achievements in clinical trials, AAV gene therapy can be expanded to more incurable diseases in humans. Though the small packaging capacity of AAV vehicles limits their applicability, multiple strategies may pose a solution to deal with this issue. As a skillful specialist in the biomedical area, Creative Biolabs concentrates on the development of AAV vectors to deliver large transgenes and offers a range of comprehensive large gene delivery services to achieve the expression of the oversized fragments. For more information, please feel free to contact us.

Reference

1. Trapani, I. (2019). Adeno-associated viral vectors as a tool for large gene delivery to the retina. Genes (Basel). 10(4).