Alternative Splicing Cassettes for AAV Gene Therapy Regulation

Uses Tissue-Specific Exons for Precise Control Therapeutic Cargo Delivery

These alternative splicing cassettes use tissue specific exons to precisely deliver adeno-associated virus (AAV) cargo. AAVs are a commonly used gene therapy vector for delivering genetic material into cells. Current technology relies upon tissue specific promoters to guide and activate the delivery of genetic material. Targeted promoters are commonly expressed in more than one part of the body, making this method unreliable and prone to failed deliveries or deliveries to the wrong tissue. AAV delivered genetic material is also always expressed, even in situations where expression of the delivered genetic material is unnecessary, or harmful. In addition, synthetic material is commonly used in current AAV technology, leading to a potential immune response to the AAV therapy.

Researchers at the University of Florida have developed an AAV gene therapy regulation method that exploits alternative gene-splicing mechanisms in humans, addressing the aforementioned issues. Highly specific exons used to guide the AAV allow for successful delivery of genetic material at ahigh rate. In 2024, the gene therapeutics market was valued at USD 3.19 billion and is projected to grow annually by 19.22%, to a value of USD 18.50 billion in 2034.

Application

Tissue-specific exons to regulate the AAV gene therapy for safe and accurate genetic material delivery

Advantages

• Uses only naturally occurring elements and no synthetic regulatory proteins or other foreign agents, reducing the chances of an immune response to AAV therapy
• Transgene can be activated or deactivated by external conditions, like inflammation or stress levels, allowing for a dynamic switch-like treatment that only activates when stimulated
• Nonsense media decay is implemented if an alternative exon is wrongly included or excluded, providing a reliable fail-safe mechanism to protect against wrongful expression
• Utilizes highly specific splicing genes in tissues as the guide for the AAV, preventing off-target expression that is commonly seen with today’s technologies
• Flexible platform, enabling technology to be molded into the needs of the user

Technology

This technology exploits tissue-specific alternative splicing mechanisms to deliver genetic payloads with AAVs. These mechanisms allow researchers to reliably target certain sites for AAV therapy. Because these splicing mechanisms are not always used in the tissue, researchers can also control which genes get transcribed into the final mRNA product, and under what conditions. Two different methods are used to complete this task.

The first method uses a start codon in the alternative exon. If the exon is included in the gene after splicing is carried out, the therapeutic gene will be transcribed into the mRNA final product. If the exon is not included, the gene will not be transcribed. The second method utilizes stop codons in the alternative exon. If that exon is transcribed, nonsense-mediated decay occurs, preventing expression of that gene. These methods allow for a switch-like mechanism for expression of the delivered genetic material. Different exon-intron pairs were then researched, creating a library of exons that show strong switch-like mechanisms in different tissues.