Adeno-associated viruses (AAVs) are non-pathogenic viruses that can be engineered to deliver DNA to drive the endogenous production of therapeutic proteins within specific target tissues. By programming cells to produce particular proteins, AAV gene therapies can convert transduced cells into local “biofactories” for these therapeutic proteins to achieve the desired clinical effect.
The ITRs demarcate the DNA sequence and play a key role in its packaging into the AAV capsid particles. The ITRs also play a critical role in the formation of concatemers that ensure long term transgene expression following AAV delivery to human subjects. Maintaining consistency and integrity of the ITRs is critical throughout the production process.
Minimizing the presence of immunostimulatory features through targeted sequence modification
The open reading frame (ORF) includes the transgene that encodes for the desired protein product. In engineering the ORF, it is important to minimize the presence of immunostimulatory motifs, utilize strong and appropriate signaling elements, and ensure the generation of optimally folded and functionally active proteins.
The Polyadenylation (PolyA) signal is located at the end of the expression cassette and promotes the stability and longevity of the transcript.
The backbone is a part of the plasmid DNA that is used in the production of AAV but does not serve a functional purpose in the final drug product. Nevertheless, portions of the backbone often get packaged into the final product and are considered impurities. Rational engineering of the backbone can help reduce the degree of plasmid backbone packaging and the potential immunogenicity associated with such impurities.