Unfortunately there are some hurdles for in vivo use of siRNA. siRNA is relatively unstable and the quantity of siRNA necessary for efficient silencing is incompatible with scale-up to larger preclinical models. Other methods of RNAi delivery, such as the liposomal packing method and polyethylene glycol (PEG) conjugated methods, require large amounts of RNAi and financially non-viable techniques.
Traditional retroviral vectors randomly integrate into genome and generate insertional mutagenesis. Adenoviral vectors trigger unacceptable levels of immune responses. Both vectors had serious limitations for human trials and FDA restricted their usage.
Due to the safety, efficacy and potency provided by Adeno-associated virus (AAV), it makes it a better alternative vector.Besides AAV has various serotypes that are tissue specific and that makes it useful for targeted therapy. AAV delivery of RNAi makes it expressed within the cells, unlike liposomal or PEG methods. AAV is the safest, FDA approved and non-pathogenic vector.
However, one of the drawbacks of AAV vectors is that they often have low transduction efficiencies requiring large doses of vectors to achieve desired effect. This is because the AAV capsids are phosphorylated at tyrosine residues in the cell, which leads to ubiquitin-proteasome degradation of majority of AAV particles leading to inefficient transduction.
AAV represents an attractive vector choice owing to its low immunogenicity and small size (making it ideal for applications requiring diffusion into other target areas). Another advantage is its ability to exist as a stable episomal form, resulting in lasting gene expression.
AAV is a 4, 7 kb non-pathogenic single stranded DNA virus, smallest virus, initially discovered as contaminant in Adenoviral preparations. Compared to 50 kb Adenovirus and 12 kb retrovirus, the other popular gene therapy vectors, AAV is very small ( as can be seen in the picture below), mostly remaining in episomal form – thus avoiding insertional mutagenesis as in the case of retroviral vectors. AAV is structurally simple, non-enveloped virus – thus reducing immunological response in the host unlike adenoviral vectors. At least eleven AAV serotypes have been identified that have different tissue specificity – a better targeting vector.
Adeno-Associated Virus (AAV) is the only human gene therapy vector which is non-pathogenic to humans with negligible immune response and negligible random integration.