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.

Two strategies can introduce siRNAs into the cytoplasm of cells,where they are active – a drug approach where double-stranded RNAs are administered in complexes designed for intracellular delivery and a gene therapy approach to express precursor RNAs from viral vectors.

At Transgene, we have mutated capsid and generated recombinant virus that has significant resistance to ubiquitin-proteasome degradation.

We provide evidence for the effective usage of our proprietary, genetically engineered, new generation self-complimentary AAV vectors (scAAV) that are not only efficient in gene silencing but also have capsid mutations to enhance the transduction by 20 fold. Self-complimentary adeno-associated viral (scAAV) vectors are safe, clinically proven and efficient that ensures continuous production of RNAi molecules within the cell.

Challenge 1:
Initially requires co-infection with adenovirus to produce virus

Solution:
We cloned the required Adenoviral genes in a plasmid, hence no adenoviral contaminations in the purified AAV.


Challenge 2:
Limitation on vector size – limited to express less than 2 kb gene.
Solution:
RNAi is only 22 nt hence size limitation is not a factor for gene silence therapy with RNAi.


Challenge 3:
Inability to produce a stable packaging cell line, hence purification is difficult with many steps.

Solution:
We streamlined the process of purification, which not only avoids empty viral particles, but is as simple as having cell lines to secrete.


Challenge 4:
Low transduction efficiency since the genome is single stranded and there is a lag phase for second strand synthesis in order to express gene.

Solution:
We made double stranded AAV, thus avoiding the lag phase of second strand synthesis.


Challenge 5:
Requires large amounts of vector for gene expression since majority of the vector is degraded by proteasome-ubiquitin pathway in the cell.

Solution:
We generated mutant AAV that is resistant to degradation by proteasome-ubiquitin pathway.