Viral RNAi Delivery

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Evolutionary fine-tuning has given viruses the ability to infiltrate specific cells and deliver genetic material for expression, making them an attractive option for delivering RNAi effectors therapeutically. Viral delivery systems typically carry shRNA expression cassettes [59]. Non-essential regions within the virus genome can be modified to incorporate a promoter and shRNA of interest, to achieve long-term shRNA expression and hence repression of targeted genes in transfected cells. Adenoviruses, adeno-associated viruses, herpesviruses and lentiviruses are among those that have been tested as shRNA delivery vectors [60].

Adenoviruses are widely used in gene therapy studies due to their ability to incorporate large genes and deliver them to the nucleus [61]. Adenoviral delivery of shRNAs has been demonstrated in several human cancer cell lines, in which delivered shRNAs targeting the mRNA of the tumour suppressor protein, p53, successfully triggered gene silencing [62]. Despite these encouraging results from in vitro studies, the inherent, high immunogenicity of adenoviruses has severely limited their use for shRNA delivery in vivo [63, 64].

Systemic administration of adenoviral vectors to human patients leads to high liver uptake and activation of innate immunity, causing severe acute inflammatory responses and even fatalities [65]. Moreover, the development of neutralising anti-adenoviral antibodies prohibits repeated drug dosing [66]. These findings seriously question the suitability of adenoviral vectors for in vivo applications. However, work is under way to address some of these issues, in particular the immunogenicity and poor targeting of adenoviruses. Various approaches, including addition of polyethylene glycol (PEG) to the virus surface [67], liposomal modification [68], and genetic incorporation of tumour-targeting ligands [61], have shown promising results.

Lentiviruses, of the Retroviridae family, have also been explored for therapeutic shRNA delivery. Lentiviruses are single stranded RNA viruses with the ability to reverse transcribe their RNA genome into DNA, before inserting the viral DNA into the host genome for expression [69]. Brummelkamp and coworkers successfully used lentiviruses to deliver shRNA vectors that targeted an activated K-RAS oncogene to pancreatic cancer cells. Effective knockdown of the oncogene was achieved, resulting in the elimination of the tumorigenic capacity of transfected cancer cells in mice [12].

Another lentiviral shRNA delivery system is currently under clinical evaluation for autologous cell therapy against AIDS-related non-Hodgkin’s lymphoma [70]. This involves ex vivo shRNA delivery. Haematopoietic progenitor cells are removed from patients via apheresis, transfected with lentiviral shRNA vectors and then reinfused into the patient. Early results revealed no short-term toxicity associated with the transfected haematopoietic progenitor cells, and two of four patients exhibited persistent expression of the shRNA [71].

The major drawback of retroviral shRNA delivery is that random retroviral genome insertion may cause major disruptions to the host genome, resulting in a multitude of problems, including the activation of proto-oncogenes [72]. This could be particularly dangerous if a large amount of virus is taken up by untargeted healthy tissues. Thus, retroviruses are not ideal candidates for shRNA delivery in vivo.

             

Hong and coworkers demonstrated that Herpesvirus saimiri can deliver shRNA targeting the endothelin converting enzyme 1 (ECE-1) into prostate cancer cells [73]. ECE-1 was selected as the target due to its involvement in the invasion and migration of several cancers, including prostate, lung, breast, colorectal and ovarian [74]. The herpesvirus vector was tested in vitro in several cell lines and ex vivo in primary cells from three different forms of prostate cancer. Significant levels of ECE-1 knockdown were observed in all cases, which severely limited the capacity of the cancer cells to migrate and invade [73]. However, much in vivo testing is still needed to evaluate the safety of herpesviruses for therapeutic applications, especially considering the well reported tendency of vectors based on herpes simplex virus 1, another member of the herpesviridae family, to trigger severe inflammatory responses in the central nervous system [75, 76].