The number of reports of pseudoviral-mediated siRNA delivery remains relatively small, due primarily to the fact that the majority of pseudovirion systems to date have been used for exogenous gene transfer applications. However, as the systems and studies discussed here demonstrate, pseudoviral systems have been used to successfully deliver a number of different siRNA effector molecules, including single-stranded and duplex siRNA, and or siRNA-encoding DNA. They have achieved gene-specific knockdown in a variety of cell types in vitro and in vivo (see ). For the majority of systems, direct evidence of the ability to deliver siRNA or an shRNA-encoding vector was shown. More importantly, specific and efficient silencing was achieved over the course of several days in many different model systems.
Summary of RNAi studies using pseudoviral systems
The basic requirement for any delivery vector is that it delivers its cargo and, once delivered, that the cargo can exert its intended effect. Ideally, a delivery vehicle should also be efficient, targeted, easy to prepare and modify, and non-immunogenic. The unique combination of characteristics possessed by pseudoviral systems make them extremely attractive as delivery vehicles. For example, pseudovirions are very flexible. Envelope-type vectors are able to deliver multiple cargos, such as combinations of siRNA and small molecule drugs. Also, SV40 in vitro-packaged vectors can encapsulate any number of expression plasmids without modification, greatly simplifying the generation of vectors for new target genes.
There are inherent problems associated with any given pseudoviral vector. For example, systems such as HSV amplicons and phagemid particles are only able to deliver DNA, which is inherently more risky than siRNA oligomers due to the possibility of integration into the host genome and insertional mutagenesis. One of the most important concerns when using pseudovirions is immunogenicity. For several of the pseudoviral systems presented here, such as the influenza virosome, HVJ-E, and SV40 in vitro-packaged pseudovirions, the extent to which the vectors are inherently immunogenic is still in question and ways to mitigate cellular and host immune responses are under investigation. Additionally, systems in which the RNAi is derived from DNA (i.e., phagemid particles and HSV amplicons) are unable to package siRNA effector molecules that have been chemically modified to reduce susceptibility to degradation by nucleases and avoid eliciting an innate immune response.
The other most pressing technical problem is that of cell- or tissue-specific targeting, an issue which is not unique to pseudoviral vectors. Fortunately, techniques for addressing this, such as the development of high-affinity ligands through the phage-display system in the case of phagemid particles or functionalized biopolymers in the case of virosomes, already exist. Furthermore, ongoing work to create targeted versions of the parent viral systems is likely to be directly applicable to the virally-derived systems (i.e., pseudovirions), such as the work to create chimeric F-peptides in the HVJ system.
In conclusion, pseudoviral vectors may prove useful in broadening the current scope of siRNA therapeutic strategies and in vitro delivery techniques.