Virus-like Particles (VLPs)

Similar to viral delivery, RNAi delivery by VLPs relies on capsids for the encapsidation, nuclease protection and intracellular delivery of RNAs. However, several important distinctions exist between the two types of delivery system.

Firstly, VLP delivery utilises capsids of simple viruses, typically bacteriophages, to deliver siRNAs to cells, rather than complex, genetically-modified human viruses to deliver shRNA. Because siRNAs can directly enter the RNAi pathway and do not require expression from a vector, the virus genome can be removed, avoiding problems associated with host genome integration, as seen in retroviral shRNA delivery. Secondly, the components of a VLP delivery system are relatively simple and cheap to prepare. Coat protein subunits that constitute capsids can be produced in large quantities and purified from recombinant E. coli expression systems [88, 89]. In vitro assembly can then be performed to trigger capsid formation and package sxRNA cargo [90, 91] (Figure 1). Thirdly, VLPs can be surface-modified to incorporate a range of useful ligands for immunomasking and targeting [77, 89, 90, 92]. Thus, the resultant semi-synthetic particles, whilst retaining desirable viral attributes for RNAi delivery, exhibit enhanced functionalities.

Research on VLP-mediated siRNA delivery has mostly been based on bacteriophage MS2 (Figure 1) [90, 91]. MS2 is a well characterised T=3 icosahedral virus, with a 180-subunit capsid encapsidating its single-stranded RNA genome [93]. The ease of producing MS2 coat proteins, the robust nature of its capsids, and the abundance of reactive amines on its surface for multivalent ligand display are some reasons for its selection as a model VLP delivery system. MS2 capsids are pH-sensitive, readily disassembling at acidic pH and reassembling upon mixing with a packaging signal, TR, at neutral pH (Figure 1). This not only enables simple siRNA packaging, but also provides a mechanism of siRNA release upon entering the acidic environment of the endosome. With a diameter of 26 nm, MS2 capsids are ideal to benefit from enhanced permeability and retention (EPR). Further, MS2 capsids have a zeta potential of -25 mV [94], thus are unlikely to have strong electrostatic interactions with cells; this may also be adjusted via surface modifications if required.

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MS2 VLPs have recently been used to deliver siRNA targeted against the anti-apoptotic factor, BCL2, to HeLa cells [90]. Tf was conjugated to the VLP surface as a targeting ligand. VLPs successfully delivered siRNAs to the cancer cells in a Tf receptor-dependent manner, with significant levels of BCL2 knockdown and cell death observed at nanomolar siRNA concentrations. The efficiency of siRNA delivery by VLPs was comparable to that by a commercially available liposomal agent, although VLP-delivered siRNAs appeared to be more active.   

MS2 VLPs have also been targeted to hepatocellular carcinoma (HCC) cells for siRNA delivery [91]. A cocktail of siRNAs targeting the mRNA of several cyclins, whose overexpression is associated with hepatocarcinogenesis [95], were packaged inside MS2 VLPs. The VLP surface was then decorated with (1) PEG for immunomasking, (2) SP94, a targeting peptide previously identified by affinity selection from a phage display library against HCC cell surface targets [96], and (3) a histidine-rich peptide to promote endosomal escape. Gene silencing of targeted cyclins, growth arrest and apoptosis were observed at picomolar siRNA concentrations in HCC cells, but not in normal hepatocyte cells. This demonstrated the ability of VLPs to simultaneously deliver different siRNAs to targeted cells, and to achieve multiple gene knockdowns. Such combination therapies may play a significant role in the treatment of tumours in which a diverse set of mutations are present [97].

Preliminary in vivo testing of VLP-mediated RNAi delivery has recently been carried out [98, 99]. MS2 VLPs packaging a pre-miRNA, pre-miR146a, were surface-decorated with HIV Tat peptides for cell penetration; no cell-targeting ligands were used. Upon intravenous delivery to mice, these VLPs displayed widespread biodistribution in the plasma, lung, spleen and kidney, where high levels of mature miR146a were detected and knockdown of known targets of miR146a was observed. Additionally, no off-target toxicities in VLP-treated mice were reported.

Despite these promising data, more is required, in particular detailed pharmacokinetic and long-term safety profiles of VLPs in vivo, to fully assess their suitability for entering clinical trials as an RNAi delivery system.