In Vivo Application of RNA Interference: From Functional Genomics to Therapeutics

Abstract

RNAi has rapidly become a powerful tool for drug target discovery and validation in cell culture, and now has largely displaced efforts with antisense and ribozymes. Consequently, interest is rapidly growing for extension of its application to in vivo systems, such as animal disease models and human therapeutics. Studies on RNAi have resulted in two basic methods for its use for gene selective inhibition: 1) cytoplasmic delivery of short dsRNA oligonucleotides (siRNA), which mimics an active intermediate of an endogenous RNAi mechanism and 2) nuclear delivery of gene expression cassettes that express a short hairpin RNA (shRNA), which mimics the micro interfering RNA (miRNA) active intermediate of a different endogenous RNAi mechanism. Non-viral gene delivery systems are a diverse collection of technologies that are applicable to both of these forms of RNAi. Importantly, unlike antisense and ribozyme systems, a remarkable trait of siRNA is a lack of dependence on chemical modifications blocking enzymatic degradation, although chemical protection methods developed for the earlier systems are being incorporated into siRNA and are generally compatible with non-viral delivery systems. The use of siRNA is emerging more rapidly than for shRNA, in part due to the increased effort required to construct shRNA expression systems before selection of active sequences and verification of biological activity are obtained. In contrast, screens of many siRNA sequences can be accomplished rapidly using synthetic oligos. It is not surprising that the use of siRNA in vivo is also emerging first. Initial in vivo studies have been reported for both viral and non-viral delivery but viral delivery is limited to shRNA. This review describes the emerging in vivo application of non-viral delivery systems for RNAi for functional genomics, which will provide a foundation for further development of RNAi therapeutics. Of interest is the rapid adaptation of ligand-targeted plasmid-based nanoparticles for RNAi agents. These systems are growing in capabilities and beginning to pose a serious rival to viral vector based gene delivery. The activity of siRNA in the cytoplasm may lower the hurdle and thereby accelerate the successful development of therapeutics based on targeted non-viral delivery systems.

Plasmid-based Stat3 siRNA delivered by hydroxyapatite nanoparticles suppresses mouse prostate tumour growth in vivo

“…Delivering siRNA to animal tissues in vivo is a complicated process and involves Plasmid-based Stat3 siRNA ZW Liang et al 484 physical, chemical or biological approaches or a combination of them in some cases. 26 The major weaknesses limiting the wide application of most delivery systems are the potential for mutagenesis or oncogenesis, host immune responses and high cost. 27 Non-viral siRNA delivery to tissues does not elicit an immune response, has a great advantage in drug target validation and allows multiple administrations of siRNA, attributes that are crucial for the therapeutic application of siRNA.…”

Therapeutic RNA interference of malignant melanoma by electrotransfer of small interfering RNA targeting Mitf

“…Nonviral transfection with synthetic siRNA is free from virusassociated adverse effects, while the RNAi effect may be transient. 43,53,54 Although the most appropriate delivery system should be selected for each RNAi therapeutic application, electrotransfer of synthetic siRNA may be suitable for Mitf-knockdown therapy for melanoma, because long-lasting silencing effects are not always required to kill tumor cells that have been successfully transfected with siRNA.…”

“…Further frequent treatments with siRNA may lead to more prominent therapeutic outcomes. Other nonviral procedures, including sonoporation 67 and synthetic vectors, 54,68 may also allow repetitive treatments for a melanoma patient.…”

Matrix metalloproteinase 12 silencing: A therapeutic approach to treat pathological lung tissue remodeling?

“…Stabilization can be a challenge to take up as naked siRNA are theorically not protected against nuclease digestion. Nevertheless, stabilization does not appear to be necessary in most cases, as rapid excretion seems to occur prior to degradation [116] and double strand RNA are much more resistant to nucleases than single strand RNA. These considerations are supported by longlasting effect (for weeks) that has been observed in non-dividing cells in vitro and in vivo [117].…”