Codelivering siRNA/Ago-2 Complexes Improve RNA interference

G Sharath Chandra, Ph.D. is a postdoctoral fellow at the University of Kentucky and is interested in RNA interference (RNAi)-mediated management of insect pests.
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A recent paper by Li et al (2018) published in The Proceedings of the National Academy of Sciences, demonstrated an effective method for enhancing the RNA interference (RNAi) by codelivery of siRNA and Argonaute 2 (Ago-2) protein complex through different delivery carries.

RNAi emerging as a potential therapeutic strategy to rapidly validate drug inhibitors and testing of undrug-able genes in many diseases. It is also a technical approach to investigating insect functional genomics that has been of variable effectiveness for numerous reasons. RNAi is achieved by delivery of double-stranded RNAs of 21-23 nucleotide length – small interfering RNAs (siRNAs) – to cells that enable the specific Ago-2 mediated degradation of the target mRNA and eventually cause the loss-of-function of the target gene. Achieving efficient knockdown of  target genes is essential for clinical translation of therapeutic drugs as well as insect functional genomics, however, this remains challenging.

The major barrier to siRNA based gene silencing is inefficient formation of siRNA/Ago-2 protein complexes in the cytoplasm. Ago-2 protein acts as a molecular scissor that is loaded with siRNA. It retains one strand of the siRNA – the guide strand – and cleaves the remaining passenger strand of the siRNA. The resulting guide strand containing complex then searches for complementary sequences in target mRNA, leading directly to their cleavage.

To deliver preassembled siRNA and Ago-2 complexes into cells, the authors tested different carriers such as cationic lipids and structurally defined synthetic polypeptides. In this regard, the authors expressed human Ago-2 protein in SF9 insect cells and purified the recombinant protein.  Preassembled single-stranded (ss)/double stranded (ds) siRNA of specific to GFP and Ago-2 protein complex was delivered using the commercially available lipid-based transfection reagent, TransIT-X2 into GFP expressing human 293T cells.  GFP express was used as a metric for gene silencing.

The authors observed significantly more silencing of GFP expression when ds siRNA/Ago-2 preassembled complex is delivered compared to siRNA alone or single-stranded siRNA/Ago-2 complex was delivered in the same way.

Li et al, also  synthesized a series of lengths and charge variants of the side chain polyamines with a polyaspartamide backbone used for delivering the preassembled siRNA/Ago-2 complex human 293T cells. The polyamines are derived using N-carboxyanhydride polymerization of L-benzyl aspartate, followed by exhaustive amination of side-chain with one to four repeats of aminoethylene. These polyamines are biocompatible and biodegradable, thus offers protection, efficient cellular uptake and sustained delivery of siRNA into target cells.

Image result for endosomal escape

Endosomal escape is a general issue when trying to deliver materials into cells via endocytosis. If endosomal contents are shunted to lysosomes then delivery fails because the material is degraded. When shunted away from lysosomes materials can be delivered to the cytoplasm and possibly effect the desired change.

The authors observed that the extent of GFP silencing was proportional to the number of amine moieties in the polyamine side-chain used for codelivery of siRNA/Ago-2. This indicates that the degree of silencing is impacted by the structure of polyamine transfection reagent also.

Li et al applied their strategy to silence the oncogene, Signal Transducer and Activator of Transcription 3 (STAT3), as its overexpression is linked to tumor progression, drug resistance and formation of cancer stem cells in melanoma induced mice. Their results revealed that polyamine based codelievry of preassembled siRNA and Ago-2 complex induced highly efficient STAT3 gene silencing compared to naked siRNA delivery. This also resulted in a pronounced growth inhibition of melanoma cells and a significant increase in the survival of mice challenged with melanoma.

Interestingly, the authors observed a majority of siRNA/Ago-2 complex molecules delivered through polyamine escaped the late endosomes and lysosomes which are the main barriers causing less efficient RNAi in some species.

Despite powerful genome editing technologies like CRISPR/Cas, the majority of clinical drug validations require temporary knockdown of the target gene. The method demonstrated by the authors gives a ray of hope to enhance the RNAi efficiency in clinical research and could also be applied in RNAi-refractive insect systems like Lepidopteran insects where the delivered siRNA/dsRNA trapped in endosomes that resulting in less efficient RNAi.

Li, J., Wu, C., Wang, W., He, Y., Elkayam, E., Joshua-Tor, L., Hammond, P.T., 2018. Structurally modulated codelivery of siRNA and Argonaute 2 for enhanced RNA interference. Proceedings of the National Academy of Sciences of the United States of America 115, E2696-e2705. https://doi.org/10.1073/pnas.1719565115

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