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Toward New Design Principles for Superior Gene Silencing Rangaramanujam M

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Toward New Design Principles for Superior Gene Silencing Rangaramanujam M COMMENTARY COMMENTARY Toward new design principles for superior gene silencing Rangaramanujam M. Kannana,b,1 Delivery Is the Key Challenge in siRNA rapidly degraded by nucleases in bodily fluids, experi- Therapies ence rapid clearance by the kidney and liver, and have RNA interference (RNAi) or “gene silencing” is a fas- the potential to trigger unfavorable innate immune re- cinating mechanism in which a cell utilizes a gene’s sponses (6, 7). Many promising siRNA delivery strate- own RNA sequence to shut down expression of that gies are being explored in preclinical stages and gene. This process can be employed to investigate beyond, including the use of lipid nanoparticles and the role of genes in disease and to design therapeutic N-acetylgalactosamine (GalNAc)-based conjugates, approaches involving gene silencing (1, 2). During the with some success. There is a critical need for ap- process of RNAi, “dicer” enzymes cut the RNA into proaches that offer improved efficacy and safety, and small fragments, called small-interfering RNA (siRNA) deliver siRNAs to organs other than the liver (6, 7). or microRNA (miRNA), which bind to a special class of proteins called Argonaute proteins, particularly Codeilvery of siRNA with Ago2 Produces Argonaute 2 (Ago2), enabling the inhibition of target Superior Efficacy messenger RNA translation. RNAi is often used by cells The PNAS paper by Li et al. (8) provides important as a defense mechanism against the infiltration of foreign insights toward addressing the challenges in siRNA nucleic acids from viruses and bacteria, but can be therapies, especially relating to improving their cel- engineered in vitro to selectively silence genes. While lular delivery and functional efficacy, by offering novel genome editing technologies, such as CRISPR, “per- design principles at the nanoscale level. Building on the manently” modify a gene, “temporary” silencing of recent studies on the important role of the combined genes at the mRNA level by siRNAs is desirable in local presence of siRNA and Ago2 protein (Ago2- many diseases and would have fewer associated off- bound siRNA), Li et al. developed an approach to target effects. Since the discovery of RNAi more than package both the duplex siRNA and Ago2 protein 20 years ago, the approach has attracted tremendous onto “structurally defined” polyamines for codelivery attention, due to the sheer versatility it provides in the to the cytoplasm (9). Ago2 was found to be more manipulation of specific undruggable genes, with critical compared with the other three Argonaute significant implications in many indications, including proteins in mammalian cells. Once in the cells, pas- viral diseases and cancer (1–5). Unlike small-molecule senger strands of the siRNA get cleaved and detach inhibitors or antibodies, siRNAs can act at the specific from the Ago2 during RNA-induced gene silencing gene level (3). The delivery of siRNA may be more complex formation and mRNA translation, while the desirable as it can be delivered to the cytosol and antisense siRNA strand-loaded Ago2 can continue does not require transport into the nucleus or in- to recognize and cleave target mRNA over a sus- teraction with chromosomal DNA, unlike miRNA (2). tained period of time. Several promising proof-of- A large library of siRNAs for many genes has been concept efficacy results are offered showing that: identified, and can be scaled up at relatively low costs. (i) double-stranded siRNA (ds siRNA)/Ago2 is better Many siRNA products are undergoing clinical trans- than single-stranded siRNA alone, and (ii)codelivery lation, with significant commercial investments, with the of a preassembled siRNA/Ago2 is superior to in- rate-limiting step for successful translation being the creasing the inherent Ago2 levels in the cells and design of appropriate and effective delivery vehicles (6, delivering just the ds siRNA. The improved efficacy 7). However, as in the case of other DNA and RNA drug associated with the preassembled siRNA/Ago2 may candidates, siRNAs are facing major challenges of offer novel approaches to improve efficacy of RNAi “delivery.” These “charged” macromolecules are technology. aCenter for Nanomedicine, Wilmer Eye Institute, The Johns Hopkins School of Medicine, Baltimore, MD 21287; and bDepartment of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD 21218 Author contributions: R.M.K. wrote the paper. The author declares no conflict of interest. Published under the PNAS license. See companion article on page E2696 in issue 12 of volume 115. 1Email: [email protected]. Published online March 16, 2018. 3200–3201 | PNAS | March 27, 2018 | vol. 115 | no. 13 www.pnas.org/cgi/doi/10.1073/pnas.1801554115 Downloaded by guest on October 1, 2021 This raises the question of how to design delivery vehicles that and in a mouse model. When administered intratumorally, the codeliver both components, which is also addressed by Li et al. “optimized” polyamine/anti-STAT3 siRNA/Ago2 complex led to (8). To study the design parameters of the synthetic polyamine reduced tumor burden and improved survival compared with delivery vehicle that govern the coassembly and the resulting polymer/anti-STAT3 siRNA complexes and additional controls, gene-silencing efficiency of siRNA/Ago2, Li et al. synthesized a suggesting that the codelivery of structurally optimized siRNA/ series of synthetic polypeptides derived from N-carboxyanhydride Ago2 has significant potential as a therapy. polymerization of an L-benzyl aspartate backbone, with varying aminoethylene side chain substitutions and additional methylene Considerations for Clinical Translation groups between the amines. This polymer backbone has been This PNAS paper from the Hammond group is a promising effort shown to be relatively safe in vitro and in vivo, indicating that ap- in the challenging field of siRNA therapeutics (8). It offers ap- propriate manipulation of the side chains may enable superior proaches to enhance the potential of RNAi in preclinical models delivery without negatively affecting toxicity (10). For a fixed N/P and beyond, attempting to define important nanoscale aspects in ratio of 20:1 (ratio of the number of protonatable amine groups in the assembly, cellular cytosolic delivery, and disassembly of two the polyamine side chain to that in the siRNA), Li et al. (8) show that critical molecules (siRNA and Ago2) that must work together in- the gene-silencing efficacy was higher: (i) when the extent of amine timately. Gene-silencing technologies could be a key component protonation at the cytoplasmic pH (∼7.4) was higher, and the of precision medicine when harnessed appropriately. Over the number of amine groups in the side chain was higher, due to the last 15 years substantial efforts have been made to bring siRNA increased colocalization between siRNA and Ago2 inside cells; and technologies to the clinic. These were met with many challenges (ii) when there is more spacing between the protonatable amine associated with fast clearance, liver accumulation, toxicities as- side groups. These studies suggest that amine group distribution sociated with on-target, off-target, innate immune activation, and on the polymer side chain, their spacing, and the relative stoichi- delivery vehicles, and a lack of robust efficacy with siRNA alone (4, ometries between the polymer/siRNA/Ago2 can be tunable nano- 5). However, Alnylam/Sanofi recently reported the first Phase III scale parameters that can be toolsets in the context of systems success for RNAi therapy with their drug Patisiren (APOLLO trial) biology, eventually enabling us to tune different genes simulta- for the treatment of a rare nerve disorder called familial amyloid neously. This is enabled by the fact that codelivered Ago2 plays polyneuropathy (11). Several trials, with many targeting liver such a crucial role in gene silencing. Two sets of complexes could genes, are currently underway (5). It is clear that significant in- contain different amounts of siRNA/Ago2 levels modulating the creases in robustness and efficacy at the cellular/tissue level are genes differently. Even though these effects are attributed to critical. Li et al. (8) demonstrate that they could address this to changing electrostatic interactions between the polymer, siRNA, some extent when the complexes are administered locally. These and Ago2, more detailed studies on the mechanism of these must be translated to delivery systems that can overcome the nanoscale effects, and the subsequent impact on the in vivo sta- challenges that accompany systemic administration, especially bility would be helpful. Li et al. also provide elegant experimental relating to reaching target tissues. Improved siRNA packaging tools to investigate this within cells. and cellular delivery, through well-defined design principles, can Having established the proof-of-concept GFP silencing using open new avenues for RNAi therapies, enabling personalized, anti-GFP siRNA, Li et al. (8) evaluate the therapeutic efficacy using precision medicines, which appear more attainable due in part to a STAT3 oncogene as the target for silencing in melanoma cells the advances published here. 1 Ozcan G, Ozpolat B, Coleman RL, Sood AK, Lopez-Berestein G (2015) Preclinical and clinical development of siRNA-based therapeutics. Adv Drug Deliv Rev 87:108–119. 2 Wang J, Lu Z, Wientjes MG, Au JL (2010) Delivery of siRNA therapeutics: barriers and carriers. AAPS J 12:492–503. 3 Zhang P, et al. (January 31, 2018) Recent advances in siRNA delivery for cancer therapy using smart nanocarriers. Drug Discov Today, 10.1016/j.drudis.2018.01.042. 4 Chakraborty C, Sharma AR, Sharma G, Doss CGP, Lee SS (2017) Therapeutic miRNA and siRNA: Moving from bench to clinic as next generation medicine. Mol Ther Nucleic Acids 8:132–143. 5 Anthiya S, et al. (February 9, 2018) MicroRNA-based drugs for brain tumors. Trends Cancer, 10.1016/j.trecan.2017.12.008. 6 Chery J (2016) RNA therapeutics: RNAi and antisense mechanisms and clinical applications. Postdoc J 4:35–50. 7 Wittrup A, Lieberman J (2015) Knocking down disease: A progress report on siRNA therapeutics.
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