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Taking charge of siRNA delivery Anastasia Khvorova, Maire F Osborn, and Matthew R Hassler

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Delivery of siRNA into cells is achieved by neutralizing the negative charge of the phosphate backbone in a reversible manner. Short interfering RNAs (siRNAs) show promise for treating a wide range of human diseases and could become a third major class of drugs, on par with small molecules and proteins. The main hurdle in developing these molecules as drugs has been delivery. Because siRNAs are large and negatively charged, they must be chemically modified or formulated to promote tissue distribution and cellular uptake, but in a way that does not interfere with their biological function. In this issue, Meade et al.1 describe a set of modifications that facilitates siRNA delivery into cells and is then removed by intracellular enzymes (Fig. 1). Their technology, called short interfering ribonucleic neutrals (siRNNs), opens up an array of new possibilities for oligonucleotide delivery. Current methods for delivering siRNAs to target tissues are too inefficient to support clinical translation except in a few tissues, such as the liver and kidney, that can be targeted systemically, or tissues amenable to local administration. For delivery to the liver, the preferred approach is fully stabilized siRNA or antisense triple GalNAc (acetylgalactosamine) conjugates, mostly owing to their efficacy, ease of manufacturing and wide therapeutic index2. The technology developed by Meade et al.1 combines and extends two previous approaches—backbone neutralization and cell-penetrating peptides—to generate novel compounds capable of unassisted cellular internalization. Backbone neutralization is provided by acyl-2-thioethyl phosphotriester (SATE) groups, which are conjugated to phosphate groups of an siRNA, creating an siRNN. The SATE groups are then further modified with cell-penetrating peptides from the HIV trans-activating transcriptional activator (TAT) protein3. RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA. e-mail: [email protected]

The SATE groups partially neutralize the backbone phosphate charge and add hydrophobicity. Importantly, they are also bio­ cleavable. After siRNNs enter cells, the SATE groups are cleaved by cellular thioesterases, restoring the siRNA to its native, functional state. The SATE group is a biologically cleavable version of a protection group initially developed as a pro-drug approach to enhance cell internalization of nucleotide-based HIV and hepatitis B virus inhibitors4 and is a version of a thioester-protecting group used by the Caruthers laboratory5. However, none of these previous approaches were robust enough to achieve efficient oligo internalization while maintaining potent biological function. The main contribution of Meade et al.1 is optimization of the number, type and position of SATE modifications in the siRNA to enable efficient delivery without compromising enzymatic function. The role of the TAT peptides is to facilitate transport of the siRNA across cellular membranes (Fig. 1b and Supplementary Video 1). Attachment of TAT peptides to uncharged molecules such as PNAs (peptide nucleic acids)6 and proteins is known to promote their efficient cellular uptake. But direct linkage of TAT peptides to charged oligonucleotides such as siRNA has produced mixed results, probably because the peptides can be inactivated through binding to the oligonucleotide. In previous work from the same group7, this issue was partially resolved by making a hybrid of a TAT peptide with a double-stranded (ds)RNA binding domain. The dsRNA RNA binding domain provided a handle for the siRNA to bind, leaving the TAT peptide free for cellular interactions. However, for therapeutic applications such a protein-based construct would be too complex and potentially immunogenic. Meade et al.1 systematically evaluate a large number of constructs using more than 40 novel phosphoramidites (RNA synthesis precursors) and identify the type, number and

nature biotechnology volume 32 number 12 DECEMBER 2014

positional location of SATE modifications that do not interfere with duplex formation and improve uptake. They show that direct conjugation of four TAT peptides to an siRNA of which only 11 of 40 phosphodiester groups are modified by SATE is sufficient to produce compounds that enter cells passively (Supplementary Video 1). Once inside a cell, the SATE groups are efficiently cleaved by intracellular phosphodiesterases, creating molecules capable of gene silencing, with apparent EC50 values in the low nano­molar range (Fig. 1a). The discovery that siRNA delivery is enabled by neutralizing only ~25% of the phosphate charges and adding only four TAT peptides is one of the most striking findings of the study. With fully neutral PNAs, a single TAT conjugate enables uptake, whereas with siRNNs, four peptides are necessary for optimal delivery. This suggests that the extra TAT peptides are binding to the SATE-modified siRNA and thereby providing additional charge neutralization. Modification of ~65% of backbone phosphate was an upper limit for maintaining duplex integrity and thus biological efficacy. In addition to neutralizing the charge of the backbone, SATE modifications are highly hydrophobic, affecting oligonucleotide lipophilicity, which by itself might be affecting cellular internalization properties. Indeed, the authors demonstrate that incorporation of 18 SATE groups (42%) per siRNA results in poor aqueous solubility, which might be rescued by more hydrophilic (O-SATE) variants. In the future, more extensive neutralization may be possible, specifically in a context of singlestranded oligonucleotides. It will be interesting to determine whether SATE modifications improve cellular uptake of other classes of therapeutic oligonucleotides, such as antisense, antagomiRs, GalNac and cholesterol conjugates. It is likely that in each case the winning combination of the number, type and position of SATE modifications will have to be optimized separately. 1197

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Figure 1 Internalization and cellular processing of TAT-conjugated siRNN. (a) Internalization of siRNNs is mediated by four TAT peptides conjugated to SATE groups. Upon internalization, the TAT-SATE modification is cleaved by intracellular thioesterases, releasing a functional negatively charged siRNA molecule. The deprotected siRNA is subsequently loaded into the RISC complex for target mRNA silencing. (b) TAT-conjugated siRNNs include four highly cationic TAT peptides (red) bound to a SATE-modified passenger strand RNA (white) containing four A-SATE linkers (dark gray) and a single irreversible 5′-dimethylbutyl (DMB) modification (light gray). The guide strand (teal) is decorated with six t-butyl SATE groups (black) and a 5′-phosphate (blue). Both RNA strands contain 2′-O-methyl purine and 2′-fluoro pyrimidine substitutions. The SATE modifications partially neutralize the highly negative charge of the 20-nucleotide RNA duplex, which is restored upon intracellular thioesterase conversion of the phosphotriester to a phosphatediester (mechanism shown in insert).

The most clinically advanced RNAi platform today is GalNAc conjugates. This technology— which combines fully stabilized siRNA8 and triple GalNac conjugation (first considered for oligonucleotide delivery in the early 90s9)— works extremely well in the liver because uptake depends on the cellular expression levels of asialoglycoprotein receptors, which are highly expressed on hepatocytes. In contrast, uptake of siRNNs is not cell-type specific and therefore might have much wider clinical applications. The ease of incorporating siRNN modifications will allow the community to perform a myriad of novel conjugations without the need for complicated chemistry. This capability will undoubtedly lead to the creation of novel classes of oligonucleotides. Naturally, further optimization will be needed to achieve ideal pharmacokinetic and pharmacodynamic behavior and acceptable toxicity, as indicated by the moderate in vivo efficacy shown by Meade et al.1 But clearly this work inaugurates a fundamentally new approach to overcome the challenge of cellular delivery of therapeutic oligonucleotides. Note: Any Supplementary Information and Source Data files are available in the online version of the paper .

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COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Meade, V. et al. Nat. Biotechnol. 32, 1256–1261 (2014). 2. Anonymous. RNAi roundtable: advances in delivery of RNAi therapeutics with enhanced stabilization chemistry (ESC)-GalNAc-siRNA conjugates http:// www.alnylam.com/capella/roundtables/esc-galnacsirna-conjugates/ (Alnylam, 2014). 3. Fawell, S. et al. Proc. Natl. Acad. Sci. USA 91, 664–668 (1994).

4. Périgaud, C. et al. Bioorg. Med. Chem. 3, 2521–2526 (1993). 5. Wiesler, W.T. & Caruthers, M.H. J. Org. Chem. 61, 4272–4281 (1996). 6. Gait, M.J. Cell. Mol. Life Sci. 60, 844–853 (2003). 7. Eguchi, A. et al. Nat. Biotechnol. 27, 567–571 (2009). 8. Morrissey, D.V. et al. Hepatology 41, 1349–1356 (2005). 9. Hangeland, J.J. et al. Bioconjug. Chem. 6, 695–701 (1995).

Lightening the load in synthetic biology Eric Klavins A new biological device known as a ‘load driver’ improves the performance of synthetic circuits by insulating genetic parts from each other. Synthetic biologists dream of designing genetic circuits as easily as electrical engineers design electronic circuits. Yet, unlike microchips, genetic parts such as transcription factors and Eric Klavins is at the Department of Electrical Engineering, University of Washington, Seattle, Washington, USA. e-mail: [email protected]

promoters rarely behave as intended when they are interconnected. Predictable performance is undermined by the nature of the cellular environment, including the genetic context of sequences encoding parts, the unanticipated effects of parts on each other and their nonspecific interactions with other molecules in the cell. These effects are compounded as the size of a circuit increases, preventing the

volume 32 number 12 DECEMBER 2014 nature biotechnology

Taking charge of siRNA delivery.

Delivery of siRNA into cells is achieved by neutralizing the negative charge of the phosphate backbone in a reversible manner...
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