Structural Optimization of siRNA Conjugates for Albumin Binding Achieves Effective MCL1-Targeted Cancer Therapy

Abstract

The high potential for therapeutic application of siRNAs to silence traditionally undruggable oncogenic drivers remains largely untapped due to the challenges of tumor cell delivery. Here, siRNAs were optimized for in situ binding to albumin through C18 lipid modifications to improve pharmacokinetics and tumor delivery. Systematic variation of siRNA conjugates revealed a lead structure with divalent C18 lipids each linked through three repeats of hexaethylene glycol connected by phosphorothioate bonds. Importantly, we discovered that locating the branch site of the divalent lipid structure proximally (adjacent to the RNA) rather than at a more distal site (after the linker segment) promotes association with albumin, while minimizing self-assembly and lipoprotein association. Comparison to higher albumin affinity (diacid) lipid variants and siRNA directly conjugated to albumin underscored the importance of conjugate hydrophobicity and reversibility of albumin binding for siRNA delivery and bioactivity in tumors. The lead conjugate increased tumor siRNA accumulation 12-fold in orthotopic mouse models of triple negative breast cancer over the parent siRNA. When applied for silencing of the anti-apoptotic oncogene MCL-1, this structure achieved approximately 80% MCL1 silencing in orthotopic breast tumors. Furthermore, application of the lead conjugate structure to target MCL1 yielded better survival outcomes in three independent, orthotopic, triple negative breast cancer models than an MCL1 small molecule inhibitor. These studies provide new structure-function insights on optimally leveraging siRNA-lipid conjugate structures that associate in situ with plasma albumin for molecular-targeted cancer therapy.

Albumin-binding RNAi Conjugate for Carrier Free Treatment of Arthritis

“…Notably, >80% of the total siRNA<(EG 18 L) 2 detected by FPLC was found in the albumin-containing fraction, higher than any other siRNA-lipid conjugate tested. This observation was consistent with the previously-measured high affinity binding of siRNA<(EG 18 L) 2 to albumin ( K D = 30 nM) 49 .…”

“…The zipper siRNAs were end-modified with bivalent C18 lipids intended to promote binding of the siRNA into the natural fatty acid (FA) binding pockets of albumin ( Figure 1I ) 26 . A library of bivalent lipid structures were synthesized as recently reported 49 . Briefly, a splitter phosphoramidite (<) was added at the 5D end of the sense strand, followed by addition of 0 (EG 0 ), 1 (EG 6 ), 3 (EG 18 ), or 5 (EG 30 ) hexa-ethylene glycol (EG 6 ) phosphoramidite spacers to each branch.…”

“…Although covalent conjugation of siRNA to albumin has been explored to some extent 47,48 , lipid-siRNA conjugates that enable reversible, in situ association with native circulating albumin has been proof-of-concept tested only in cancer studies 26,49,50 . We show here that systemic delivery of an albumin-hitchhiking siRNA-lipid conjugate targeting MMP13 achieves robust delivery, MMP13 silencing, and therapeutic efficacy in joints afflicted by OA and RA.…”

Programable Albumin-Hitchhiking Nanobodies Enhance the Delivery of STING Agonists to Potentiate Cancer Immunotherapy

“…While several promising albumin-binding molecules have been described, including small molecules, fatty acids, and peptides, 27,29 we elected to build our platform from a nanobody with high a nity for albumin because nanobodies are modular and programmable via genetic engineering, are molecularly well-de ned, are amenable to scalable industrial manufacturing, and are components of approved and clinically-advanced therapeutics, including ozoralizumab, which contains an anti-albumin nanobody domain. 31 Additionally, we sought to avoid the potential risk of accelerated albumin clearance that can occur due to direct covalent drug conjugation strategies 27,29 and to minimize the liver accumulation associated with the use of lipid-based albumin binders 30 , a challenge also faced by many promising nanoparticle-based STING agonists. 19,21,33,34 We therefore recombinantly expressed a previously described nanobody domain -termed nAlb -that binds with nanomolar a nity to serum albumin (Fig.…”

“…26 Albumin is a promising drug carrier based on its long circulation half-life and proclivity to accumulate at tumor sites via both passive and active transport mechanisms. [27][28][29] Albumin and albumin-binding chaperones have been widely employed to improve the delivery of chemotherapeutics, exempli ed by albumin-bound paclitaxel (Abraxane®) 30 , as well as protein 31 , peptide 32 , and nucleic acid therapeutics 30 . Inspired by this previous work that motivates the unexplored potential of albumin as a carrier for STING agonists, we engineered a high-a nity anti-albumin nanobody (i.e., single-domain antibody) for site-selective enzymatic bioconjugation of STING agonists via biorthogonal chemistry.…”