(E-200) Albumin-binding siRNA conjugates targeted against Mcl-1 effectively reduce metastatic tumor burden

Sub-Track: Cancer Drug Delivery

Triple-Negative Breast Cancer (TNBC) comprises approximately 15-20% of breast cancers. Because TNBC lacks targetable surface receptors, patients are relegated to more toxic and less effective chemotherapies. In addition to limited treatment options, outcomes for these patients are also worse than in patients with other breast cancer subtypes, because TNBCs are highly invasive, prone to relapse, and have high metastatic potential. Short interfering RNA (siRNA)-based therapies are a promising class of therapeutics capable of silencing the genes of traditionally ‘undruggable’ oncogenic targets. However, effective tumor delivery of siRNA faces many challenges including nuclease degradation and short circulation times. The recent development of enhanced stabilization chemistries such as 2ʹ ribose modifications and phosphorothioate linkages has enabled carrier-free delivery of serum-stable siRNA. Beyond these chemical modifications, we have developed an siRNA lipid end-modification strategy that  promotes in situ albumin binding following in vivo delivery as a means to greatly enhance the siRNA circulation time and tumor delivery. Here, we describe a chemically modified siRNA capable of albumin binding and tumor gene silencing and demonstrate its therapeutic efficacy in treating both solid tumors and metastatic disease by targeting the anti-apoptotic protein Mcl-1, which is amplified in many TNBCs and actively promotes tumor cell survival.

Synthesis of siRNA-L₂ lipid conjugates was performed in house on an oligonucleotide synthesizer using 2′-F and 2′-O-Me phosphoramidites in an alternating “zipper” pattern, combined with use of phosphorothioate linkages to the final two bases on both ends of both strands. To evaluate the blood plasma pharmacokinetic profile, mice were injected i.v. with 20 mg/kg siRNA-L₂, and 10 µL blood was collected by lateral tail vein puncture at specified timepoints. A peptide nucleic acid (PNA) hybridization HPLC assay was use for absolute, label-free quantification from blood samples. To establish solid tumors, HCC70, HCC1187, or MDA-MB-231 cells (1 x 10⁶) in 50% Matrigel were injected into the inguinal mammary fat pads of female athymic (nu/nu) mice. Mice were treated once weekly with 10 mg/kg siMCL1-L₂ or control conjugate, and tumors were measured with calipers. Metastases were established by injecting female athymic (nu/nu) mice with MDA-MB-231.Luc cells (1 x 10⁶) in saline either i.v. to form lung metastases or i.p. to form visceral metastases. To monitor tumor growth, D-luciferin salt was injected i.p. followed by IVIS bioluminescence imaging to establish baseline radiance measurements. Mice were treated with siMCL1-L₂ or control once weekly and luminescence imaging was performed 7 days after each injection.

We have successfully developed and optimized a chemically modified, serum-stable siRNA conjugated to a divalent-lipid moiety that enables albumin hitch-hiking and an enhanced pharmacokinetic profile. The PNA assay revealed a distribution (alpha phase) half-life of 30 minutes and clearance (beta phase) half-life of 9 hours in the circulation. In long term efficacy studies in three different orthotopic mouse models of triple-negative breast cancer, siMCL1-L₂ showed a significant reduction in tumor volume over time and inhibited growth in 8/8 HCC70 tumors, 4/6 HCC1187 tumors, and 4/10 MDA-MB-231 tumors. Excitingly, in models of lung and visceral metastatic triple-negative breast cancer, once weekly treatments of siMCL1-L₂ markedly reduced tumor growth compared to controls.

These promising results highlight the immense potential of siRNA therapeutics as a new treatment option for TNBC. In addition to therapeutic efficacy, we expect that siMCL1-L₂ will also avoid the toxicities associated with small molecule inhibitors of Mcl-1. In our in-progress studies, we are comparing the therapeutic effect of weekly treatment with siMCL1-L₂ and an Mcl-1 inhibitor in three different TNBC models of lung metastases. Additionally, we are evaluating the acute and chronic toxicities of both therapeutics in a mouse genetically engineered to express the human isoform of Mcl-1. While our siRNA sequence targets both the human and mouse isoforms of Mcl-1, the small molecule inhibitor has a higher affinity for human Mcl-1, and it has been shown that these mice are more sensitive to Mcl-1 inhibitors and may represent a more accurate evaluation of Mcl-1 related toxicities. Overall, we expect these paired studies to highlight the benefits and widened therapeutic window of siRNA therapeutics versus traditional small molecules.