The central goal of the Siegwart Lab is to use materials chemistry to solve challenges in cancer therapy (1-10) and imaging (11-14). In particular, we are focused on the development of new materials that can deliver ribonucleic acids (RNAs) to improve cancer outcomes. An array of coding and non-coding RNAs can now be used as cancer therapeutics (siRNA, miRNA, mRNA, CRISPR RNAs) because they are able to manipulate and edit expression of the essential genes that drive cancer development and progression. For example, gene silencing via the RNA Interference (RNAi) mechanism is a promising strategy to treat cancer. However, the success of short interfering RNA (siRNA) or microRNA (miRNA)-based therapies has been limited by the difficulty of delivering these highly anionic biomacromolecular drugs into cells. The delivery of longer RNAs, such as messenger RNA (mRNA) and single guide RNA (sgRNA) poses an even greater challenge. Overall, we seek to discover and define the critical physical and chemical properties of synthetic carriers required for therapeutic delivery of small (e.g. ~22 base pair miRNA) to large (e.g. ~5,000 nucleotide mRNA) RNAs.
Enabling non-viral CRISPR/Cas gene eding.
The Siegwart lab reported the first successful non-viral system for in vivo co-delivery of Cas9 mRNA and sgRNA (1). CRISPR/Cas is a revolutionary gene editing technology with wide-ranging utility. Future therapeutic use depends on the ability to safely and effectively deliver CRISPR/Cas components to mammalian cells. Prior to our work, delivery had largely been accomplished using viruses, which have limited translational potential. We reported the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to deliver long RNAs (Cas9 mRNA and targeted single guide RNA (sgRNA)) from a single ZAL nanoparticle (ZNP) to enable gene editing. Delivery of low sgRNA doses (15 nM) can reduce reporter protein expression by >90% in cells. In contrast to transient therapies (e.g. RNAi-mediated mRNA degradation), we showed that ZNP delivery of sgRNA resulted in permanent DNA modification, where the 95% decrease in protein expression is sustained indefinitely even after multiple rounds of cellular division. ZNP delivery of mRNA results in high protein expression at low doses in vitro (<600 pM) and in vivo (1 mg/kg). In mice, intravenous co-delivery of Cas9 mRNA and sgLoxP (4:1 wt ratio; 5 mg/kg total RNA) induced expression of floxed tdTomato in the liver, kidneys, and lungs of genetically engineered mice. The effectiveness of ZNPs for delivery of long RNAs provides a chemical guide for the rational design of future carriers (1). Moreover, this development of gene editing using synthetic nanoparticles is a promising step towards improving the safety, efficacy, and utility of CRISPR/Cas.
Studying and enabling cancer cell selectivity.
Conventional chemotherapeutics nonselectively kill all rapidly dividing cells, which produces numerous side effects. To address this challenge, we reported discovery of functional polyesters that are capable of delivering small RNA drugs selectively to lung cancer cells and not to normal lung cells (2). Selective polyplex nanoparticles (NPs) were identified by high-throughput library screening on a unique pair of matched cancer/normal cell lines obtained from a single patient. Selective NPs promoted rapid endocytosis into HCC4017 cancer cells, but were arrested at the membrane of HBEC30-KT normal cells during the initial transfection period. When injected into tumor xenografts in mice, cancer-selective NPs were retained in tumors for over 1 week, whereas nonselective NPs were cleared within hours. This translated to improved siRNA-mediated cancer cell apoptosis and significant suppression of tumor growth. Selective NPs were also able to mediate gene silencing in xenograft and orthotopic tumors via i.v. injection or aerosol inhalation, respectively (2, 5).
The finding that cells respond differently to the same nanoparticle has profound implications for gene therapy because cell-type specificity of drug carriers in vivo could alter clinical patient outcomes. Our data suggest that selectivity is an underappreciated reality that should be carefully considered when evaluating drug carriers. The combination of both well-defined molecular targets and nanoparticle delivery to targeted cells is likely required to improve cancer drug accuracy in the clinic.
Improving the tolerability of nanoparticle carriers to enhance miRNA therapeutics.
The Siegwart lab has developed synthetic strategies towards polyester-based dendrimers that can function in aggressive, late-stage disease (4). Liver cancer is a leading cause of death and a global health problem. Unfortunately, seven small-molecule drugs for hepatocellular carcinoma (HCC) have failed phase III clinical trials largely because late-stage liver dysfunction amplifies drug toxicity. MicroRNAs present a promising alternative treatment strategy but require development of delivery vehicles that can avoid this cancer-induced dysfunction, which exacerbates toxicity (10). We overcame this challenge by developing dendrimer nanoparticles that mediate miRNA delivery to late-stage liver tumors with low hepatotoxicity (4).
The chemical design was based on the hypothesis that through introduction of ester bonds and molecular diversity at each expansion step, modular degradable dendrimers would possess a critical balance of low toxicity and high delivery potency to function in aggressive cancer models where the carrier’s own toxicity can negate the benefit of on-target small RNA therapies. The chemical modulation of cores, peripheries, and generations was accelerated by utilization of efficient click reactions to enable a large increase in both the total number (>1,500) and chemical diversity of dendrimers. An aggressive, MYC-driven transgenic liver cancer model was used to examine let-7g tumor suppressor efficacy, resulting in a significant survival benefit. These dendrimer carriers provide high potency in tumors without negatively affecting normal tissues, solving a critical issue in treating aggressive liver cancer (4).
Advancing fundamental reactions for the synthesis of functional polyesters.
The Siegwart lab has developed a series of synthetic strategies towards functional polyesters (2-9). Aliphatic polyesters including polyglycolide, polylactide, polycaprolactone, and their copolymers are useful materials for drug delivery and tissue engineering that have been utilized in a number of FDA-approved products. In order to improve physical properties and to render these materials applicable to delivery of emerging biomacromolecular drugs including RNAs, there is significant interest in synthetic strategies that can be used to prepare side chain functionalized polyesters. Notably, the Siegwart lab developed the first large library of lipocationic polyesters that were synthesized directly from functional monomers without protecting groups (3). A library of lipocationic polyesters was synthesized from functional monomers in high yield, fast time (~2 minutes), and in gram scale. This was accomplished with precise monomer incorporation ratios to enable tunable hydrophobicity and pKa. Formulated nanoparticles (NPs) could localize to tumors in vivo after intravenous administration and were able to silence gene expression in tumor-bearing mice.
Detecting cancer early.
The Siegwart lab has developed tumor imaging agents based on fundamental physical chemistry properties including charge transfer (11), photon upconversion (12), and pH-responsive fluorescence activation (HOMO/LUMO) (13). These imaging modalities can be combined with RNA therapies for the creation of theranostic nanoparticles. Among cancer types, breast cancer metastasis is a major cause of cancer death in women worldwide. Early detection would save many lives, but current fluorescence imaging probes are limited in their detection ability, particularly of bone and liver micrometastases. The Siegwart lab reported development of probes that are capable of imaging tiny (<1 mm) micrometastases in the liver, lung, pancreas, kidneys, and bone, that have disseminated from the primary site (14). The influence of the poly(ethylene glycol) (PEG) chain length on the performance of water-soluble, pH-responsive, near-infrared 4,4′-diﬂuoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) probes was systematically investigated to demonstrate that PEG tuning can provide control over micrometastasis tracking with high tumor-to-background contrast (up to 12/1). Optimized probes effectively visualized tumor boundaries and successfully detected micrometastases with diameters <1 mm. The bone-metastasis-targeting ability of these probes was further enhanced by covalent functionalization with bisphosphonate. This improved detection of both bone and liver micrometastases (<2 mm) with excellent tumor-to-normal contrast (5.2/1). Through a single intravenous injection, these materials can image micrometastases in multiple organs with spatiotemporal resolution (14). They thus hold promise for metastasis diagnosis, image-guided surgery, and theranostic PEGylated drug therapies.
Studies are ongoing to examine therapeutic potential of these RNA delivery systems in liver, ovarian, and lung cancer models. We are currently employing genetic and patient-derived xenograft (PDX) models to better capture the biological complexity of cancer. These results will eventually yield a comprehensive understanding of how and why manipulation of specific biological pathways that drive cancer are effective or not effective in patients.
(1) “Non-viral CRISPR/Cas gene editing in vitro and in vivo enabled by synthetic nanoparticle co-delivery of Cas9 mRNA and sgRNA.” Miller JB, Zhang S, Kos P, Xiong H, Zhou K, Perelman SS, Zhu H, Siegwart DJ* Angew. Chem. Int. Ed. 2017, 56, 1059-1063.
(2) “Functional polyesters enable selective siRNA delivery to lung cancer over matched normal cells.” Yan Y, Liu L, Xiong H, Miller JB, Zhou K, Kos P, Huffman KE, Elkassih S, Norman JW, Carstens R, Kim J, Minna JD, Siegwart DJ.* Proc. Natl. Acad. Sci. U.S.A. 2016, 113, E5702–E5710.
(3) “Rapid synthesis of a lipocationic polyester library via ring-opening polymerization of functional valerolactones for efficacious siRNA delivery.” Hao J, Kos P, Zhou K, Miller JB, Xue L, Yan Y, Xiong H, Elkassih S, Siegwart DJ.* J. Am. Chem. Soc. 2015, 137, 9206-9209.
(4) “Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model.” Zhou K, Nguyen LH, Miller JB, Yan Y, Kos P, Xiong H, Li L, Hao J, Minnig JT, Zhu H, Siegwart DJ.* Proc. Natl. Acad. Sci. U.S.A. 2016, 113, 520-525.
(5) “Aerosol delivery of stabilized polyester-siRNA nanoparticles to silence gene expression in orthotopic lung tumors.” Yan Y, Zhou K, Xiong H, Miller JB, Motea EA, Boothman DA, Liu L,* Siegwart DJ.* Biomaterials 2017, 118, 84-93.
(6) “Scalable synthesis and derivation of functional polyesters bearing ene and epoxide side chains.” Yan Y, Siegwart DJ.* Polym. Chem. 2014, 5, 1362-1371.
(7) “One-pot synthesis of functional poly(amino ester sulfide)s and utility in delivering pDNA and siRNA.” Yan Y, Xue L, Miller JB, Zhou K, Kos P, Elkassih S, Nagai A, Xiong H, Siegwart DJ.* Polymer 2015, 72, 271-280.
(8) “Intercalation-mediated nucleic acid nanoparticles for siRNA delivery.” Zhou K, Kos P, Yan Y, Xiong H, Min Y, Minnig JT, Miller JB, Siegwart DJ.* Chem. Commun. 2016, 52, 12155-12158.
(9) “Progress towards the synthesis of amino polyesters via ring-opening polymerization (ROP) of functional lactones.” Hao J, Elkassih S, Siegwart DJ.* Synlett, 2016, 27, 2285-2292.
(10) “Precise regulation of let-7 levels balance organ regeneration against tumor suppression.” Wu L, Nguyen LH, Zhou K, de Soysa Y, Li L, Miller JB, Tian J, Locker J, Zhang S, Shinoda G, Seligson MT, Zeitels LR, Acharya A, Wang SC, Mendell JT, Nishino J, Morrison SJ, Siegwart DJ, Daley GQ, Shyh-Chang N, Zhu H.* eLife 2015, 4, e09431.
(11) “Biocompatible organic charge transfer complex nanoparticles based on a semi-crystalline cellulose template.” Nagai A, Miller JB, Du J, Kos P, Stefan MC, Siegwart DJ.* Chem. Commun. 2015, 15, 11868-11871.
(12) “Tumor imaging based on photon upconversion of Pt(II) porphyrin rhodamine co-modified NIR excitable cellulose enhanced by aggregation.” Nagai A, Miller JB, Kos P, Elkassih S, Xiong H, Siegwart DJ.* ACS Biomat. Sci. Eng. 2015, 1, 1206-1210.
(13) “Activatable water soluble probes enhance tumor imaging by responding to dysregulated pH and exhibiting high tumor-to-liver fluorescence emission contrast.” Xiong H, Kos P, Yan Y, Zhou K, Miller JB, Elkassih S, Siegwart DJ.* Bioconj. Chem. 2016, 27, 1737–1744.
(14) “High-contrast fluorescence detection of metastatic breast cancer including bone and liver micrometastases via size-controlled pH-activatable water-soluble probes.” Xiong H, Zuo H, Yan Y, Occhialini G, Zhou K, Wan Y, Siegwart DJ.* Advanced Materials, 2017, 29, 1700131.