Justin H. Lo

Targeted Tumor-Penetrating siRNA Nanocomplexes for Credentialing the Ovarian Cancer Oncogene ID4

Tumor-penetrating siRNA nanocomplexes credential ID4 as a therapeutic oncogene target in human ovarian cancer. Nanotechnology Sets Sights on Ovarian Tumors In the world of anticancer research, targeting tumor cells is one challenge; penetrating the cells to deliver therapeutics is another. The combination of specific targeting and efficient delivery is the clinical holy grail, wherein optimization of this approach could lead to highly effective cancer therapy in humans. Ren et al. have now developed a nanotechnology platform that allows for just that: targeted intracellular delivery of RNA-based therapeutics to ovarian cancer cells, which halts the oncogenic activity of a potent gene, in this case ID4. In a screen of overexpressed and essential genes in human ovarian cancer, the authors first identified a potential oncogene, ID4. They then confirmed ID4 tumorigenicity and mechanism in vitro in cell lines. After confirming that ID4 was an oncogene, Ren et al. reasoned that “silencing” the gene using small interfering RNA (siRNA) would prevent tumor growth in vivo. The trick was to make sure the siRNA could cross the cell membrane to exert its silencing effects. To accomplish this, the authors designed a tumor-penetrating nanocomplex (TPN) that could not only bind a protein overexpressed on the surface of human cancer cells but also pass through the membrane via a cell-penetrating peptide. Once inside the cells, the TPN could release the siRNA directed against ID4. Tumor homing was confirmed in mouse models of human melanoma and ovarian cancer. In mice harboring subcutaneous ovarian tumors, TPN/siRNA decreased ID4 expression by up to 90% and suppressed tumor growth by 82%. In mice bearing disseminated intra-abdominal tumors, TPN/siRNA allowed 80% of the animals to live 60 or more days. Control treatments did not prevent tumor growth in either study, and the TPN/siRNA therapy did not elicit any immunogenic side effects. Locked and loaded with siRNA, these TPNs are ready to target and kill cancer cells. The authors envision this to be a platform for credentialing oncogenes and for validating RNA interference in preclinical models before development of therapeutics. However, before moving this TPN/siRNA approach to patients, some additional preclinical optimization is necessary, including pharmacokinetics, testing in human cancer models, and increasing siRNA efficiency at lower doses. The comprehensive characterization of a large number of cancer genomes will eventually lead to a compendium of genetic alterations in specific cancers. Unfortunately, the number and complexity of identified alterations complicate endeavors to identify biologically relevant mutations critical for tumor maintenance because many of these targets are not amenable to manipulation by small molecules or antibodies. RNA interference provides a direct way to study putative cancer targets; however, specific delivery of therapeutics to the tumor parenchyma remains an intractable problem. We describe a platform for the discovery and initial validation of cancer targets, composed of a systematic effort to identify amplified and essential genes in human cancer cell lines and tumors partnered with a novel modular delivery technology. We developed a tumor-penetrating nanocomplex (TPN) that comprised small interfering RNA (siRNA) complexed with a tandem tumor-penetrating and membrane-translocating peptide, which enabled the specific delivery of siRNA deep into the tumor parenchyma. We used TPN in vivo to evaluate inhibitor of DNA binding 4 (ID4) as a novel oncogene. Treatment of ovarian tumor–bearing mice with ID4-specific TPN suppressed growth of established tumors and significantly improved survival. These observations not only credential ID4 as an oncogene in 32% of high-grade ovarian cancers but also provide a framework for the identification, validation, and understanding of potential therapeutic cancer targets.

Identification and Characterization of Receptor-Specific Peptides for siRNA Delivery

Tumor-targeted delivery of siRNA remains a major barrier in fully realizing the therapeutic potential of RNA interference. While cell-penetrating peptides (CPP) are promising siRNA carrier candidates, they are universal internalizers that lack cell-type specificity. Herein, we design and screen a library of tandem tumor-targeting and cell-penetrating peptides that condense siRNA into stable nanocomplexes for cell type-specific siRNA delivery. Through physiochemical and biological characterization, we identify a subset of the nanocomplex library of that are taken up by cells via endocytosis, trigger endosomal escape and unpacking of the carrier, and ultimately deliver siRNA to the cytosol in a receptor-specific fashion. To better understand the structure–activity relationships that govern receptor-specific siRNA delivery, we employ computational regression analysis and identify a set of key convergent structural properties, namely the valence of the targeting ligand and the charge of the peptide, that help transform ubiquitously internalizing cell-penetrating peptides into cell type-specific siRNA delivery systems.

In Vivo Gene Expression Dynamics of Tumor-Targeted Bacteria.

The engineering of bacteria to controllably deliver therapeutics is an attractive application for synthetic biology. While most synthetic gene networks have been explored within microbes, there is a need for further characterization of in vivo circuit behavior in the context of applications where the host microbes are actively being investigated for efficacy and safety, such as tumor drug delivery. One major hurdle is that culture-based selective pressures are absent in vivo, leading to strain-dependent instability of plasmid-based networks over time. Here, we experimentally characterize the dynamics of in vivo plasmid instability using attenuated strains of S. typhimurium and real-time monitoring of luminescent reporters. Computational modeling described the effects of growth rate and dosage on live-imaging signals generated by internal bacterial populations. This understanding will allow us to harness the transient nature of plasmid-based networks to create tunable temporal release profiles that reduce dosage requirements and increase the safety of bacterial therapies.

Smart nanosystems: Bio-inspired technologies that interact with the host environment.

Nanoparticle technologies intended for human administration must be designed to interact with, and ideally leverage, a living host environment. Here, we describe smart nanosystems classified in two categories: (i) those that sense the host environment and respond and (ii) those that first prime the host environment to interact with engineered nanoparticles. Smart nanosystems have the potential to produce personalized diagnostic and therapeutic schema by using the local environment to drive material behavior and ultimately improve human health.

Self-Titrating Anticoagulant Nanocomplexes That Restore Homeostatic Regulation of the Coagulation Cascade

Antithrombotic therapy is a critical portion of the treatment regime for a number of life-threatening conditions, including cardiovascular disease, stroke, and cancer; yet, proper clinical management of anticoagulation remains a challenge because existing agents increase the propensity for bleeding in patients. Here, we describe the development of a bioresponsive peptide–polysaccharide nanocomplex that utilizes a negative feedback mechanism to self-titrate the release of anticoagulant in response to varying levels of coagulation activity. This nanoscale self-titrating activatable therapeutic, or nanoSTAT, consists of a cationic thrombin-cleavable peptide and heparin, an anionic polysaccharide and widely used clinical anticoagulant. Under nonthrombotic conditions, nanoSTATs circulate inactively, neither releasing anticoagulant nor significantly prolonging bleeding time. However, in response to life-threatening pulmonary embolism, nanoSTATs locally release their drug payload and prevent thrombosis. This autonomous negative feedback regulator may improve antithrombotic therapy by increasing the therapeutic window and decreasing the bleeding risk of anticoagulants.

Drug-induced amplification of nanoparticle targeting to tumors

Nanomedicines have the potential to significantly impact cancer therapy by improving drug efficacy and decreasing off-target effects, yet our ability to efficiently home nanoparticles to disease sites remains limited. One frequently overlooked constraint of current active targeting schemes is the relative dearth of targetable antigens within tumors, which restricts the amount of cargo that can be delivered in a tumor-specific manner. To address this limitation, we exploit tumor-specific responses to drugs to construct a cooperative targeting system where a small molecule therapeutic modulates the disease microenvironment to amplify nanoparticle recruitment in vivo. We first administer a vascular disrupting agent, ombrabulin, which selectively affects tumors and leads to locally elevated presentation of the stress-related protein, p32. This increase in p32 levels provides more binding sites for circulating p32-targeted nanoparticles, enhancing their delivery of diagnostic or therapeutic cargos to tumors. We show that this cooperative targeting system recruits over five times higher doses of nanoparticles to tumors and decreases tumor burden when compared with non-cooperative controls. These results suggest that using nanomedicine in conjunction with drugs that enhance the presentation of target antigens in the tumor environment may be an effective strategy for improving the diagnosis and treatment of cancer.

Influence of vascular network design on gas transfer in lung assist device technology.

Blood oxygenators are vital for the critically ill, but their use is limited to the hospital setting. A portable blood oxygenator or a lung assist device for ambulatory or long-term use would greatly benefit patients with chronic lung disease. In this work, a biomimetic blood oxygenator system was developed which consisted of a microfluidic vascular network covered by a gas permeable silicone membrane. This system was used to determine the influence of key microfluidic parameters—channel size, oxygen exposure length, and blood shear rate—on blood oxygenation and carbon dioxide removal. Total gas transfer increased linearly with flow rate, independent of channel size and oxygen exposure length. On average, CO2 transfer was 4.3 times higher than oxygen transfer. Blood oxygen saturation was also found to depend on the flow rate per channel but in an inverse manner; oxygenation decreased and approached an asymptote as the flow rate per channel increased. These relationships can be used to optimize future biomimetic vascular networks for specific lung applications: gas transfer for carbon dioxide removal in patients with chronic obstructive pulmonary disease or oxygenation for premature infants requiring complete lung replacement therapy.

Gas Transfer in Cellularized Collagen-Membrane Gas Exchange Devices

Chronic lower respiratory disease is highly prevalent in the United States, and there remains a need for alternatives to lung transplant for patients who progress to end-stage lung disease. Portable or implantable gas oxygenators based on microfluidic technologies can address this need, provided they operate both efficiently and biocompatibly. Incorporating biomimetic materials into such devices can help replicate native gas exchange function and additionally support cellular components. In this work, we have developed microfluidic devices that enable blood gas exchange across ultra-thin collagen membranes (as thin as 2 μm). Endothelial, stromal, and parenchymal cells readily adhere to these membranes, and long-term culture with cellular components results in remodeling, reflected by reduced membrane thickness. Functionally, acellular collagen-membrane lung devices can mediate effective gas exchange up to ∼288 mL/min/m2 of oxygen and ∼685 mL/min/m2 of carbon dioxide, approaching the gas exchange efficienc...

Abstract 5088: Tumor penetrating RNA delivery for therapeutic benefit of pancreatic cancer

Pancreatic cancer remains a deadly disease with a median survival of less than one year, in part because we lack effective therapeutic strategies. Mutations of the oncogene KRAS and the tumor suppressor genes TP53, INK4A, and DPC4 play important roles in the pathogenesis of pancreatic ductal adenocarcinoma (PDAC); however, these driver events have proven to be challenging targets for drug development. RNA interference through small interfering RNA (siRNA) delivery has the potential to have an impact on silencing ‘undruggable’ targets; however, siRNA delivery in tumors has been challenging in general and worsened in PDAC by the stromal barriers to drug delivery. Herein, we integrated biomedical engineering, materials science, and pre-clinical investigation to establish a novel class of tumor-penetrating nanoparticles (TPNs) to enhance delivery of siRNAs in PDAC for therapeutic benefit. Specifically, we adapted self-assembled nanoparticles comprised of siRNA complexed with tandem tumor-penetrating and membrane-translocating peptides, which enabled the specific delivery of siRNA deep into the tumor parenchyma. Specifically, to establish efficacious siRNA delivery vehicles, we characterized and selected tandem peptides that incorporate novel tumor-penetrating peptide domains known to target PDAC in mice. In parallel, we optimized the particle stability through incorporation of hydrophilic molecules, focusing on achieving desirable pharmacokinetic properties which will reduce off-target delivery and increase potency of a given siRNA dose. The resulting TPNs carry siRNA into the cytosol of monolayer cultures of tumor cells, and also exhibit penetration in pancreas organoids derived from primary mouse and human tumors. As a proof-of-concept, we used TPN in vivo to knockdown KRAS in a murine PDAC model, using subcutaneously-implanted, autochthonous KRASG12D/+p53-/- tumors. Treatment of tumor-bearing mice with systemically administrated KRAS-specific TPNs suppressed the growth of established tumors. With the delivery platform established, the ongoing goals of this project are to identify siRNA targets that synergize with the suppression of oncogenic KRAS, and to validate synthetic lethal targets by interfering with signature PDAC driver events uncovered by functional genomic efforts. Together, these studies will provide a foundation for translational studies involving new targets and delivery technology in pancreatic cancer. Citation Format: Liangliang Hao, Justin Lo, Sangeeta Bhatia. Tumor penetrating RNA delivery for therapeutic benefit of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5088. doi:10.1158/1538-7445.AM2017-5088

Abstract PR5: Treatment of ovarian cancer with targeted tumor-penetrating siRNA nanocomplexes

Abstract Whole-genome analysis of cancer samples is identifying many potential therapeutic targets, by virtue of their being frequently mutated or functionally essential in specific types of cancer. However, we lack efficient ways to test the therapeutic benefit of modulating targets in vivo. RNAi offers one potential solution; however, approaches to deliver siRNA in vivo have been challenging due to their susceptibility to serum nucleases, endosomal entrapment, and stimulation of innate immunity. Furthermore, nanoparticle- and antibody-based siRNA delivery approaches have historically suffered from limited tumor penetration and low transvascular transit, thereby limiting the applicability of parenchymal siRNA targets. Here we describe a tumor penetrating nanocomplex (TPN) comprised of siRNA complexed to a tandem tumor-penetrating and membrane-translocating peptide, which enables the homing of siRNA deep into tumor parenchyma. Upon complexation with siRNA, the resulting nanocomplex is stable, non-immunostimulatory, displays homing peptides in a multivalent fashion that increases their binding avidity and delivers siRNA to the cytosol of tumor cells through receptor-specific interactions and membrane translocation. Upon systemic administration into mice, this nanocomplex penetrates into the parenchyma of metastatic peritoneal tumors and silences target genes in cells of interest in a receptor-specific manner. We employed TPNs in vivo to evaluate ID4, a novel candidate oncogene in ovarian cancer, which we identified by combining genome-scale RNAi screening of cancer cell lines with genome-scale sequence analysis of patient tumors. We show that treatment of tumor-bearing mice with ID4-specific TPNs suppresses tumor growth and significantly improved survival. These findings provide a framework for the identification, credentialing, and understanding of novel cancer targets as well as validating a specific therapeutic target in ovarian cancer. This abstract is also presented as Poster B2. Citation Format: Yin Ren, Hiu Wing Cheung, Ronny Drapkin, David Root, Justin Lo, Valentina Fogal, Erkki Ruoslahti, William Hahn, Sangeeta Bhatia, Geoffrey von Maltzahn, Amit Agrawal, Glenn Cowley, Barbara Weir, Jesse Boehm, Pablo Tamayo, Jill Mesirov, Alison Karst. Treatment of ovarian cancer with targeted tumor-penetrating siRNA nanocomplexes [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr PR5.