This Small Business Innovation Research (SBIR) Phase II project will advance the development of new drug compounds for the treatment of glioma, which have been designed and constructed with an innovative combination of grid-powered, computer-aided design (CAD) software and DNA nano-fabrication technology. The compounds are self-assembling DNA nanostructures functionalized with molecular subcomponents for targeting and destroying malignant glioma (brain) tumors. Prognosis for glioma is poor because complete surgical resection is impossible and chemotherapy (being poorly selective) leads to collateral brain damage, hence treatments are needed that target and destroy glioma cells with high specificity.
The broader impacts of this research are the societal benefits associated with improved disease outcomes through the creation of revolutionary new nano-pharmaceuticals. The Company's efforts under this project are focused initially on creating an effective treatment for glioma, but the Company's Essemblix platform has the potential to be used to create compounds for a wide variety of indications. The ability to "plug and play" at the molecular level, made possible by PNL's computational and nano-fabrication technology, opens the door to the deliberate design and development of entirely new types of pharmaceutical materials that could address indications across a vast and diverse number of pharmaceutical and biotechnology market segments.
A. Project Overview & Intellectual Merit In this project, Parabon NanoLabs (PNL) designed, produced and tested a new class of nano-therapeutics using the company’s Essemblix™ Drug Development Platform - an innovative combination of proprietary computer-aided design (CAD) software for designing macromolecules and nanoscale fabrication technology for their production. Enhancement of the CAD software that supports Essemblix, which is called the Parabon inSequio Design Studio, was a major accomplishment under this project. Essemblix uses self-assembling DNA nanotechnology to enable "plug and play" molecular engineering. With Essemblix, PNL scientists are able to employ a "molecular breadboard" approach to drug design that provides precise control over many aspects of a custom compound: the size, shape, surface charge, choice of functional elements, even their spatial arrangement, are all attributes that are present by design. This level of nano-engineering control conveys at least five benefits for medical applications: Increased margination, cell surface interaction and slower clearance from deliberately planar shaped nanocarriers; Highly selective tumor targeting, which enables: Delivery of extremely potent cytotoxic payloads, which can reduce systemic toxicity risks; Efficient and straightforward means of optimizing therapeutic designs to safely maximize efficacy. Controllable immunogenicity. The immunogenicity of a DNA nanostructure is highly sequence-dependent. By controlling the sequences of the DNA oligos used to self-assemble the DNA nanostructure, the DNA nanostructure can be non-immunogenic (good for therapeutics and diagnostics) or highly immunogenic (good for synthetic vaccines). In this project, PNL successfully demonstrated in vitro and in vivo luciferase knockdown with a targeted Essemblix DNA nanocarrier (P24) that employed luciferase siRNA payloads. Based on these results, PNL designed, produced and tested a targeted P24 nanocarrier and cytotoxic siRNA payloads for the treatment of glioblastoma (GBM), one of the most lethal human cancers. The most efficacious compound, P24RDN, prolonged median survival of mice implanted intracranially with GBM tumor cells (U87MG cell line) by 36% (p<0.01). This success against GBM eventually led to an NSF TECP project with industry partner Janssen Pharmaceuticals, Inc. Under the TECP project, PNL designed and developed an Essemblix compound to treat drug-resistant prostate cancer. At Janssen’s request, the lead compound, PJ-01, was designed to employ docetaxel (DTX) as the cytotoxic payload, with the aim of improving DTX’s native performance. PJ-01 showed strong in vitro efficacy against LNCaP, a drug-resistant prostate cancer cell line. At the maximum concentration of DTX that is soluble in aqueous solution (50nM equiv.), PJ-01 killed 92.5% of the LNCaP cells in vitro while free DTX killed only 78% (p<0.005). In an LNCaP xenograft mouse model, PJ-01, labeled with fluorescent dye, demonstrated greater accumulation in tumors than in controls receiving non-targeted constructs. Although these results are encouraging, additional in vivo testing will be required to determine whether PJ-01 has sufficient efficacy or needs further optimization (e.g., by inclusion of a different cytotoxic payload). B. Outcomes The major goal of this project was to prove our hypothesis that DNA nanostructures, specifically those designed with Essemblix, could be used to produce safe and effective nanotherapeutics. Collecively, the outcomes below strongly confirm this hypothesis. In addition to developing the methods and protocols for the synthesis and purification of Essemblix compounds, PNL successfully demonstrate that: 1. An Essemblix compound carrying luciferase siRNA could knock down expression of luciferase in a U87mg-Luc2 cell line in vitro; 2. An Essemblix compound carrying luciferase siRNA can knock down the expression of luciferase in a U87mg-Luc2 cell line in vivo 3. An Essemblix compound with cytotoxic siRNA payloads can effectively inhibit tumor growth and extend animal survival in U87mg xenograft mouse model. 4. An Essemblix compound with docetaxel payloads can effectively eliminate drug-resistant LnCAP prostate cancer cells in vitro. 5. A targeted Essemblix compound with docetaxel payloads and siRNA sensitizer shows better accumulation in xenograft tumor in vivo over non-targeted control. At the conclusion of the project, PNL consulted drug development experts and devised a plan for further development of P24RDN as a GBM treatment slated for an eventual IND filing.
