Jason Fuller

A combinatorial library of lipid-like materials for delivery of RNAi therapeutics

The safe and effective delivery of RNA interference (RNAi) therapeutics remains an important challenge for clinical development. The diversity of current delivery materials remains limited, in part because of their slow, multi-step syntheses. Here we describe a new class of lipid-like delivery molecules, termed lipidoids, as delivery agents for RNAi therapeutics. Chemical methods were developed to allow the rapid synthesis of a large library of over 1,200 structurally diverse lipidoids. From this library, we identified lipidoids that facilitate high levels of specific silencing of endogenous gene transcripts when formulated with either double-stranded small interfering RNA (siRNA) or single-stranded antisense 2′-O-methyl (2′-OMe) oligoribonucleotides targeting microRNA (miRNA). The safety and efficacy of lipidoids were evaluated in three animal models: mice, rats and nonhuman primates. The studies reported here suggest that these materials may have broad utility for both local and systemic delivery of RNA therapeutics.

Triggered release of siRNA from poly(ethylene glycol)-protected, pH-dependent liposomes.

The ability of small interfering RNA (siRNA) to regulate gene expression has potential therapeutic applications, but its use is limited by inefficient delivery. Triggered release of adsorbed poly(ethylene glycol) (PEG)-b-polycation polymers from pH-dependent (PD) liposomes enables protection from immune recognition during circulation (pH 7.4) and subsequent intracellular delivery of siRNA within the endosome (pH ~5.5). Polycationic blocks, based on either poly[2-(dimethylamino)ethyl methacrylate] (31 or 62 DMA repeat units) or polylysine (21 K repeat units), act as anchors for a PEG (113 ethylene glycol repeat units) protective block. Incorporation of 1,2-dioleoyl-3-dimethylammonium-propane (DAP), a titratable lipid, increases the liposomes net cationic character within acidic environments, resulting in polymer desorption and membrane fusion. Liposomes encapsulating siRNA demonstrate green fluorescent protein (GFP) silencing in genetically-modified, GFP-expressing HeLa cells and glyceraldehyde-3-phosphate dehydrogenase (GAPD) knockdown in human umbilical vein endothelial cells (HUVEC). Bare and PD liposomes coated with PEG113-DMA31 exhibit a 0.16+/-0.2 and 0.32+/-0.3 fraction of GFP knockdown, respectively. In contrast, direct siRNA administration and Oligofectamine complexed siRNA reduce GFP expression by 0.06+/-0.02 and 0.14+/-0.02 fractions, respectively. Our in vitro data indicates that polymer desorption from PD liposomes enhances siRNA-mediated gene knockdown.

An implantable microdevice to perform high-throughput in vivo drug sensitivity testing in tumors

An implantable microdevice is demonstrated to release microdoses of multiple drugs into confined regions of tumors and allows for assessment of each drug’s efficacy to identify optimal therapy. Drug-releasing implant tests cancer’s resolve Predicting whether a patient will respond to a drug is not easy, often relying on empirical evidence. Toward personalized medicine, animal models of patient tumors have been developed as well as engineered cell-material combinations meant to replicate a tumor in vitro. But the true test of whether a tumor responds to a drug will be by evaluating the tumor itself, within its own microenvironment. To this end, Jonas et al. created miniature drug delivery vessels that can be implanted with a standard biopsy needle directly into the tumor. These vessels, less than 1 mm in diameter, contained up to 16 microwells that each released a bolus of drug into the surrounding tumor tissue. The device and its surrounding tissue were then removed with a larger coring needle to see if the cancer cells had responded to the drug—or combination of drugs. In mouse models of melanoma, breast, or prostate cancers, the local response to a common chemotherapeutic, doxorubicin, matched the tumor response to systemic therapy. Furthermore, in a mouse model of triple-negative breast cancer, tumor sensitivity to five different locally delivered cancer drugs was identical to tumor response after intravenous administration of drug; for instance, tumors were most responsive to paclitaxel and least responsive to lapatinib. Such tiny drug-releasing devices can be implanted at different locations within the tumor, overcoming issues with tumor heterogeneity, and allowing for reproducible evaluation of drug sensitivity directly within the patient. Current anticancer chemotherapy relies on a limited set of in vitro or indirect prognostic markers of tumor response to available drugs. A more accurate analysis of drug sensitivity would involve studying tumor response in vivo. To this end, we have developed an implantable device that can perform drug sensitivity testing of several anticancer agents simultaneously inside the living tumor. The device contained reservoirs that released microdoses of single agents or drug combinations into spatially distinct regions of the tumor. The local drug concentrations were chosen to be representative of concentrations achieved during systemic treatment. Local efficacy and drug concentration profiles were evaluated for each drug or drug combination on the device, and the local efficacy was confirmed to be a predictor of systemic efficacy in vivo for multiple drugs and tumor models. Currently, up to 16 individual drugs or combinations can be assessed independently, without systemic drug exposure, through minimally invasive biopsy of a small region of a single tumor. This assay takes into consideration physiologic effects that contribute to drug response by allowing drugs to interact with the living tumor in its native microenvironment. Because these effects are crucial to predicting drug response, we envision that these devices will help identify optimal drug therapy before systemic treatment is initiated and could improve drug response prediction beyond the biomarkers and in vitro and ex vivo studies used today. These devices may also be used in clinical drug development to safely gather efficacy data on new compounds before pharmacological optimization.

A Comparison Between Polymeric Microsphere and Bacterial Vectors for Macrophage P388D1 Gene Delivery

PurposeThe purpose of this study was to compare bacterial and polymeric gene delivery devices for the ability to deliver plasmid DNA to a murine macrophage P388D1 cell line.MethodsAn 85:15 ratio of poly(lactic-co-glycolic acid) (PLGA) and poly(β-amino ester) polymers were formulated into microspheres that physically entrapped plasmid DNA encoding for the firefly luciferase reporter gene; whereas, the same plasmid was biologically transformed into a strain of Escherichia coli engineered to produce recombinant listeriolysin O. The two delivery devices were then tested for gene delivery and dosage effects using a macrophage cell line with both assays taking advantage of a 96-well high throughput format to quantify and compare each vector type.ResultsGene delivery was comparable for both vectors at higher vector dosages while lower dosages showed an improved delivery for the microsphere vectors. Delivery efficiency (defined as luciferase measurement/mg cellular protein/ng DNA delivered) was 881 luminescence mg−1 ng−1 for polymeric microspheres compared to 171 luminescence mg−1 ng−1 for the bacterial vectors.ConclusionA first head-to-head comparison between polymeric and bacterial gene delivery vectors shows a delivery advantage for polymeric microspheres that must also be evaluated in light of vector production, storage, and future potential.

Entrepreneurship in Biomaterials

Start-up companies are an integral part of the biomedical product development landscape. Although most medical devices are manufactured, marketed, and sold by large companies, their fundamental technologies are often born in the university setting, and incubated in small start-up companies. Starting a company can be a great opportunity to raise the awareness of academic work, and also to have a significant impact on the industry and on patients’ lives. It is a complex process of bringing together people, ideas, money, and business. The start-up environment is a fast-paced, dynamic, and collaborative home for a project, and with the right vision, team, technology, and plan, a start-up company can be highly efficient at creating significant development progress and creating considerable value. However, we want the reader to understand the risks inherent in starting a company: a relatively small percentage of start-up companies get a product to market, and the effort required can be substantial. Therefore, the decision to spin out one’s research into a company should not be taken lightly.