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.

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.

We propose a new methodology to enhance the vascular differentiation of human embryonic stem cells (hESCs) by encapsulation in a bioactive hydrogel. hESCs were encapsulated in a dextran-based hydrogel with or without immobilized regulatory factors: a tethered RGD peptide and microencapsulated VEGF 165 . The fraction of cells expressing vascular endothelial growth factor (VEGF) receptor KDR/Flk-1, a vascular marker, increased up to 20-fold, as compared to spontaneously differentiated embryoid bodies (EBs). The percentage of encapsulated cells in hydrogels with regulatory factors expressing ectodermal markers including nestin or endodermal markers including α-fetoprotein decreased 2 or 3 fold, respectively, as compared to EBs. When the cells were removed from these networks and cultured in media conditions conducive for further vascular differentiation, the number of vascular cells was higher than the number obtained through EBs, using the same media conditions. Functionalized dextran-based hydrogels could thus enable derivation of vascular cells in large quantities, particularly endothelial cells, for potential application in tissue engineering and regenerative medicine. 1-INTRODUCTIONDuring normal embryogenesis, human embryonic stem cells (hESCs) differentiate along different lineages in the context of complex three-dimensional tissue structures, where the extracellular matrix (ECM) and different growth factors play an important role in this process. The three-dimensional ECM provides structural support for higher level of tissue organization and remodelling [1]. Significant differences were found in the differentiation profile of ESCs when cultured in a three-dimensional (3D) versus two-dimensional (2D) system [2,3] or 3D scaffold system versus embryoid body (EB) system [4,5]. In this last case, mouse ESCs cultured within tantalum scaffolds differentiate at higher extent into hematopoietic cells than EBs [4], while hESCs cultured in alginate scaffolds express significantly more vascular markers than EBs [5]. Moreover, the culture of hESCs within 3D poly(α-hydroxy esters) scaffolds with media containing different growth factors induced their differentiation into 3D structures with || To whom reprint requests should be addressed at: Department of Chemical Engineering, E25-342 Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139. E-mail: rlanger@mit.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [1,6]. However, these growth factors were supplied from outside the scaffold, and thus their activity may be affected by diffusion lim...

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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 liposome's 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.