Nucleic Acid Carriers Based on Precise Polymer Conjugates

Polymer polydispersity, random conjugation of functional groups, and poorly understood structure–activity relationships have constantly hampered progress in the development of nucleic acid carriers. This review focuses on the synthetic concepts for the generation of precise polymers, site-specific conjugation strategies, and multifunctional conjugates for nucleic acid transport. Dendrimers, defined peptide carriers, sequence-defined polyamidoamines assembled by solid-phase supported synthesis, and precise lipopeptides or lipopolymers have been characterized for pDNA and siRNA delivery. Conjugation techniques such as click chemistries and peptide ligation are available for conjugating polymers with functional transport elements such as targeting or shielding domains and for direct covalent modification of therapeutic nucleic acids in a site-specific mode

In Vitro-In Vivo Translation of Lipid Nanoparticles for Hepatocellular siRNA Delivery

A significant challenge in the development of clinically viable siRNA delivery systems is a lack of in vitro–in vivo translatability: many delivery vehicles that are initially promising in cell culture do not retain efficacy in animals. Despite its importance, little information exists on the predictive nature of in vitro methodologies, most likely due to the cost and time associated with generating in vitro–in vivo data sets. Recently, high-throughput techniques have been developed that have allowed the examination of hundreds of lipid nanoparticle formulations for transfection efficiency in multiple experimental systems. The large resulting data set has allowed the development of correlations between in vitro and characterization data and in vivo efficacy for hepatocellular delivery vehicles. Consistency of formulation technique and the type of cell used for in vitro experiments was found to significantly affect correlations, with primary hepatocytes and HeLa cells yielding the most predictive data. Interestingly, in vitro data acquired using HeLa cells were more predictive of in vivo performance than mouse hepatoma Hepa1-6 cells. Of the characterization parameters, only siRNA entrapment efficiency was partially predictive of in vivo silencing potential, while zeta-potential and, surprisingly, nanoparticle size (when <300 nm) as measured by dynamic light scattering were not. These data provide guiding principles in the development of clinically viable siRNA delivery materials and have the potential to reduce experimental costs while improving the translation of materials into animals.Alnylam Pharmaceuticals (Firm)National Institutes of Health (U.S.) (Fellowship Award F32EB009623

Organ-targeted high-throughput in vivo biologics screen identifies materials for RNA delivery

Therapies based on biologics involving delivery of proteins, DNA, and RNA are currently among the most promising approaches. However, although large combinatorial libraries of biologics and delivery vehicles can be readily synthesized, there are currently no means to rapidly characterize them in vivo using animal models. Here, we demonstrate high-throughput in vivo screening of biologics and delivery vehicles by automated delivery into target tissues of small vertebrates with developed organs. Individual zebrafish larvae are automatically oriented and immobilized within hydrogel droplets in an array format using a microfluidic system, and delivery vehicles are automatically microinjected to target organs with high repeatability and precision. We screened a library of lipid-like delivery vehicles for their ability to facilitate the expression of protein-encoding RNAs in the central nervous system. We discovered delivery vehicles that are effective in both larval zebrafish and rats. Our results showed that the in vivo zebrafish model can be significantly more predictive of both false positives and false negatives in mammals than in vitro mammalian cell culture assays. Our screening results also suggest certain structure–activity relationships, which can potentially be applied to design novel delivery vehicles.National Institutes of Health (U.S.) (Transformative Research Award R01 NS073127)National Institutes of Health (U.S.) (Director's Innovator Award DP2 OD002989)David & Lucile Packard Foundation (Award in Science and Engineering)Sanofi Aventis (Firm)Foxconn International Holdings Ltd.Hertz Foundation (Fellowship)University Grants Committee (Hong Kong, China) (Early Career Award 125012)National Natural Science Foundation (China) (81201164)ITC (ITS/376/13)Chinese University of Hong Kong (Grant 9610215)Chinese University of Hong Kong (Grant 7200269

Organ-targeted high-throughput in vivo biologics screen identifies materials for RNA delivery

Therapies based on biologics involving delivery of proteins, DNA, and RNA are currently among the most promising approaches. However, although large combinatorial libraries of biologics and delivery vehicles can be readily synthesized, there are currently no means to rapidly characterize them in vivo using animal models. Here, we demonstrate high-throughput in vivo screening of biologics and delivery vehicles by automated delivery into target tissues of small vertebrates with developed organs. Individual zebrafish larvae are automatically oriented and immobilized within hydrogel droplets in an array format using a microfluidic system, and delivery vehicles are automatically microinjected to target organs with high repeatability and precision. We screened a library of lipid-like delivery vehicles for their ability to facilitate the expression of protein-encoding RNAs in the central nervous system. We discovered delivery vehicles that are effective in both larval zebrafish and rats. Our results showed that the in vivo zebrafish model can be significantly more predictive of both false positives and false negatives in mammals than in vitro mammalian cell culture assays. Our screening results also suggest certain structure–activity relationships, which can potentially be applied to design novel delivery vehicles.National Institutes of Health (U.S.) (Transformative Research Award R01 NS073127)National Institutes of Health (U.S.) (Director's Innovator Award DP2 OD002989)David & Lucile Packard Foundation (Award in Science and Engineering)Sanofi Aventis (Firm)Foxconn International Holdings Ltd.Hertz Foundation (Fellowship)University Grants Committee (Hong Kong, China) (Early Career Award 125012)National Natural Science Foundation (China) (81201164)ITC (ITS/376/13)Chinese University of Hong Kong (Grant 9610215)Chinese University of Hong Kong (Grant 7200269

Avoiding the Pitfalls of siRNA Delivery to the Retinal Pigment Epithelium with Physiologically Relevant Cell Models

Inflammation is involved in the pathogenesis of several age-related ocular diseases, such as macular degeneration (AMD), diabetic retinopathy, and glaucoma. The delivery of anti-inflammatory siRNA to the retinal pigment epithelium (RPE) may become a promising therapeutic option for the treatment of inflammation, if the efficient delivery of siRNA to target cells is accomplished. Unfortunately, so far, the siRNA delivery system selection performed in dividing RPE cells in vitro has been a poor predictor of the in vivo efficacy. Our study evaluates the silencing efficiency of polyplexes, lipoplexes, and lipidoid-siRNA complexes in dividing RPE cells as well as in physiologically relevant RPE cell models. We find that RPE cell differentiation alters their endocytic activity and causes a decrease in the uptake of siRNA complexes. In addition, we determine that melanosomal sequestration is another significant and previously unexplored barrier to gene silencing in pigmented cells. In summary, this study highlights the importance of choosing a physiologically relevant RPE cell model for the selection of siRNA delivery systems. Such cell models are expected to enable the identification of carriers with a high probability of success in vivo, and thus propel the development of siRNA therapeutics for ocular disease.Peer reviewe

Avoiding the Pitfalls of siRNA Delivery to the Retinal Pigment Epithelium with Physiologically Relevant Cell Models

Inflammation is involved in the pathogenesis of several age-related ocular diseases, such as macular degeneration (AMD), diabetic retinopathy, and glaucoma. The delivery of anti-inflammatory siRNA to the retinal pigment epithelium (RPE) may become a promising therapeutic option for the treatment of inflammation, if the efficient delivery of siRNA to target cells is accomplished. Unfortunately, so far, the siRNA delivery system selection performed in dividing RPE cells in vitro has been a poor predictor of the in vivo efficacy. Our study evaluates the silencing efficiency of polyplexes, lipoplexes, and lipidoid-siRNA complexes in dividing RPE cells as well as in physiologically relevant RPE cell models. We find that RPE cell differentiation alters their endocytic activity and causes a decrease in the uptake of siRNA complexes. In addition, we determine that melanosomal sequestration is another significant and previously unexplored barrier to gene silencing in pigmented cells. In summary, this study highlights the importance of choosing a physiologically relevant RPE cell model for the selection of siRNA delivery systems. Such cell models are expected to enable the identification of carriers with a high probability of success in vivo, and thus propel the development of siRNA therapeutics for ocular disease

Formulation of Small Activating RNA Into Lipidoid Nanoparticles Inhibits Xenograft Prostate Tumor Growth by Inducing p21 Expression

Application of RNA interference (RNAi) in the clinic has improved with the development of novel delivery reagents (e.g., lipidoids). Although RNAi promises a therapeutic approach at silencing gene expression, practical methods for enhancing gene production still remain a challenge. Previously, we reported that double-stranded RNA (dsRNA) can activate gene expression by targeting promoter sequence in a phenomenon termed RNA activation (RNAa). In the present study, we investigate the therapeutic potential of RNAa in prostate cancer xenografts by using lipidoid-based formulation to facilitate in vivo delivery. We identify a strong activator of gene expression by screening several dsRNAs targeting the promoter of tumor suppressor p21WAF1/ Cip1 (p21). Chemical modification is subsequently implemented to improve the medicinal properties of the candidate duplex. Lipidoid-encapsulated nanoparticle (LNP) formulation is validated as a delivery vehicle to mediate p21 induction and inhibit growth of prostate tumor xenografts grown in nude mice following intratumoral injection. We provide insight into the stepwise creation and analysis of a putative RNAa-based therapeutic with antitumor activity. Our results provide proof-of-principle that RNAa in conjunction with lipidioids may represent a novel approach for stimulating gene expression in vivo to treat disease

Giving genes the silent treatment: Lipid-like materials for siRNA delivery

Despite the promise of RNA interference therapeutics, progress towards the clinic has been slowed by the difficulty of delivering short interfering RNA (siRNA) into cellular targets within the body. siRNA is large (~13 kDa) and negatively charged; it does not have favorable biodistribution properties in vivo nor an ability to cross the cellular membrane of target cells. In order to facilitate these transport processes, a class of lipid-like materials termed ‘lipidoids’ has been synthesized and studied for applications in siRNA-mediated gene silencing. Although efficacious, initial lipidoids identified for siRNA delivery applications in vivo can have limited utility in therapeutic settings due to toxicity and non-degradability issues. In response to these challenges, a library of biodegradable lipidoids was synthesized and novel high-throughput methodologies were employed to demonstrate lipidoid gene silencing potential both in vitro and in vivo. Degradable lipidoids induced near-complete gene silencing at low siRNA doses in a variety of biological systems, including hepatocytes, myeloid and lymphoma cells, and ovarian cancer tumors. Furthermore, structure-function analysis has revealed material design criteria that reliably predict in vivo delivery efficacy without the need for any biological testing. Together, these results indicate that lipidoid materials can achieve potent, specific and non-toxic siRNA delivery in a variety of biological contexts and have the potential to hasten the advent of RNA interference therapeutics in the clinic

Nucleic acid-mediated intracellular protein delivery by lipid-like nanoparticles

Intracellular protein delivery has potential biotechnological and therapeutic application, but remains technically challenging. In contrast, a plethora of nucleic acid carriers have been developed, with lipid-based nanoparticles (LNPs) among the most clinically advanced reagents for oligonucleotide delivery. Here, we validate the hypothesis that oligonucleotides can serve as packaging materials to facilitate protein entrapment within and intracellular delivery by LNPs. Using two distinct model proteins, horseradish peroxidase and NeutrAvidin, we demonstrate that LNPs can yield efficient intracellular protein delivery in vitro when one or more oligonucleotides have been conjugated to the protein cargo. Moreover, in experiments with NeutrAvidin in vivo, we show that oligonucleotide conjugation significantly enhances LNP-mediated protein uptake within various spleen cell populations, suggesting that this approach may be particularly suitable for improved delivery of protein-based vaccines to antigen-presenting cells.National Heart, Lung, and Blood Institute (Contract HHSN268201000045C)National Institutes of Health (U.S.) (Grant R01-EB000244-27)National Institutes of Health (U.S.) (Grant 5-R01-CA132091-04)National Science Foundation (U.S.)Juvenile Diabetes Research Foundation International (Grant 17–2007-1063)United States. Dept. of Defense. Congressionally Directed Medical Research Programs (Grant W81XWH-13-1-0215