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1.  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 nearly perfect 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 relationship, which can potentially be applied to design novel delivery vehicles.
PMCID: PMC4350364  PMID: 25184623
high-throughput screening; microinjection; zebrafish larva; biologics; drug delivery vehicles; lipidoid materials
2.  Lipidoid-coated Iron Oxide Nanoparticles for Efficient DNA and siRNA delivery 
Nano letters  2013;13(3):1059-1064.
The safe, targeted and effective delivery of gene therapeutics remains a significant barrier to their broad clinical application. Here we develop a magnetic nucleic acid delivery system composed of iron oxide nanoparticles and cationic lipid-like materials termed lipidoids. Coated nanoparticles are capable of delivering DNA and siRNA to cells in culture. The mean hydrodynamic size of these nanoparticles was systematically varied and optimized for delivery. While nanoparticles of different sizes showed similar siRNA delivery efficiency, nanoparticles of 50–100 nm displayed optimal DNA delivery activity. The application of an external magnetic field significantly enhanced the efficiency of nucleic acid delivery, with performance exceeding that of the commercially available lipid-based reagent, Lipofectamine 2000. The iron oxide nanoparticle delivery platform developed here offers the potential for magnetically guided targeting, as well as an opportunity to combine gene therapy with MRI imaging and magnetic hyperthermia.
PMCID: PMC3992974  PMID: 23394319
siRNA delivery; DNA delivery; iron oxide nanoparticle; magnetofection; gene therapy
3.  Molecularly Self-Assembled Nucleic Acid Nanoparticles for Targeted In Vivo siRNA Delivery 
Nature nanotechnology  2012;7(6):389-393.
Nanoparticles are employed for delivering therapeutics into cells1,2. However, size, shape, surface chemistry and the presentation of targeting ligands on the surface of nanoparticles can affect circulation half-life and biodistribution, cell specific internalization, excretion, toxicity, and efficacy3-7. A variety of materials have been explored for delivering small interfering RNAs (siRNAs) - a therapeutic agent that suppresses the expression of targeted genes8,9. However, conventional delivery nanoparticles such as liposomes and polymeric systems are heterogeneous in size, composition and surface chemistry, and this can lead to suboptimal performance, lack of tissue specificity and potential toxicity10-12. Here, we show that self-assembled DNA tetrahedral nanoparticles with a well-defined size can deliver siRNAs into cells and silence target genes in tumours. Monodisperse nanoparticles are prepared through the self-assembly of complementary DNA strands. Because the DNA strands are easily programmable, the size of the nanoparticles and the spatial orientation and density of cancer targeting ligands (such as peptides and folate) on the nanoparticle surface can be precisely controlled. We show that at least three folate molecules per nanoparticle is required for optimal delivery of the siRNAs into cells and, gene silencing occurs only when the ligands are in the appropriate spatial orientation. In vivo, these nanoparticles showed a longer blood circulation time (t1/2 ∼ 24.2 min) than the parent siRNA (t1/2 ∼ 6 min).
PMCID: PMC3898745  PMID: 22659608
4.  Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling 
Nature biotechnology  2013;31(7):653-658.
Despite substantial efforts to understand the interactions between nanoparticles and cells, the cellular processes that determine the efficiency of intracellular drug delivery remain largely unclear. Here we examined cellular uptake of siRNA delivered in lipid nanoparticles (LNPs) using cellular trafficking probes in combination with automated high-throughput confocal microscopy as well as defined perturbations of cellular pathways paired with systems biology approaches to uncover protein-protein and protein-small molecule interactions. We show that multiple cell signaling effectors are required for initial cellular entry of LNPs through macropinocytosis, including proton pumps, mTOR, and cathepsins. SiRNA delivery is substantially reduced as ≅70% of the internalized siRNA undergoes exocytosis through egress of LNPs from late endosomes/lysosomes. Niemann Pick type C1 (NPC1) is shown to be an important regulator of the major recycling pathways of LNP-delivered siRNAs. NPC1-deficient cells show enhanced cellular retention of LNPs inside late endosomes/lysosomes and increased gene silencing of the target gene. Our data suggests that siRNA delivery efficiency might be improved by designing delivery vehicles that can escape the recycling pathways.
PMCID: PMC3814166  PMID: 23792629
5.  Alkane-modified short polyethyleneimine for siRNA delivery 
RNA interference (RNAi) is a highly specific gene-silencing mechanism triggered by small interfering RNA (siRNA). Effective intracellular delivery requires the development of potent siRNA carriers. Here, we describe the synthesis and screening of a series of siRNA delivery materials. Short polyethyleneimine (PEI, Mw 600) was selected as a cationic backbone to which lipid tails were conjugated at various levels of saturation. In solution these polymer–lipid hybrids self-assemble to form nanoparticles capable of complexing siRNA. The complexes silence genes specifically and with low cytotoxicity. The efficiency of gene knockdown increased as the number of lipid tails conjugated to the PEI backbone increased. This is explained by reducing the binding affinity between the siRNA strands to the complex, thereby enabling siRNA release after cellular internalization. These results highlight the importance of complexation strength when designing siRNA delivery materials.
PMCID: PMC3814168  PMID: 22155553
Lipid; Conjugation; Complexation; Affinity; Polyethyleneimine (PEI); Cationic
6.  FRET-Labeled siRNA Probes for Tracking Assembly and Disassembly of siRNA-Nanocomplexes 
ACS Nano  2012;6(7):6133-6141.
The assembly, stability and timely disassembly of short interfering RNA (siRNA) nanocomplexes all have the potential to affect the efficiency of siRNA delivery and gene silencing. As such, the design of new probes that can measure these properties without significantly perturbing the nanocomplexes or their environment may facilitate the study and further development of new siRNA nanocomplexes. Herein, we study Förster resonance energy transfer (FRET)-labeled siRNA probes that can track the assembly, stability and disassembly of siRNA nanocomplexes in different environments. The probe is composed of two identical siRNAs, each labeled with a fluorophore. Upon nanocomplex formation, the siRNA-bound fluorophores become locally aggregated within the nanocomplex and undergo FRET. A key advantage of this technique is that the delivery vehicle (DV) need not be labeled, thus enabling the characterization of a large variety of nanocarriers, some of which maybe difficult or even impossible to label. We demonstrate proof-of-concept by measuring the assembly of various DVs with siRNAs and show good agreement with gel electrophoresis experiments. As a consequence of not having to label the DV, we are able to determine nanocomplex biophysical parameters such as the extracellular apparent dissociation constants (KD) and intracellular disassembly half-life for several in-house and proprietary commercial DV’s. Furthermore, the lack of DV modification allows for a true direct comparison between DVs as well as correlation between their biophysical properties and gene silencing.
PMCID: PMC3404193  PMID: 22693946
siRNA; FRET; Nanocomplex; Fluorescent Probe; polycation; lipid; lipidoid
7.  YY1 Regulates Melanocyte Development and Function by Cooperating with MITF 
PLoS Genetics  2012;8(5):e1002688.
Studies of coat color mutants have greatly contributed to the discovery of genes that regulate melanocyte development and function. Here, we generated Yy1 conditional knockout mice in the melanocyte-lineage and observed profound melanocyte deficiency and premature gray hair, similar to the loss of melanocytes in human piebaldism and Waardenburg syndrome. Although YY1 is a ubiquitous transcription factor, YY1 interacts with M-MITF, the Waardenburg Syndrome IIA gene and a master transcriptional regulator of melanocytes. YY1 cooperates with M-MITF in regulating the expression of piebaldism gene KIT and multiple additional pigmentation genes. Moreover, ChIP–seq identified genome-wide YY1 targets in the melanocyte lineage. These studies mechanistically link genes implicated in human conditions of melanocyte deficiency and reveal how a ubiquitous factor (YY1) gains lineage-specific functions by co-regulating gene expression with a lineage-restricted factor (M-MITF)—a general mechanism which may confer tissue-specific gene expression in multiple lineages.
Author Summary
Skin and hair pigmentation is among the most identifiable human traits. Disorders of pigment cells, melanocytes, result in multiple hypopigmentation conditions. Here, we described the phenotype of loss of a ubiquitous transcription factor YY1 in mouse melanocytes, which is reminiscent of certain human hypopigmentation conditions. We revealed at a molecular level that YY1 cooperates with a melanocyte-specific transcription factor M-MITF to regulate survival and pigmentation gene expression. This study is the first report of YY1 function in melanocyte lineage, and it reveals how a ubiquitous transcription factor gains lineage-specific functions by co-regulating gene expression with a lineage-restricted transcription factor.
PMCID: PMC3342948  PMID: 22570637
8.  Therapeutic siRNA silencing in inflammatory monocytes 
Nature biotechnology  2011;29(11):1005-1010.
Inflammatory monocytes -- but not the non-inflammatory subset -- depend on the chemokine receptor CCR2 for distribution to injured tissue and stimulate disease progression. Precise therapeutic targeting of this inflammatory monocyte subset could spare innate immunity's essential functions for maintenance of homeostasis and thus limit unwanted effects. Here we developed siRNA nanoparticles targeting CCR2 expression in inflammatory monocytes. We identified an optimized lipid nanoparticle and silencing siRNA sequence that when administered systemically, had rapid blood clearance, accumulated in spleen and bone marrow and showed high cellular localization of fluorescently tagged siRNA inside monocytes. Efficient degradation of CCR2 mRNA in monocytes prevented their accumulation in sites of inflammation. Specifically, the treatment attenuated their number in atherosclerotic plaques, reduced infarct size following coronary artery occlusion, prolonged normoglycemia in diabetic mice after pancreatic islet transplantation and resulted in reduced tumor volumes and lower numbers of tumor-associated macrophages. Taken together, siRNA nanoparticle-mediated CCR2 gene silencing in leukocytes selectively modulates functions of innate immune cell subtypes and may allow for the development of specific anti-inflammatory therapy.
PMCID: PMC3212614  PMID: 21983520
9.  Systemic RNAi-mediated Gene Silencing in Nonhuman Primate and Rodent Myeloid Cells 
Leukocytes are central regulators of inflammation and the target cells of therapies for key diseases, including autoimmune, cardiovascular, and malignant disorders. Efficient in vivo delivery of small interfering RNA (siRNA) to immune cells could thus enable novel treatment strategies with broad applicability. In this report, we develop systemic delivery methods of siRNA encapsulated in lipid nanoparticles (LNP) for durable and potent in vivo RNA interference (RNAi)-mediated silencing in myeloid cells. This work provides the first demonstration of siRNA-mediated silencing in myeloid cell types of nonhuman primates (NHPs) and establishes the feasibility of targeting multiple gene targets in rodent myeloid cells. The therapeutic potential of these formulations was demonstrated using siRNA targeting tumor necrosis factor-α (TNFα) which induced substantial attenuation of disease progression comparable to a potent antibody treatment in a mouse model of rheumatoid arthritis (RA). In summary, we demonstrate a broadly applicable and therapeutically relevant platform for silencing disease genes in immune cells.
PMCID: PMC3381593  PMID: 23344621
delivery; immune cell; siRNA
10.  A combinatorial approach to determine functional group effects on lipidoid-mediated siRNA delivery 
Bioconjugate chemistry  2010;21(8):1448-1454.
The application of RNA interference (RNAi), either in the clinic or laboratory, requires safe and effective delivery methods. Here we develop a combinatorial approach to synthesize a library of delivery vectors based on two lipid-like substrates with known siRNA delivery capabilities. Members of this library have a mixture of lipid-like tails and feature appendages containing hydroxyl, carbamate, ether or amine functional groups as well as variations in alkyl chain length and branching. Using a luciferase reporter system in HeLa cells, we study the relationship between lipid chemical modification and delivery performance in vitro. The impact of the functional group was shown to vary depending on the overall amine content and tail number of the delivery vector. Additionally, in vivo performance was evaluated using a Factor VII knockdown assay. Two library members, each containing ether groups, were found to knock down the target protein at levels comparable to the parent delivery vector. These results demonstrate that small chemical changes to the delivery vector impact knockdown efficiency and cell viability both in vitro and in vivo. The work described here identifies new materials for siRNA delivery, as well as provides new insight into the parameters for optimized chemical makeup of lipid-like siRNA delivery materials.
PMCID: PMC2931596  PMID: 20715849
11.  Gold, Poly(β-amino ester) Nanoparticles for Small Interfering RNA Delivery 
Nano letters  2009;9(6):2402-2406.
The safe and effective delivery of RNA therapeutics remains the major barrier to their broad clinical application. Here we develop a new nanoparticulate delivery system based on inorganic particles and biodegradable polycations. First, gold nanoparticles were modified with the hydrophilic polymer poly(ethylene glycol) (PEG), and then small interfering RNA (siRNA) was conjugated to the nanoparticles via biodegradable disulfide linkages, with ~30 strands of siRNA per nanoparticle. The particles were then coated with a library of end-modified poly(β-amino ester)s (PBAEs), previously identified as capable of facilitating intracellular DNA delivery. Nanoparticulate formulations developed here facilitate high levels of in vitro siRNA delivery, facilitating delivery as good or better than the commercially available lipid reagent, Lipofectamine 2000.
PMCID: PMC2728206  PMID: 19422265

Results 1-11 (11)