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author:("akin, Akin")
1.  Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNA Lipid Nanoparticles 
Lipid nanoparticles (LNPs) encapsulating short interfering RNAs that target hepatic genes are advancing through clinical trials, and early results indicate the excellent gene silencing observed in rodents and nonhuman primates also translates to humans. This success has motivated research to identify ways to further advance this delivery platform. Here, we characterize the polyethylene glycol lipid (PEG-lipid) components, which are required to control the self-assembly process during formation of lipid particles, but can negatively affect delivery to hepatocytes and hepatic gene silencing in vivo. The rate of transfer from LNPs to plasma lipoproteins in vivo is measured for three PEG-lipids with dialkyl chains 14, 16, and 18 carbons long. We show that 1.5 mol % PEG-lipid represents a threshold concentration at which the chain length exerts a minimal effect on hepatic gene silencing but can still modify LNPs pharmacokinetics and biodistribution. Increasing the concentration to 2.5 and 3.5 mol % substantially compromises hepatocyte gene knockdown for PEG-lipids with distearyl (C18) chains but has little impact for shorter dimyristyl (C14) chains. These data are discussed with respect to RNA delivery and the different rates at which the steric barrier disassociates from LNPs in vivo.
doi:10.1038/mtna.2013.66
PMCID: PMC3894582  PMID: 24345865
drug delivery; hepatocyte; lipid nanoparticles; polyethylene glycol; siRNA; prenatal diagnosis
3.  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.
doi:10.1038/mtna.2011.3
PMCID: PMC3381593  PMID: 23344621
delivery; immune cell; siRNA
4.  Influence of Cationic Lipid Composition on Gene Silencing Properties of Lipid Nanoparticle Formulations of siRNA in Antigen-Presenting Cells 
Molecular Therapy  2011;19(12):2186-2200.
Lipid nanoparticles (LNPs) are currently the most effective in vivo delivery systems for silencing target genes in hepatocytes employing small interfering RNA. Antigen-presenting cells (APCs) are also potential targets for LNP siRNA. We examined the uptake, intracellular trafficking, and gene silencing potency in primary bone marrow macrophages (bmMΦ) and dendritic cells of siRNA formulated in LNPs containing four different ionizable cationic lipids namely DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA. LNPs containing DLinKC2-DMA were the most potent formulations as determined by their ability to inhibit the production of GAPDH target protein. Also, LNPs containing DLinKC2-DMA were the most potent intracellular delivery agents as indicated by confocal studies of endosomal versus cytoplamic siRNA location using fluorescently labeled siRNA. DLinK-DMA and DLinKC2-DMA formulations exhibited improved gene silencing potencies relative to DLinDMA but were less toxic. In vivo results showed that LNP siRNA systems containing DLinKC2-DMA are effective agents for silencing GAPDH in APCs in the spleen and peritoneal cavity following systemic administration. Gene silencing in APCs was RNAi mediated and the use of larger LNPs resulted in substantially reduced hepatocyte silencing, while similar efficacy was maintained in APCs. These results are discussed with regard to the potential of LNP siRNA formulations to treat immunologically mediated diseases.
doi:10.1038/mt.2011.190
PMCID: PMC3242662  PMID: 21971424
5.  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.
doi:10.1021/bc100041r
PMCID: PMC2931596  PMID: 20715849
6.  Effective RNAi-mediated gene silencing without interruption of the endogenous microRNA pathway 
Nature  2007;449(7163):745-747.
Systemic administration of synthetic small interfering RNAs (siRNAs) effectively silences hepatocyte gene expression in rodents and primates1–3. Whether or not in vivo gene silencing by synthetic siRNA can disrupt the endogenous microRNA (miRNA) pathway remains to be addressed. Here we show that effective target-gene silencing in the mouse and hamster liver can be achieved by systemic administration of synthetic siRNA without any demonstrable effect on miRNA levels or activity. Indeed, siRNA targeting two hepatocyte-specific genes (apolipo-protein B and factor VII) that achieved efficient (~80%) silencing of messenger RNA transcripts and a third irrelevant siRNA control were administered to mice without significant changes in the levels of three hepatocyte-expressed miRNAs (miR-122, miR-16 and let-7a) or an effect on miRNA activity. Moreover, multiple administrations of an siRNA targeting the hepatocyte-expressed gene Scap in hamsters achieved long-term mRNA silencing without significant changes in miR-122 levels. This study advances the use of siRNAs as safe and effective tools to silence gene transcripts in animal studies, and supports the continued advancement of RNA interference therapeutics using synthetic siRNA.
doi:10.1038/nature06179
PMCID: PMC3019095  PMID: 17898712
7.  A combinatorial library of lipid-like materials for delivery of RNAi therapeutics 
Nature biotechnology  2008;26(5):561-569.
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′-O Me) 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.
doi:10.1038/nbt1402
PMCID: PMC3014085  PMID: 18438401
8.  Assessing siRNA Pharmacodynamics in a Luciferase-expressing Mouse 
A significant barrier to the successful general development of small-interfering RNA (siRNA) therapeutics is the ability to deliver them systemically to target organs and cell types. In this study, we have developed a mouse strain that will facilitate the evaluation of the efficacy of siRNA delivery strategies. This strain contains robust ubiquitous expression of firefly luciferase from germ line Cre-mediated recombination of the ROSA26-LSL-Luc allele. We show that luciferase is highly and uniformly expressed in all tissues examined. Using this mouse model, we describe a facile assay that enables the assessment of the pharmacodynamics of a systemically delivered siRNA formulation. These mice can also be used as universal donors, enabling the efficient and sensitive monitoring of cell trafficking or tissue transplantation. The primary advantage of this approach is that siRNA efficacy against a nonessential target can be easily evaluated in any tissue. This strain should generally enhance the ability to rapidly screen, compare and optimize various siRNA formulations for tissue-targeted or -enhanced systemic delivery in a preclinical development setting.
doi:10.1038/mt.2008.187
PMCID: PMC3014086  PMID: 18781145
9.  In vivo quantification of formulated and chemically modified small interfering RNA by heating-in-Triton quantitative reverse transcription polymerase chain reaction (HIT qRT-PCR) 
Silence  2010;1:16.
Background
While increasing numbers of small interfering RNA (siRNA) therapeutics enter into clinical trials, the quantification of siRNA from clinical samples for pharmacokinetic studies remains a challenge. This challenge is even more acute for the quantification of chemically modified and formulated siRNAs such as those typically required for systemic delivery.
Results
Here, we describe a novel method, heating-in-Triton quantitative reverse transcription PCR (HIT qRT-PCR) that improves upon the stem-loop RT-PCR technique for the detection of formulated and chemically modified siRNAs from plasma and tissue. The broad dynamic range of this assay spans five orders of magnitude and can detect as little as 70 pg duplex in 1 g of liver or in 1 ml of plasma. We have used this assay to quantify intravenously administrated siRNA in rodents and have reliably correlated target reduction with tissue drug concentrations. We were able to detect siRNA in rat liver for at least 10 days post injection and determined that for a modified factor VII (FVII) siRNA, on average, approximately 500 siRNA molecules per cell are required to achieve a 50% target reduction.
Conclusions
HIT qRT-PCR is a novel approach that simplifies the in vivo quantification of siRNA and provides a highly sensitive and reproducible tool to measure the silencing efficiency of chemically modified and formulated siRNAs.
doi:10.1186/1758-907X-1-16
PMCID: PMC2939650  PMID: 20731861

Results 1-10 (10)