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ON-TARGETplus siRNA | Dharmacon

Six-to eight-week-old, female CD-1 mice (Charles River Laboratories, Wilmington, MA) were administered ILL-siRNA formulations via tail vein injection at an siRNA concentration of 5 mg/kg (15:1 w:w ratio of ILL:siRNA) in a total volume of 200 μL. Control mice received an injection of 200 μL of phosphate buffered saline without calcium or magnesium salts (PBS). 48h after administration, animals were anesthetized with isoflurane. Blood was collected by submandibular cheek bleeding. Serum samples were obtained by allowing the blood to clot for 30 min and then centrifuging for 15 min at 15,000 rpm at 4°C. The supernatant was collected and analyzed for serum levels of Factor VII protein using the Biophen VII chromogenic assay (Aniara, Mason, OH) according to manufacturer’s instructions. A standard curve was generated using serially diluted concentrations of PBS-treated animals. Serum Factor VII levels of mice treated with ILL-siRNA formulations were expressed as percentage of PBS-control. Each group consisted of n=3 mice. DLinDMA liposomes (40:40:10:10 DLinDMA:Chol:DSPC:PEG-DMG) served as a positive control.

  Option A  We recommend using  from the University of Tokyo for the design of your siRNAs.

Here, we have described the synthesis and characterization of ionizable lysine-based lipids (ILL), a novel class of lipids with pH-dependent biophysical behavior. The goal of this work was to investigate how ionizable lipids with polyvalent, zwitterionic head groups behave compared to ionizable cationic systems. Four distinct ILLs were synthesized, and structural variations between ILLs impacted their biophysical behavior as well as their ability to deliver siRNA in vitro. All ILLs became increasingly cationic with a reduction in pH; however, only LOA-LysC1 was neutral at physiological pH. The ionization curves of ILL differ significantly from DLinDMA, likely due to the more complex, polyvalent head groups. All ILLs show potent siRNA mediated luciferase knockdown in vitro in a stably transfected HeLa-Luc cell line, with LOA-LysC2-OMe and LOA-LysC2 exhibiting IC50 values of roughly 1 nM. Interestingly, our in vitro data correlated well with our membrane lysis results, indicating that electrostatically driven membrane disruption promotes transfection. All ILLs form small diameter, monodisperse liposomes across multiple liposomal formulations, and efficiently encapsulate siRNA. However, ILL liposomes showed no in vivo knockdown in hepatocytes using a mouse Factor VII model. Given their potent transfection capabilities in vitro, the lack of in vivo efficacy may be due to poor targeting of liposomes to hepatocytes. Further optimization of these systems, through systemic manipulation of liposomal formulation or chemical modification of the ILL structures, may lead to higher in vivo efficacy.

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New England Biolabs provides many products useful for working with miRNA and siRNA.

A report is generated indicating the position of the AA dinucleotide, the 21 base target and the corresponding sense and antisense siRNA oligonucleotides.

We report the synthesis and characterization of a series of ionizable lysine-based lipids (ILL), novel lipids containing a lysine head group linked to a long-chain dialkylamine through an amide linkage at the lysine α-amine. These ILLs contain two ionizable amines and a carboxylate, and exhibit pH-dependent lipid ionization that varies with lipid structure. The synthetic scheme employed allows for the simple, orthogonal manipulation of lipids. This provides a method for the development of a compositionally diverse library with varying ionizable headgroups, tail structures, and linker regions. A focused library of four ILLs was synthesized to determine the impact of hydrophobic fluidity, lipid net charge, and lipid pKa on the biophysical and siRNA transfection characteristics of this new class of lipids. We found that manipulation of lipid structure impacts the protonation behavior, electrostatic driven membrane disruption, and ability to promote siRNA mediated knockdown in vitro. ILL-siRNA liposomal formulations were tested in a murine Factor VII model; however, no significant siRNA-mediated knockdown was observed. These results indicate that ILL may be useful in vitro transfection reagents, but further optimization of this new class of lipids is required to develop an effective in vivo siRNA delivery system.

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Despite their potency in vitro, ILL show no in vivo knockdown in hepatocytes. The difficulty of translating effective in vitro transfection reagents to viable in vivo siRNA delivery systems is well documented; cationic systems are rapidly cleared by the RES, preventing accumulation and transfection in hepatocytes., Additionally, structure-activity relationships for ionizable lipids have shown that slight changes in lipid structure significantly impact their in vivo efficacy.,, Though our ionization data indicates that ILL are more cationic at low pH, the two most membrane active ILL (LOA-LysC2-OMe and LOA-LysC2) show no pH dependent lysis behavior. Despite their promising in vitro transfection data, this indicates that these two ILL may be too cationic at pH = 7.4 to avoid protein or membrane interactions in circulation, stimulating RES uptake and preventing hepatocyte targeting. LOA-LysC1, which exhibits pH-dependent membrane lysis, has a lower extent of lysis at low pH that may prevent transfection in vivo. DOA-LysC2 appears to be less membrane active than all other ILL, possibly explaining its lack of efficacy. Changes to the liposomal formulation of LOA-LysC2 and LOA-LysC2-OMe did not improve results (data not shown). However, a broad, systemic manipulation of liposomal formulation, particularly the percentage of ILL as well as the choice of helper lipids or inclusion of targeting moieties may help to improve efficacy in these ILL systems.

All ILL were tested for their ability to deliver siRNA in vivo. The mouse Factor VII model was chosen as the primary in vivo screen for ILL liposome mediated delivery of siRNA to hepatocytes via systemic injection. Stable siRNA encapsulating liposomes suitable for systemic delivery were formulated using 40:40:10:10 ILL:Chol:DSPC:PEG-DMG at a 15:1 lipid:siRNA (w:w) ratio. DSPC and PEG-DMG were used in place of DOPE in these formulations to promote liposomal stability in vivo. The encapsulation efficiency and particle sizes of the resulting particles were 83% to 93% and 86 nm to 104 nm (). siRNA against the blood clotting protein Factor VII was administered at 5 mg/kg via tail vein injection. At 48 hrs post injection, blood was drawn and protein levels were assayed. Factor VII levels were unchanged compared to a PBS control for all tested ILL formulations (). A positive control formulation containing DLinDMA showed potent knockdown, thereby verifying the validity of the assay. Additional formulations containing LOA-LysC2 or LOA-LysC2-OMe with varying helper lipids and lipid ratios also showed no significant knockdown of Factor VII in vivo ().

Selected candidates are then screened to identify those siRNA sequences that are specific to the gene of interest.
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Recommended checks and controls for siRNA experiments | Abcam

Membrane lysis activity at low-pH is expected to correlate with transfection efficiency, and may help identify lipids with favorable properties for siRNA delivery in vitro and in vivo. Understanding the pH-dependent interactions between ILL containing liposomes and anionic membranes provides insight into their expected behavior in circulation. We investigated the capacity of ILL containing liposomes to lyse biomembrane mimicking vesicles (BMV) encapsulating the fluorophore/quencher ANTS/DPX as a function of pH. BMV consisted of 45:20:20:15 DOPC:DOPE:DOPG:cholesterol, and ILL liposomes contained 40:60 ILL:POPC. pH values of 7.4, 6.5, and 5.5 were chosen to assay lipid behavior under physiologically relevant conditions.

New Reagents for Synthesis of High Potency siRNA | …

The synthesized ILLs provide a platform for investigating how changes in lipid net charge (LOA-LysC2-OMe), hydrophobic group fluidity (DOA-LysC2), and lipid ionization behavior (LOA-LysC1) impact membrane lysis compared to LOA-LysC2 liposomes. Additionally, we compare the behavior of ILL with DLinDMA, a well characterized ionizable lipid that is effective for in vivo siRNA delivery, and DOPC, a zwitterionic membrane phospholipid that is neutral across the tested pH range. DOPC liposomes show no lysis across the tested pH-range, as expected. DLinDMA liposomes show strong pH-dependent lysis characteristics, with a 5-fold increase in lysis at pH = 5.5 (86%) compared to pH = 7.4 (16%). Interestingly, DLinDMA still shows significantly more lysis at pH = 7.4 than DOPC, indicating that these lipids are not fully deprotonated at physiological pH ().

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Ionizable lysine-based lipids (ILL) contain a lysine head group linked to a long-chain dialkylamine through an amide linkage at the lysine α-amine (). The resulting lipid structure contains a primary and tertiary amine, as well as a single carboxylate. As such, the overall net charge of ILL depends upon the ionization state of these protonatable moieties (). At low pH, all ionizable groups should be protonated, yielding a net charge of +2. As the pH increases, deprotonation of the carboxylate reduces the overall net charge to +1, and subsequent deprotonation of the amines results in a net charge of 0 or −1. We hypothesize that, similar to other ionizable delivery systems, this pH-dependent ionization behavior will promote siRNA encapsulation at low pH and membrane destabilization along the endocytic pathway, while preventing protein opsonization at physiological pH. The presence of the anionic moiety gives these lipids zwitterionic characteristics that may reduce the immunostimulatory effects that are characteristic of cationic lipid systems.

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