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1.
Cell Rep ; 22(9): 2227-2235, 2018 02 27.
Article in English | MEDLINE | ID: mdl-29490262

ABSTRACT

The development of clinically viable delivery methods presents one of the greatest challenges in the therapeutic application of CRISPR/Cas9 mediated genome editing. Here, we report the development of a lipid nanoparticle (LNP)-mediated delivery system that, with a single administration, enabled significant editing of the mouse transthyretin (Ttr) gene in the liver, with a >97% reduction in serum protein levels that persisted for at least 12 months. These results were achieved with an LNP delivery system that was biodegradable and well tolerated. The LNP delivery system was combined with a sgRNA having a chemical modification pattern that was important for high levels of in vivo activity. The formulation was similarly effective in a rat model. Our work demonstrates that this LNP system can deliver CRISPR/Cas9 components to achieve clinically relevant levels of in vivo genome editing with a concomitant reduction of TTR serum protein, highlighting the potential of this system as an effective genome editing platform.


Subject(s)
CRISPR-Associated Protein 9/metabolism , CRISPR-Cas Systems/genetics , Gene Editing , Gene Transfer Techniques , Lipids/chemistry , Nanoparticles/administration & dosage , Nanoparticles/chemistry , Animals , Base Sequence , Liver/metabolism , Mice , RNA, Guide, Kinetoplastida/chemistry , RNA, Guide, Kinetoplastida/genetics , Rats
2.
Mol Ther Nucleic Acids ; 5(8): e342, 2016 Aug 02.
Article in English | MEDLINE | ID: mdl-27483025

ABSTRACT

Short interfering RNAs (siRNAs) are a valuable tool for gene silencing with applications in both target validation and therapeutics. Many advances have recently been made to improve potency and specificity, and reduce toxicity and immunostimulation. However, siRNA delivery to a variety of tissues remains an obstacle for this technology. To date, siRNA delivery to muscle has only been achieved by local administration or by methods with limited potential use in the clinic. We report systemic delivery of a highly chemically modified cholesterol-conjugated siRNA targeting muscle-specific gene myostatin (Mstn) to a full range of muscles in mice. Following a single intravenous injection, we observe 85-95% knockdown of Mstn mRNA in skeletal muscle and >65% reduction in circulating Mstn protein sustained for >21 days. This level of Mstn knockdown is also accompanied by a functional effect on skeletal muscle, with animals showing an increase in muscle mass, size, and strength. The cholesterol-conjugated siRNA platform described here could have major implications for treatment of a variety of muscle disorders, including muscular atrophic diseases, muscular dystrophy, and type II diabetes.

3.
Bioorg Med Chem Lett ; 26(18): 4513-4517, 2016 09 15.
Article in English | MEDLINE | ID: mdl-27503684

ABSTRACT

Single-stranded silencing RNAs (ss siRNA), while not as potent as duplex RNAs, have the potential to become a novel platform technology in RNA interference based gene silencing by virtue of their simplicity and plausibly favorable characteristics in pharmacokinetics and biodistribution. Like other therapeutic pharmaceutical agents, ss siRNA can be optimized to achieve higher potency through a structure-activity based approach. Systematic chemical modification at each position of a 21-mer oligonucleotide identified 2',5'-linked 3'-deoxythymidine (3dT) at position 1 and locked nucleic acids (LNAs) at the seed region as key components to afford significant enhancement in knockdown activity both in vitro and in vivo. Further optimization by additional chemical modifications should enable ss siRNA as an alternative gene silencing modality.


Subject(s)
Gene Silencing , RNA, Messenger/genetics , RNA, Small Interfering/genetics , beta Catenin/genetics , HEK293 Cells , Humans
4.
Pharmacol Res Perspect ; 4(1): e00207, 2016 Feb.
Article in English | MEDLINE | ID: mdl-26977298

ABSTRACT

The benefits of novel oral anticoagulants are hampered by bleeding. Since coagulation factor IX (fIX) lies upstream of fX in the coagulation cascade, and intermediate levels have been associated with reduced incidence of thrombotic events, we evaluated the viability of fIXa as an antithrombotic target. We applied translational pharmacokinetics/pharmacodynamics (PK/PD) principles to predict the therapeutic window (TW) associated with a selective small molecule inhibitor (SMi) of fIXa, compound 1 (CPD1, rat fIXa inhibition constant (Ki, 21 nmol/L) relative to clinically relevant exposures of apixaban (rat fXa Ki 4.3 nmol/L). Concentrations encompassing the minimal clinical plasma concentration (C min) of the 5 mg twice daily (BID) dose of apixaban were tested in rat arteriovenous shunt (AVS/thrombosis) and cuticle bleeding time (CBT) models. An I max and a linear model were used to fit clot weight (CW) and CBT. The following differences in biology were observed: (1) antithrombotic activity and bleeding increased in parallel for apixaban, but to a lesser extent for CPD1 and (2) antithrombotic activity occurred at high (>99%) enzyme occupancy (EO) for fXa or moderate (>65% EO) for fIXa. translational PK/PD analysis indicated that noninferiority was observed for concentrations of CPD1 that provided between 86% and 96% EO and that superior TW existed between 86% and 90% EO. These findings were confirmed in a study comparing short interfering (si)RNA-mediated knockdown (KD) modulation of fIX and fX mRNA. In summary, using principles of translational biology to relate preclinical markers of efficacy and safety to clinical doses of apixaban, we found that modulation of fIXa can be superior to apixaban.

5.
Thromb Haemost ; 113(6): 1300-11, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25790442

ABSTRACT

Haemophilia A and B are characterised by a life-long bleeding predisposition, and several lines of evidence suggest that risks of atherothrombotic events may also be reduced. Establishing a direct correlation between coagulation factor levels, thrombotic risks and bleeding propensity has long been hampered by an inability to selectively and specifically inhibit coagulation factor levels. Here, the exquisite selectivity of gene silencing combined with a gene knockout (KO) approach was used to define the relative contribution of factor IX (fIX) to thrombosis and primary haemostasis in the rat. Using a lipid nanoparticle (LNP) formulation, we successfully delivered fIX siRNAs to the liver by intravenous administration. The knockdown (KD) of target gene mRNA was achieved rapidly (within 24 hour post-siRNA dosing), sustained (maintained for at least 7 days post dosing) and not associated with changes in mRNA expression levels of other coagulation factors. We found that intermediate levels of liver fIX mRNA silencing (60-95 %) translating into a 50-99 % reduction of plasma fIX activity provided protection from thrombosis without prolonging the cuticle bleeding time. Over 99 % inhibition of fIX activity was required to observe increase in bleeding, a phenotype confirmed in fIX KO rats. These data provide substantial evidence of a participation of fIX in the mechanisms regulating thrombosis prior to those regulating primary haemostasis, therefore highlighting the potential of fIX as a therapeutic target. In addition, hepatic mRNA silencing using LNP-encapsulated siRNAs may represent a promising novel approach for the chronic treatment and prevention of coagulation-dependent thrombotic disorders in humans.


Subject(s)
Factor IX/genetics , Hemophilia B/genetics , Hemorrhage/genetics , Liver/metabolism , RNA Interference , RNA, Small Interfering/genetics , RNAi Therapeutics , Thrombosis/prevention & control , Animals , Cell Line , Chlorides , Disease Models, Animal , Factor IX/metabolism , Ferric Compounds , Gene Expression Regulation , Genotype , Hemophilia B/blood , Hemorrhage/blood , Hemostasis/genetics , Male , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Rats , Rats, Sprague-Dawley , Rats, Transgenic , Thrombosis/blood , Thrombosis/chemically induced , Thrombosis/genetics , Time Factors , Transfection
6.
Pharm Res ; 31(12): 3445-60, 2014 Dec.
Article in English | MEDLINE | ID: mdl-24980206

ABSTRACT

PURPOSE: To develop a tool based on siRNA-mediated knockdown of hepatic P450 oxidoreductase (POR) to decrease the CYP-mediated metabolism of small molecule drugs that suffer from rapid metabolism in vivo, with the aim of improving plasma exposure of these drugs. METHODS: siRNA against the POR gene was delivered using lipid nanoparticles (LNPs) into rats. The time course of POR mRNA knockdown, POR protein knockdown, and loss of POR enzyme activity was monitored. The rat livers were harvested to produce microsomes to determine the impact of POR knockdown on the metabolism of several probe substrates. Midazolam (a CYP3A substrate with high intrinsic clearance) was administered into LNP-treated rats to determine the impact of POR knockdown on midazolam pharmacokinetics. RESULTS: Hepatic POR mRNA and protein levels were significantly reduced by administering siRNA and the maximum POR enzyme activity reduction (~85%) occurred 2 weeks post-dose. In vitro analysis showed significant reductions in metabolism of probe substrates due to POR knockdown in liver, and in vivo POR knockdown resulted in greater than 10-fold increases in midazolam plasma concentrations following oral dosing. CONCLUSIONS: Anti-POR siRNA can be used to significantly reduce hepatic metabolism by various CYPs as well as greatly increase the bioavailability of high clearance compounds following an oral dose, thus enabling it to be used as a tool to increase drug exposure in vivo.


Subject(s)
Cytochrome P-450 Enzyme System/genetics , Gene Knockdown Techniques/methods , RNA, Small Interfering/pharmacology , Animals , Chemistry, Pharmaceutical , Diclofenac/metabolism , In Vitro Techniques , Male , Microsomes/drug effects , Microsomes/enzymology , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Midazolam/metabolism , Nanoparticles , Protein Binding , Rats
7.
Mol Ther Nucleic Acids ; 2: e65, 2013 Jan 15.
Article in English | MEDLINE | ID: mdl-23322014

ABSTRACT

Lung pathology in cystic fibrosis is linked to dehydration of the airways epithelial surface which in part results from inappropriately raised sodium reabsorption through the epithelial sodium channel (ENaC). To identify a small-interfering RNA (siRNA) which selectively inhibits ENaC expression, chemically modified 21-mer siRNAs targeting human ENaCα were designed and screened. GSK2225745, was identified as a potent inhibitor of ENaCα mRNA (EC(50) (half maximal effective concentration) = 0.4 nmol/l, maximum knockdown = 85%) and protein levels in A549 cells. Engagement of the RNA interference (RNAi) pathway was confirmed using 5' RACE. Further profiling was carried out in therapeutically relevant human primary cells. In bronchial epithelial cells, GSK2225745 elicited potent suppression of ENaCα mRNA (EC(50) = 1.6 nmol/l, maximum knockdown = 82%). In human nasal epithelial cells, GSK2225745 also produced potent and long-lasting (≥72 hours) suppression of ENaCα mRNA levels which was associated with significant inhibition of ENaC function (69% inhibition of amiloride-sensitive current in cells treated with GSK2225745 at 10 nmol/l). GSK2225745 showed no evidence for potential to stimulate toll-like receptor (TLR)3, 7 or 8. In vivo, topical delivery of GSK2225745 in a lipid nanoparticle formulation to the airways of mice resulted in significant inhibition of the expression of ENaCα in the lungs. In conclusion, GSK2225745 is a potent inhibitor of ENaCα expression and warrants further evaluation as a potential novel inhaled therapeutic for cystic fibrosis.Molecular Therapy - Nucleic Acids (2013) 2, e65; doi:10.1038/mtna.2012.57; published online 15 January 2013.

8.
J Lipid Res ; 52(6): 1084-1097, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21398511

ABSTRACT

Increased serum apolipoprotein (apo)B and associated LDL levels are well-correlated with an increased risk of coronary disease. ApoE⁻/⁻ and low density lipoprotein receptor (LDLr)⁻/⁻ mice have been extensively used for studies of coronary atherosclerosis. These animals show atherosclerotic lesions similar to those in humans, but their serum lipids are low in apoB-containing LDL particles. We describe the development of a new mouse model with a human-like lipid profile. Ldlr CETP⁺/⁻ hemizygous mice carry a single copy of the human CETP transgene and a single copy of a LDL receptor mutation. To evaluate the apoB pathways in this mouse model, we used novel short-interfering RNAs (siRNA) formulated in lipid nanoparticles (LNP). ApoB siRNAs induced up to 95% reduction of liver ApoB mRNA and serum apoB protein, and a significant lowering of serum LDL in Ldlr CETP⁺/⁻ mice. ApoB targeting is specific and dose-dependent, and it shows lipid-lowering effects for over three weeks. Although specific triglycerides (TG) were affected by ApoB mRNA knockdown (KD) and the total plasma lipid levels were decreased by 70%, the overall lipid distribution did not change. Results presented here demonstrate a new mouse model for investigating additional targets within the ApoB pathways using the siRNA modality.


Subject(s)
Apolipoproteins B/genetics , Atherosclerosis/genetics , Atherosclerosis/metabolism , Cholesterol Ester Transfer Proteins/genetics , Cholesterol, LDL/blood , Disease Models, Animal , Receptors, LDL/genetics , Animals , Apolipoproteins B/blood , Apolipoproteins E/blood , Apolipoproteins E/genetics , Atherosclerosis/pathology , Cell Line, Tumor , Cholesterol Ester Transfer Proteins/metabolism , Founder Effect , Gene Expression Profiling , Gene Knockdown Techniques , Hemizygote , Humans , Lipid Metabolism/genetics , Liposomes/metabolism , Liver/drug effects , Liver/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Nanoparticles/administration & dosage , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , RNA, Small Interfering/metabolism , RNA, Small Interfering/pharmacology , Receptors, LDL/metabolism , Triglycerides/blood
9.
Nucleic Acids Res ; 38(14): 4788-97, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20360048

ABSTRACT

The use of short interfering RNAs (siRNA) in animals for target validation or as potential therapeutics is hindered by the short physical half-life when delivered as unencapsulated material and in turn the short active half-life of siRNAs in vivo. Here we demonstrate that the character of the two 3'-overhang nucleotides of the guide strand of siRNAs is a determinant of the duration of silencing by siRNAs both in vivo and in tissue culture cells. We demonstrate that deoxyribonucleotides in the guide strand overhang of siRNAs have a negative impact on maintenance of both the in vitro and in vivo activity of siRNAs over time. Overhangs that contain ribonucleotides or 2'-O-methyl modified nucleotides do not demonstrate this same impairment. We also demonstrate that the sequence of an siRNA is a determinant of the duration of silencing of siRNAs directed against the same target even when those siRNAs have equivalent activities in vitro. Our experiments have determined that a measurable duration parameter exists, distinct from both maximum silencing ability and the potency of siRNAs. Our findings provide information on incorporating chemically modified nucleotides into siRNAs for potent, durable therapeutics and also inform on methods used to select siRNAs for therapeutic and research purposes.


Subject(s)
RNA Interference , RNA, Small Interfering/chemistry , Animals , Cell Line, Tumor , Cytokines/metabolism , Kinetics , Mice , Mice, Inbred C57BL , RNA, Small Untranslated
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