An Orthogonal Array Optimization of Lipid-like Nanoparticles for mRNA Delivery in Vivo

Li, Bin; Luo, Xiao; Deng, Binbin; Wang, Junfeng; McComb, David W.; Shi, Yimin; Gaensler, Karin; Tan, Xu; Dunn, Amy L.; Kerlin, Bryce A.; Dong, Yizhou

doi:10.1021/acs.nanolett.5b03528

Cited by 187 publications

(212 citation statements)

References 39 publications

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“…A previous study has combined lipid nanoparticle and AAV vector to deliver CRISPR/ Cas9 system components [3]. Here we show that by using an optimized formula of our newly developed lipid-like nanoparticles (LLNs) [4], we are able to effectively deliver Cas9 mRNA and single-guide RNA (sgRNA) to the liver and achieve in vivo targeting of HBV DNA and the proprotein convertase subtilisin/ kexin type 9 (pcsk9) gene, a therapeutic target for treating hypercholesterolemia [5]. To the best of our knowledge, this is the first report on non-viral delivery of CRISPR/Cas9 system components (mRNA + sgR-NA) in adult animals.…”

Section: Dear Editormentioning

confidence: 85%

“…Previously, we reported the development and optimization of TT3 LLNs (Supplementary information, Figure S1A), which are capable of efficiently delivering mRNA both in vitro and in vivo [4]. To optimize the LLN formulation for Cas9 mRNA delivery, we designed 27 formulas of TT3 LLNs (Supplementary information, Table S1) and examined their ability to delivery Cas9 mRNA into HEK293T cells stably expressing EGFP and an sgRNA targeting EGFP [3].…”

Section: Dear Editormentioning

confidence: 99%

“…Since Cas9 mRNA/ protein and sgRNA are degraded within one day in mice, our method provides a temporarily controllable way for in vivo genome editing. Based on our previous study, LLN-based delivery is highly specific to the liver [4]. Thus, LLN-mediated CRISPR/Cas9 delivery possesses the potential to treat a wide range of liver-related diseases.…”

Section: Dear Editormentioning

confidence: 99%

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A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo

et al. 2017

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Section: Dear Editormentioning

confidence: 85%

Section: Dear Editormentioning

confidence: 99%

Section: Dear Editormentioning

confidence: 99%

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A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo

et al. 2017

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“…To date, naked, chemically modified, or protamine-complexed mRNA have shown promise in phase I/II cancer trials (24)(25)(26). Recently, preclinical development of materials specific for mRNA delivery has resulted in cationic polymers such as polymethacrylates (27)(28)(29), poly(aspartamides) (30,31), and polypeptides (32), as well as multicomponent cationic lipid or lipid-like formulations (21,(33)(34)(35)(36). In many of these examples, however, transfection efficiencies can be quite low, ranging 20-80% in cells (18), with likely much lower efficiencies in vivo, which requires either high mRNA doses or hydrodynamic injections (32,37).…”

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confidence: 99%

Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals

McKinlay

Vargas

Blake

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Functional delivery of mRNA to tissues in the body is key to implementing fundamentally new and potentially transformative strategies for vaccination, protein replacement therapy, and genome editing, collectively affecting approaches for the prevention, detection, and treatment of disease. Broadly applicable tools for the efficient delivery of mRNA into cultured cells would advance many areas of research, and effective and safe in vivo mRNA delivery could fundamentally transform clinical practice. Here we report the stepeconomical synthesis and evaluation of a tunable and effective class of synthetic biodegradable materials: charge-altering releasable transporters (CARTs) for mRNA delivery into cells. CARTs are structurally unique and operate through an unprecedented mechanism, serving initially as oligo(α-amino ester) cations that complex, protect, and deliver mRNA and then change physical properties through a degradative, charge-neutralizing intramolecular rearrangement, leading to intracellular release of functional mRNA and highly efficient protein translation. With demonstrated utility in both cultured cells and animals, this mRNA delivery technology should be broadly applicable to numerous research and therapeutic applications.

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“…As the solubility of the lipids decreases, the lipids and nucleic acids are precipitated into nano-sized particles via electrostatic and hydrophobic interactions. The residual alcohol is generally removed by means of dialysis, 1,4,5) tangential flow filtration, 6) and ultrafiltration.7) In these methods, the residual alcohol is removed by solvent/small-solute selective dilution via a semipermeable membrane. These methods have technical problems associated with them when used in large-scale production; dilution of the residual alcohol to an acceptable level and the concentration of the samples are both costly.…”

mentioning

confidence: 99%

Development of an Alcohol Dilution–Lyophilization Method for Preparing Lipid Nanoparticles Containing Encapsulated siRNA

Shirane

Tanaka

Nakai³

et al. 2018

Biological & Pharmaceutical Bulletin

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Systems for delivering nucleic acids are now fundamental technologies for realizing personalized medicine. Among the various nucleic acid delivery systems that are currently available, lipid-nanoparticles (LNPs) that contain short interfering RNA (siRNA) have been extensively investigated for clinical applications. LNPs are generally prepared by an alcohol dilution method. In this method, it is necessary to remove the alcohol and then concentrate the LNP sample before they can be used. In this study, we report on the development of an "alcohol dilution-lyophilization method" for preparing siRNA-encapsulating LNPs. This method involves the use of a freeze-drying (lyophilization) method to remove the residual alcohol and to simultaneously concentrate the preparation. At first, the compositions of cryoprotectants and polyethylene glycol (PEG)-lipids that were used were optimized from the point of view of particle stabilization. A combination of sucrose and 1-(monomethoxy polyethyleneglycol5000)-2,3-dimyristoylglycerol (DMG-PEG 5000 ) was found to have the most efficient cryoprotective activity for the LNPs. The knockdown efficiency of the LNP prepared by the alcohol dilution-lyophilization method was comparable to that of an LNP prepared by the conventional ultrafiltration method. Key words lipid nano particle; freeze drying; short interfering RNA (siRNA); drug delivery system Nucleic acid delivery systems are now becoming fundamental technologies for realizing personalized medicine. Among the numerous nucleic acid delivery systems, Lipidnanoparticles (LNPs) that encapsulate short interfering RNA (siRNA) have been extensively investigated for clinical application.1) We previously reported on the development of a series of ionizable lipids, which we refer as to an SS-cleavable Proton-Activated Lipid-like Material (ssPalm), as a component of LNPs.2) The ssPalm is equipped with sensing units for the cellular environment (tertiary amines and disulfide bonding) in their structure. When the LNP containing the ssPalm (LNP ssPalm ) is taken up by cells, the pH-sensitive tertiary amines develop a positive charge in the acidic endosomal compartment and enhance endosomal escape. In the cytoplasm, the reductive cleavage of the disulfide bonding promotes the release of the siRNA from the particles.LNPs are typically prepared by a 2-step process; (1) the formation of siRNA-encapsulating LNPs by an alcohol dilution method, and (2) the removal of residual alcohol in parallel with the concentration of the samples. The alcohol dilution method is a well-established procedure for encapsulating nucleic acids in nanoparticles.3) In this method, lipids are dissolved in a water-miscible alcohol, which is then diluted by an acidic buffer containing the nucleic acid. As the solubility of the lipids decreases, the lipids and nucleic acids are precipitated into nano-sized particles via electrostatic and hydrophobic interactions. The residual alcohol is generally removed by means of dialysis, 1,4,5) tangential flow filtration, 6) and ultrafilt...

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An Orthogonal Array Optimization of Lipid-like Nanoparticles for mRNA Delivery in Vivo

Cited by 187 publications

References 39 publications

A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo

A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo

Charge-altering releasable transporters (CARTs) for the delivery and release of mRNA in living animals

Development of an Alcohol Dilution–Lyophilization Method for Preparing Lipid Nanoparticles Containing Encapsulated siRNA

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