Lipid Nanoparticle Systems for Enabling Gene Therapies
Authors: Pieter R. Cullis, Michael J. Hope
Journal: Molecular Therapy (2017)

Genetic drugs such as small interfering RNA (siRNA), mRNA, or plasmid DNA provide potential gene therapies to treat most diseases by silencing pathological genes, expressing therapeutic proteins, or through gene-editing applications. In order for genetic drugs to be used clinically, however, sophisticated delivery systems are required. Lipid nanoparticle (LNP) systems are currently the lead non-viral delivery systems for enabling the clinical potential of genetic drugs. Application will be made to the Food and Drug Administration (FDA) in 2017 for approval of an LNP siRNA drug to treat transthyretin-induced amyloidosis, presently an untreatable disease. Here, we first review research leading to the development of LNP siRNA systems capable of silencing target genes in hepatocytes following systemic administration. Subsequently, progress made to extend LNP technology to mRNA and plasmids for protein replacement, vaccine, and gene-editing applications is summarized. Finally, we address current limitations of LNP technology as applied to genetic drugs and ways in which such limitations may be overcome. It is concluded that LNP technology, by virtue of robust and efficient formulation processes, as well as advantages in potency, payload, and design flexibility, will be a dominant non-viral technology to enable the enormous potential of gene therapy. Genetic drugs such as small interfering RNA (siRNA), mRNA, or plasmid DNA provide potential gene therapies to treat most diseases by silencing pathological genes, expressing therapeutic proteins, or through gene-editing applications. In order for genetic drugs to be used clinically, however, sophisticated delivery systems are required. Lipid nanoparticle (LNP) systems are currently the lead non-viral delivery systems for enabling the clinical potential of genetic drugs. Application will be made to the Food and Drug Administration (FDA) in 2017 for approval of an LNP siRNA drug to treat transthyretin-induced amyloidosis, presently an untreatable disease. Here, we first review research leading to the development of LNP siRNA systems capable of silencing target genes in hepatocytes following systemic administration. Subsequently, progress made to extend LNP technology to mRNA and plasmids for protein replacement, vaccine, and gene-editing applications is summarized. Finally, we address current limitations of LNP technology as applied to genetic drugs and ways in which such limitations may be overcome. It is concluded that LNP technology, by virtue of robust and efficient formulation processes, as well as advantages in potency, payload, and design flexibility, will be a dominant non-viral technology to enable the enormous potential of gene therapy. The central problem preventing the widespread implementation of gene therapies based on RNA and DNA polymers is delivery.1Whitehead K.A. Langer R. Anderson D.G. Knocking down barriers: advances in siRNA delivery.Nat. Rev. Drug Discov. 2009; 8: 129-138Crossref PubMed Scopus (2421) Google Scholar The complexity of the problem is enormous. Naked RNA or DNA molecules are rapidly degraded in biological fluids, do not accumulate in target tissues following systemic administration, and cannot penetrate into target cells even if they get to the target tissue. Further, the immune system is exquisitely designed to recognize and destroy vectors containing genetic information.2Bowie A.G. Unterholzner L. Viral evasion and subversion of pattern-recognition receptor signalling.Nat. Rev. Immunol. 2008; 8: 911-922Crossref PubMed Scopus (520) Google Scholar Given these obstacles, it is remarkable that significant progress has been made over the last 30 years to develop delivery systems that enable the therapeutic use of genetic drugs. Here, we focus on non-viral delivery systems that have advantages of ease of manufacture, reduced immune responses, multi-dosing capabilities, larger payloads, and flexibility of design. The lead non-viral delivery systems are lipid nanoparticles (LNPs); more than four LNP small interfering RNA (siRNA) drugs have entered the clinic, one of which is in late stage phase III trials (see Table 1). Here, we describe the origins of LNP technology for delivery of small molecule drugs, summarize the developments leading to encapsulation and delivery of genetic drugs, and indicate the ongoing optimization process3Jayaraman M. Ansell S.M. Mui B.L. Tam Y.K. Chen J. Du X. Butler D. Eltepu L. Matsuda S. Narayanannair J.K. et al.Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.Angew. Chem. Int. Ed. Engl. 2012; 51: 8529-8533Crossref PubMed Scopus (650) Google Scholar that is leading to increasingly potent, non-toxic gene therapies with clear clinical potential.Table 1LNP siRNA Drugs in Clinical TrialsaAs of 2017.NameLNP-Encapsulated siRNAIndicationStatusCompanyPatisiransiRNA versus transthyretintransthyretin-induced amyloidosisphase IIIAlnylamARB 1467siRNAs versus 3 HBV proteinshepatitis bphase IIArbutussiRNA versus PLK1hepatocellular carcinomaphase I/IIArbutussiRNA versus KSP and VEGFliver cancerphase IAlnylamsiRNA versus PCSK9atherosclerosisphase IAlnylamLNP, lipid nanoparticle; siRNA, small interfering RNA.a As of 2017. Open table in a new tab LNP, lipid nanoparticle; siRNA, small interfering RNA. The origins of LNP systems used for genetic drugs lie in the development of liposomal drug delivery systems for small molecule drugs.4Cullis P.R. Mayer L.D. Bally M.B. Madden T.D. Hope M.J. Generating and loading of liposomal systems for drug-delivery applications.Adv. Drug Deliv. Rev. 1989; 3: 267-282Crossref Scopus (72) Google Scholar Liposomal systems are a class of LNPs containing lipids organized in a bilayer organization. Many membrane lipids, such as phosphatidylcholine (PC), adopt bilayer structures spontaneously when dispersed in an aqueous medium.5Cullis P.R. de Kruijff B. Lipid polymorphism and the functional roles of lipids in biological membranes.Biochim. Biophys. Acta. 1979; 559: 399-420Crossref PubMed Scopus (1519) Google Scholar The particular sub-class of liposomes that have proven useful for drug delivery applications are so-called large unilamellar vesicles (LUVs) exhibiting a size in the range of 100 nm and containing a single bilayer separating the interior aqueous medium from the exterior.6Hope M.J. Bally M.B. Webb G. Cullis P.R. Production of large unilamellar vesicles by a rapid extrusion procedure: characterization of size distribution, trapped volume and ability to maintain a membrane potential.Biochim. Biophys. Acta. 1985; 812: 55-65Crossref PubMed Scopus (2012) Google Scholar There are now nine liposome-based drugs for intravenous (i.v.) administration (see Table 2) that have been approved by regulatory authorities worldwide.7Allen T.M. Cullis P.R. Liposomal drug delivery systems: from concept to clinical applications.Adv. Drug Deliv. Rev. 2013; 65: 36-48Crossref PubMed Scopus (3111) Google Scholar Most of these systems contain small molecule cancer drugs and rely on the ability of small (<100 nm diameter) LNP systems to preferentially extravasate at tumor sites following i.v. administration. This preferential extravasation occurs because tumor neovasculature can contain apertures with diameters up to 200 nm, leading to penetration of small particulate systems into the tumor tissue. The preferential accumulation of small LNP into tumor tissue has been termed the "enhanced penetration and retention" (EPR) effect.8Maeda H. Macromolecular therapeutics in cancer treatment: the EPR effect and beyond.J. Control. Release. 2012; 164: 138-144Crossref PubMed Scopus (635) Google Scholar The EPR effect, in combination with LUVs with long circulation lifetimes, can improve tumor delivery by 10-fold or more compared to equivalent doses of free drug and avoid sensitive tissue, leading to therapeutic benefit.Table 2Approved LNP DrugsaAdministered by intravenous injection.NameEncapsulated DrugIndicationYear ApprovedCompanyAmBisomeamphotericin BFungal infections Leishmanaisis1990 (Europe), 1997 (USA)GileadDoxil/CaelyxdoxorubicinKaposi's sarcoma1995 (USA)Johnson & Johnsonovarian cancer1999 (USA)breast cancer2003 (Europe, Canada)DaunoXomedaunorubicinKaposi's sarcoma1996 (Europe), 1996 (USA)GalenMyocetdoxorubicinbreast cancer2000 (Europe)CephalonAbelcetamphotericin BAspergillosis1995 (USA)EnzonAmphotecamphotericin BAspergillosis1996 (USA)IntermuneVisudyneverteporphinmacular degeneration2000 (USA), 2003 (Japan)QLTLipo-DoxdoxorubicinKaposi's sarcoma, breast, and ovarian cancer2001 (Taiwan)Taiwan LiposomeMarqibovincristineacute lymphoblastic leukemia2012 (USA)Spectrum PharmaLNP, lipid nanoparticle.a Administered by intravenous injection. Open table in a new tab LNP, lipid nanoparticle. LNP systems for delivery of small molecule drugs represent a relatively mature technology and have led to rigorous design criteria, many of which carry over into the design of LNP systems for delivery of genetic drugs. These criteria include a size range of 100 nm or less, highly efficient encapsulation processes, robust, scalable manufacturing processes, and product stability of at least 1 year at 4°C. A key feature is a relatively neutral surface exterior to avoid extensive adsorption of serum proteins onto the LNP. Such adsorption leads to rapid accumulation by the fixed and free macrophages present in the circulation9Chonn A. Semple S.C. Cullis P.R. Association of blood proteins with large unilamellar liposomes in vivo. Relation to circulation lifetimes.J. Biol. Chem. 1992; 267: 18759-18765PubMed Google Scholar and consequent poor penetration to target tissue. Application of LNP technology to genetic drugs required the development of methods to achieve efficient encapsulation of RNA and DNA polymers into LNP systems. Techniques used for loading small molecule drugs into LUVs in response to transmembrane pH gradients4Cullis P.R. Mayer L.D. Bally M.B. Madden T.D. Hope M.J. Generating and loading of liposomal systems for drug-delivery applications.Adv. Drug Deliv. Rev. 1989; 3: 267-282Crossref Scopus (72) Google Scholar cannot be applied to negatively charged macromolecules because of their size and negative charge. The introduction of cationic lipids10Felgner P.L. Gadek T.R. Holm M. Roman R. Chan H.W. Wenz M. Northrop J.P. Ringold G.M. Danielsen M. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure.Proc. Natl. Acad. Sci. USA. 1987; 84: 7413-7417Crossref PubMed Scopus (4355) Google Scholar that have strong electrostatic associations with RNA and DNA polymers provided a way forward. However, as originally practiced, cationic lipids with a permanent positive charge were mixed with nucleic acid polymers to form "complexes." While these systems have proven useful for in vitro transfection purposes, their utility in vivo is limited due to their large size (>1 μm diameter), instability, positive surface charge, and dose-limiting toxic side effects.11Lv H. Zhang S. Wang B. Cui S. Yan J. Toxicity of cationic lipids and cationic polymers in gene delivery.J. Control. Release. 2006; 114: 100-109Crossref PubMed Scopus (1656) Google Scholar The introduction of ionizable cationic lipids, such as 1,2-dioleoyl-3-dimethylammonium propane (DODAP)12Bailey A.L. Cullis P.R. Modulation of membrane fusion by asymmetric transbilayer distributions of amino lipids.Biochemistry. 1994; 33: 12573-12580Crossref PubMed Scopus (92) Google Scholar combined with development of an ethanol-loading procedure, has provided methods to achieve high loading efficiencies for genetic drugs in small (<100 nm diameter) LNP systems with low surface charge, thus overcoming many issues noted for complexes. Specifically, ionizable cationic lipids such as DODAP have an apparent pK (pKa) <7 and can be used to achieve efficient encapsulation of negatively charged polymers into LNP at low pH (e.g., pH 4) where the ionizable lipids are positively charged. Subsequently, the pH can be raised to physiological pH values, leading to LNPs with a relatively neutral surface. The first expression of the ethanol loading technique involved mixing (at pH 4) preformed LNPs containing DODAP and other "structural" lipids with oligonucleotides in the presence of high (∼40% by volume) ethanol concentrations.13Semple S.C. Klimuk S.K. Harasym T.O. Dos Santos N. Ansell S.M. Wong K.F. Maurer N. Stark H. Cullis P.R. Hope M.J. Scherrer P. Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures.Biochim. Biophys. Acta. 2001; 1510: 152-166Crossref PubMed Scopus (317) Google Scholar Subsequently, a T-tube mixing process14Jeffs L.B. Palmer L.R. Ambegia E.G. Giesbrecht C. Ewanick S. MacLachlan I. A scalable, extrusion-free method for efficient liposomal encapsulation of plasmid DNA.Pharm. Res. 2005; 22: 362-372Crossref PubMed Scopus (228) Google Scholar was developed where lipids (including ionizable cationic lipids and polyethyleneglycol [PEG] lipids) dissolved in ethanol were rapidly mixed with oligonucleotides in an aqueous buffer (pH 4), again resulting in efficient loading of nucleic acid polymers into small (diameter <100 nm) LNP systems. This was followed by a microfluidic mixing technique to obtain a more rapid and controllable mixing process15Belliveau N.M. Huft J. Lin P.J. Chen S. Leung A.K. Leaver T.J. Wild A.W. Lee J.B. Taylor R.J. Tam Y.K. et al.Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA.Mol. Ther. Nucleic Acids. 2012; 1: e37Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar and the production of structurally well-defined systems. A visual depiction of LNP-oligonucleotide formulation using rapid mixing techniques is shown in Figure 1, which also provides an intuitive understanding of how high oligonucleotide encapsulation efficiencies can be achieved using the ethanol loading process. The most common embodiment of the ethanol loading approach to encapsulating oligonucleotides such as siRNA in LNP systems consists of an ethanol solution containing ionizable cationic lipid, cholesterol, distearoyl PC (DSPC), and PEG lipid (molar ratios 40-50/40-30/10/10-1, respectively) that is then mixed rapidly with an aqueous buffer (pH 4) containing the oligonucleotide at an amino lipid-to-oligonucleotide phosphate (N/P) ratio of six.16Semple S.C. Akinc A. Chen J. Sandhu A.P. Mui B.L. Cho C.K. Sah D.W. Stebbing D. Crosley E.J. Yaworski E. et al.Rational design of cationic lipids for siRNA delivery.Nat. Biotechnol. 2010; 28: 172-176Crossref PubMed Scopus (1151) Google Scholar A 3:1 ratio of aqueous media to ethanol is usually employed. Following the mixing step, the LNP dispersion is first dialyzed against a pH 4 buffer to remove residual ethanol and then against a pH 7.4 buffer to raise the pH to physiological values. The LNP systems resulting from the rapid mixing ethanol loading process satisfy basic requirements for an in vivo delivery system for genetic drugs in five ways. The formulation process is rapid, reproducible, and scalable, encapsulation efficiencies approaching 100% are readily achievable, the LNP size can be selected by adjusting the PEG-lipid content,15Belliveau N.M. Huft J. Lin P.J. Chen S. Leung A.K. Leaver T.J. Wild A.W. Lee J.B. Taylor R.J. Tam Y.K. et al.Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA.Mol. Ther. Nucleic Acids. 2012; 1: e37Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar the resulting systems exhibit adequate stability properties, and the LNP particle exhibits low surface charge as required for in vivo administration. The structure of the LNP systems for siRNA as determined by molecular modeling17Leung A.K. Hafez I.M. Baoukina S. Belliveau N.M. Zhigaltsev I.V. Afshinmanesh E. Tieleman D.P. Hansen C.L. Hope M.J. Cullis P.R. Lipid nanoparticles containing siRNA synthesized by microfluidic mixing exhibit an electron-dense nanostructured core.J. Phys. Chem. C Nanomater Interfaces. 2012; 116: 18440-18450Crossref PubMed Scopus (171) Google Scholar indicates a largely hydrophobic core consisting of inverted micelles of lipid encapsulating oligonucleotides surrounded by a coating of PEG-lipids as shown in Figure 2. This structure is supported by the "solid core" structure detected by cryogenic transmission electron microscopy (cryo-TEM) and the "currant bun" morphology seen for cryo-TEM micrographs of LNP systems containing negatively charged gold nanoparticles, also shown in Figure 2. The structure for LNP encapsulating mRNA is still to be determined; however, it should be noted that the ethanol loading technique is a generally applicable process18Leung A.K. Tam Y.Y. Chen S. Hafez I.M. Cullis P.R. Microfluidic mixing: a general method for encapsulating macromolecules in lipid nanoparticle systems.J. Phys. Chem. B. 2015; 119: 8698-8706Crossref PubMed Scopus (129) Google Scholar leading to efficient encapsulation of mRNA and plasmid DNA as well as other negatively charged entities such as the gold nanoparticles of Figure 2. The first demonstration that LNP formulations of genetic drugs containing ionizable cationic lipids could achieve significant in vivo activity was for LNP systems containing siRNA to silence genes in hepatocytes following i.v. administration.19Zimmermann T.S. Lee A.C. Akinc A. Bramlage B. Bumcrot D. Fedoruk M.N. Harborth J. Heyes J.A. Jeffs L.B. John M. et al.RNAi-mediated gene silencing in non-human primates.Nature. 2006; 441: 111-114Crossref PubMed Scopus (1192) Google Scholar This study, which employed the ionizable cationic lipid 1,2-dilinoleyloxy-3-dimethylaminopropane (DLinDMA, an ether analog of DODAP containing linoleic acyl chains), was followed by the observation that the activity of these LNP systems was highly sensitive to the particular species of cationic lipid employed.16Semple S.C. Akinc A. Chen J. Sandhu A.P. Mui B.L. Cho C.K. Sah D.W. Stebbing D. Crosley E.J. Yaworski E. et al.Rational design of cationic lipids for siRNA delivery.Nat. Biotechnol. 2010; 28: 172-176Crossref PubMed Scopus (1151) Google Scholar This observation led to an intensive lipid synthesis program to identify lipids with optimized in vivo activity when incorporated into LNP siRNA systems, resulting in the identification of dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA, usually abbreviated to MC3), which is currently the "gold standard" cationic lipid for silencing liver targets.3Jayaraman M. Ansell S.M. Mui B.L. Tam Y.K. Chen J. Du X. Butler D. Eltepu L. Matsuda S. Narayanannair J.K. et al.Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.Angew. Chem. Int. Ed. Engl. 2012; 51: 8529-8533Crossref PubMed Scopus (650) Google Scholar Remarkably, the gene-silencing activity achieved with MC3 was improved by over three orders of magnitude from that achieved for LNP containing DLinDMA.3Jayaraman M. Ansell S.M. Mui B.L. Tam Y.K. Chen J. Du X. Butler D. Eltepu L. Matsuda S. Narayanannair J.K. et al.Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.Angew. Chem. Int. Ed. Engl. 2012; 51: 8529-8533Crossref PubMed Scopus (650) Google Scholar The changes in cationic lipid structure that can so dramatically influence potency appear relatively minor (Figure 3). The dominant factors determining the cationic lipid potency were the unsaturation of the acyl chains, introduction of ether linkages, and most notably, the pKa of the amino function of the cationic lipid. Specifically, LNP siRNA systems containing cationic lipids with amino functions exhibiting a pKa between 6.2 and 6.4 are by far the most effective3Jayaraman M. Ansell S.M. Mui B.L. Tam Y.K. Chen J. Du X. Butler D. Eltepu L. Matsuda S. Narayanannair J.K. et al.Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.Angew. Chem. Int. Ed. Engl. 2012; 51: 8529-8533Crossref PubMed Scopus (650) Google Scholar for hepatocyte gene silencing (Figure 4).Figure 4Optimized Ionizable Cationic Lipids Exhibit pKa Values in the Range pKa = 6.2–6.5Show full captionThe figure presents a plot of the dose required to achieve 50% gene silencing (ED50) in hepatocytes in mice versus the pKa of the cationic lipids employed in the LNP.3Jayaraman M. Ansell S.M. Mui B.L. Tam Y.K. Chen J. Du X. Butler D. Eltepu L. Matsuda S. Narayanannair J.K. et al.Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.Angew. Chem. Int. Ed. Engl. 2012; 51: 8529-8533Crossref PubMed Scopus (650) Google Scholar Fifty-six amino lipids were synthesized and formulated in LNPs and the ED50 measured and plotted against their pKa. For 15 lipids the ED50 dose was not achieved, these are indicated by the open diamonds representing the highest dose tested for that lipid. For the remaining lipids (closed diamonds), a polynomial best-fit curve highlights the most active compounds, which exhibit an optimal pKa between 6.2 and 6.6. Each data point is derived from a four-dose response curve with groups of n = 4 mice per dose. Reproduced with permission from Jayaraman et al.3Jayaraman M. Ansell S.M. Mui B.L. Tam Y.K. Chen J. Du X. Butler D. Eltepu L. Matsuda S. Narayanannair J.K. et al.Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.Angew. Chem. Int. Ed. Engl. 2012; 51: 8529-8533Crossref PubMed Scopus (650) Google ScholarView Large Image Figure ViewerDownload Hi-res image Download (PPT) The figure presents a plot of the dose required to achieve 50% gene silencing (ED50) in hepatocytes in mice versus the pKa of the cationic lipids employed in the LNP.3Jayaraman M. Ansell S.M. Mui B.L. Tam Y.K. Chen J. Du X. Butler D. Eltepu L. Matsuda S. Narayanannair J.K. et al.Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.Angew. Chem. Int. Ed. Engl. 2012; 51: 8529-8533Crossref PubMed Scopus (650) Google Scholar Fifty-six amino lipids were synthesized and formulated in LNPs and the ED50 measured and plotted against their pKa. For 15 lipids the ED50 dose was not achieved, these are indicated by the open diamonds representing the highest dose tested for that lipid. For the remaining lipids (closed diamonds), a polynomial best-fit curve highlights the most active compounds, which exhibit an optimal pKa between 6.2 and 6.6. Each data point is derived from a four-dose response curve with groups of n = 4 mice per dose. Reproduced with permission from Jayaraman et al.3Jayaraman M. Ansell S.M. Mui B.L. Tam Y.K. Chen J. Du X. Butler D. Eltepu L. Matsuda S. Narayanannair J.K. et al.Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.Angew. Chem. Int. Ed. Engl. 2012; 51: 8529-8533Crossref PubMed Scopus (650) Google Scholar The optimization process for ionizable cationic lipids has been guided by considerations concerning the potential role of non-bilayer lipid structures in facilitating intracellular delivery of macromolecules such as siRNA. Nearly 40 years ago, we showed that membrane lipid species such as unsaturated phosphatidlyethanolamines (PEs) adopted non-bilayer structures such as the hexagonal HII phase in aqueous solution20Cullis P.R. de Kruijff B. The polymorphic phase behaviour of phosphatidylethanolamines of natural and synthetic origin. A 31P NMR study.Biochim. Biophys. Acta. 1978; 513: 31-42Crossref PubMed Scopus (371) Google Scholar and that so-called fusogens that induce inter-cellular fusion also induce HII structure in biological membranes21Cullis P.R. Hope M.J. Effects of fusogenic agent on membrane structure of erythrocyte ghosts and the mechanism of membrane fusion.Nature. 1978; 271: 672-674Crossref PubMed Scopus (233) Google Scholar, leading to the proposal that membrane fusion proceeds via non-lamellar intermediates. Some 20 years later, we made the related observation that permanently positively charged cationic lipids used as transfection reagents induce the HII phase in dispersions with naturally occurring anionic lipids.22Hafez I.M. Maurer N. Cullis P.R. On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids.Gene Ther. 2001; 8: 1188-1196Crossref PubMed Scopus (434) Google Scholar This behavior is rationalized by the "shape" hypothesis depicted in Figure 5 and led to the proposal that transfection mediated by cationic lipids results from the association of exogenous cationic lipids with endogenous anionic lipids to form membrane disruptive non-lamellar structures, allowing associated DNA to access the cell interior.16Semple S.C. Akinc A. Chen J. Sandhu A.P. Mui B.L. Cho C.K. Sah D.W. Stebbing D. Crosley E.J. Yaworski E. et al.Rational design of cationic lipids for siRNA delivery.Nat. Biotechnol. 2010; 28: 172-176Crossref PubMed Scopus (1151) Google Scholar There is considerable evidence that LNP siRNA systems containing ionizable cationic lipids are accumulated into target hepatocyte cells in vivo via endocytosis.23Akinc A. Querbes W. De S. Qin J. Frank-Kamenetsky M. Jayaprakash K.N. Jayaraman M. Rajeev K.G. Cantley W.L. Dorkin J.R. et al.Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms.Mol. Ther. 2010; 18: 1357-1364Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar Thus, the rationale for optimization of ionizable cationic lipids in LNP siRNA systems was that the pKa had to be sufficiently high so that, at endosomal pH values, a large proportion was protonated (positively charged) in order to combine with endogenous anionic lipid to induce endosome-disrupting non-bilayer structures, but also sufficiently low so that the LNP surface charge did not result in clearance by the immune system prior to accumulation by target cells. The need for polyunsaturated acyl chains could also be rationalized by this non-bilayer hypothesis as more unsaturated acyl chains assume non-bilayer structure more readily.5Cullis P.R. de Kruijff B. Lipid polymorphism and the functional roles of lipids in biological membranes.Biochim. Biophys. Acta. 1979; 559: 399-420Crossref PubMed Scopus (1519) Google Scholar Escape of genetic material resulting from non-bilayer disruption of endosomes is depicted in Figure 6. The non-bilayer hypothesis proved a productive rationale for improving the potency of the cationic lipids, however, the relative specificity of these LNP siRNA systems for hepatocytes suggested a targeting mechanism is also operating. It is here that a benefit of conducting LNP optimization using an in vivo gene-silencing model became apparent, it was found that the LNP systems containing ionizable cationic lipids take advantage of a "natural" targeting process that would have been difficult to predict a priori. Specifically, the ability of these systems to adsorb apolipoprotein E (ApoE) is vital to hepatocyte gene-silencing potency. LNP siRNA systems giving rise to essentially complete hepatocyte gene silencing in wild-type mice result in little or no gene silencing in ApoE knockout mice23Akinc A. Querbes W. De S. Qin J. Frank-Kamenetsky M. Jayaprakash K.N. Jayaraman M. Rajeev K.G. Cantley W.L. Dorkin J.R. et al.Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms.Mol. Ther. 2010; 18: 1357-1364Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar, however, potency can be restored by pre-incubation with ApoE. These results suggest that LNP siRNA systems containing ionizable cationic lipids are processed much like chylomicrons, which adsorb endogenous ApoE to trigger uptake into hepatocytes via several receptors that contain ApoE binding ligands.24Williams K.J. Chen K. Recent insights into factors affecting remnant lipoprotein uptake.Curr. Opin. Lipidol. 2010; 21: 218-228Crossref PubMed Scopus (73) Google Scholar The presence and relative amounts of LNP components other than cationic lipids also determine the potency of LNP siRNA systems. For example, while relatively complete encapsulation of siRNA can be achieved at N/P values as low as 1, the resulting systems are not effective gene-silencing agents.15Belliveau N.M. Huft J. Lin P.J. Chen S. Leung A.K. Leaver T.J. Wild A.W. Lee J.B. Taylor R.J. Tam Y.K. et al.Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA.Mol. Ther. Nucleic Acids. 2012; 1: e37Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar Optimized LNP systems exhibit N/P values in the range of 63Jayaraman M. Ansell S.M. Mui B.L. Tam Y.K. Chen J. Du X. Butler D. Eltepu L. Matsuda S. Narayanannair J.K. et al.Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo.Angew. Chem. Int. Ed. Engl. 2012; 51: 8529-8533Crossref PubMed Scopus (650) Google Scholar, 15Belliveau N.M. Huft J. Lin P.J. Chen S. Leung A.K. Leaver T.J. Wild A.W. Lee J.B. Taylor R.J. Tam Y.K. et al.Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA.Mol. Ther. Nucleic Acids. 2012; 1: e37Abstract Full Text Full Text PDF PubMed Scopus (355) Google Scholar, 16Semple S.C. Akinc A. Chen J. Sandhu A.P. Mui B.L. Cho C.K. Sah D.W. Stebbing D. Crosley E.J. Yaworski E. et al.Rational design of cationic lipids for siRNA delivery.Nat. Biotechnol. 2010; 28: 172-176Crossref PubMed Scopus (1151) Google Scholar, indicating a need for non-siRNA-associated cationic lipids to achieve endosomal escape. In addition, the type and amount of PEG-lipid employed determines the size15Belliveau N.M. Huft J. Lin P.J. Chen S. Leung A.K. Leaver T.J. Wild A.W. Lee J.B. Taylo