Lipid Nanoparticle Systems for Enabling Gene Therapies

Abstract

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 of at at is a relatively to avoid of the to rapid accumulation by the and free in the Cullis P.R. of with large unilamellar liposomes in to circulation Chem. Google Scholar and penetration to target tissue. Application of LNP technology to genetic drugs the development of to efficient encapsulation of RNA and DNA polymers into LNP used for loading small molecule drugs into LUVs in to 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 because of size and The of M. R. M. M. a highly PubMed Scopus Google Scholar that have with RNA and DNA polymers a as lipids with a with polymers to these systems have proven useful for in in is to large size and H. S. B. S. J. of lipids and polymers in gene Control. Release. PubMed Scopus Google Scholar The of lipids, such as Cullis P.R. of membrane by of PubMed Scopus Google Scholar with development of an has to loading for genetic drugs in small (<100 nm diameter) LNP systems with many for lipids such as have an and can be used to efficient encapsulation of polymers into LNP at the lipids are Subsequently, the can be to leading to LNPs with a relatively The first of the loading LNPs containing and lipids with in the of by Ansell S.M. H. Cullis P.R. Hope M.J. encapsulation of in lipid vesicles of small Biophys. Acta. PubMed Scopus Google Scholar Subsequently, a S. for efficient liposomal encapsulation of plasmid PubMed Scopus Google Scholar lipids lipids and in rapidly with in an aqueous in efficient loading of polymers into small LNP This by a to a more rapid and J. Chen S. Tam Y.K. et of highly lipid nanoparticles for in delivery of 2012; PubMed Scopus Google Scholar and the of of formulation rapid is in which an of encapsulation can be the loading The most of the loading to such as siRNA in LNP systems of an containing and lipid that is rapidly with an aqueous containing the at an of Chen J. Mui B.L. D. et design of lipids for siRNA delivery.Nat. PubMed Scopus Google Scholar of aqueous to is the the LNP is first a to and a to the to The LNP systems from the rapid loading for an in delivery system for genetic drugs in The formulation is and encapsulation are the LNP size can be by the J. Chen S. Tam Y.K. et of highly lipid nanoparticles for in delivery of 2012; PubMed Scopus Google Scholar the systems and the LNP as for in administration. The of the LNP systems for siRNA as by S. Hope M.J. Cullis P.R. Lipid nanoparticles containing siRNA by an Chem. 2012; PubMed Scopus Google Scholar a of of lipid by a of as in This is by the by and the for of LNP systems containing in The for LNP mRNA is to be however, it be that the loading is a Tam Chen S. Cullis P.R. a for in lipid nanoparticle Chem. B. PubMed Scopus Google Scholar leading to efficient encapsulation of mRNA and plasmid DNA as well as such as the nanoparticles of The first that LNP of genetic drugs containing lipids significant in for LNP systems containing siRNA to genes in hepatocytes following i.v. B. D. J. M. et gene silencing in PubMed Scopus Google Scholar This which the lipid an of containing by the that the of these LNP systems highly sensitive to the particular of lipid Chen J. Mui B.L. D. et design of lipids for siRNA delivery.Nat. PubMed Scopus Google Scholar This led to an lipid to lipids with in when into LNP siRNA in the of to which is currently the lipid for silencing 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 with by over of from that for LNP containing 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 in lipid that can potency relatively The dominant the lipid potency the of the of and most the of the of the LNP siRNA systems containing lipids with exhibiting a and are by the most 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 gene silencing in the a of the to gene silencing in hepatocytes in versus the of the lipids in the 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 lipids and in LNPs and the and lipids the not these are by the the for that the lipids a the most which an and is from a with of with from et 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 The a of the to gene silencing in hepatocytes in versus the of the lipids in the 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 lipids and in LNPs and the and lipids the not these are by the the for that the lipids a the most which an and is from a with of with from et 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 for lipids has been by the potential of lipid structures in delivery of such as years we that membrane lipid such as structures such as the phase in aqueous P.R. de Kruijff B. The phase of of and Biophys. Acta. PubMed Scopus Google Scholar and that so-called that in biological P.R. Hope M.J. of on membrane of and the of membrane PubMed Scopus Google leading to the that membrane years we made the that lipids used as the phase in with Cullis P.R. the lipids delivery of 8: PubMed Scopus Google Scholar This is by the in and led to the that by lipids from the of lipids with lipids to membrane DNA to the Chen J. Mui B.L. D. et design of lipids for siRNA delivery.Nat. PubMed Scopus Google Scholar There is that LNP siRNA systems containing lipids are into target cells in S. J. M. M. et delivery of therapeutics with and PubMed Scopus Google Scholar the for optimization of lipids in LNP siRNA systems that the to be at a large in order to with lipid to that the LNP not in by the immune system to accumulation by target The for be by as more more 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 of genetic from of is in The a for the potency of the lipids, however, the of these LNP siRNA systems for hepatocytes a is It is that a of LNP optimization an in it that the LNP systems containing lipids of a that have been to a the ability of these systems to is to LNP siRNA systems to gene silencing in in or gene silencing in S. J. M. M. et delivery of therapeutics with and PubMed Scopus Google however, potency can be by with These that LNP siRNA systems containing lipids are which to into hepatocytes that contain Chen into PubMed Scopus Google Scholar The and of LNP than lipids the potency of LNP siRNA relatively encapsulation of siRNA can be at as as the systems are not J. Chen S. Tam Y.K. et of highly lipid nanoparticles for in delivery of 2012; PubMed Scopus Google Scholar LNP systems in the range of 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 J. Chen S. Tam Y.K. et of highly lipid nanoparticles for in delivery of 2012; PubMed Scopus Google Chen J. Mui B.L. D. et design of lipids for siRNA delivery.Nat. PubMed Scopus Google a for lipids to In the and of the J. Chen S. Tam Y.K. et of highly lipid nanoparticles for in delivery of 2012; PubMed Scopus Google Scholar and the potency of LNP siRNA B.L. Tam Y.K. M. Ansell S.M. Du X. Tam Chen S. Narayanannair J.K. et of lipid on and of siRNA lipid 2013; PubMed Scopus Google S. Tam Tam Y.K. Cullis P.R. of size on the in potency of lipid nanoparticle of Control. Release. PubMed Scopus Google Scholar of lead to LNP J. Chen S. Tam Y.K. et of highly lipid nanoparticles for in delivery of 2012; PubMed Scopus Google Scholar more the of a B.L. Tam Y.K. M. Ansell S.M. Du X. Tam Chen S. Narayanannair J.K. et of lipid on and of siRNA lipid 2013; PubMed Scopus Google S. Tam Tam Y.K. Cullis P.R. of size on the in potency of lipid nanoparticle of Control. Release. PubMed Scopus Google Scholar This from a reduced potential to with cells a reduced ability to The use of a lipid containing that from LNP in with a in B.L. Tam Y.K. M. Ansell S.M. Du X. Tam Chen S. Narayanannair J.K. et of lipid on and of siRNA lipid 2013; PubMed Scopus Google Scholar The combination of the lipid and with the rapidly has led to LNP systems that can gene in hepatocytes at as as in 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 potency and for in in the clinical development of an LNP siRNA drug to treat amyloidosis, a that in the There is currently for that to and and is The LNP drug is now in phase III clinical D. J. J. S. et and of for Engl. J. 2013; PubMed Scopus Google Scholar and is to the first non-viral gene drug to be approved by the Food and Drug Administration There are many potential clinical applications of LNP siRNA drugs for silencing genes in however, of can in tissues such as the in not the to the the and to the to they are up to H. and of in the Biophys. Acta. PubMed Scopus Google Scholar It has been that LNP siRNA systems to for gene silencing are for D. J. M. Cullis P.R. Lipid nanoparticle delivery of siRNA to gene in the 2013; PubMed Scopus Google Scholar when The of these systems for target applications has been by the of a protein with following L. S.M. Cullis P.R. The of PubMed Scopus Google Scholar The potential for gene silencing in the by LNP siRNA systems following or administration for the of tissues for which gene silencing following i.v. administration of LNP siRNA systems have been include G. Tam J. B. et of lipid on gene silencing of lipid nanoparticle of siRNA in PubMed Scopus Google Scholar and in G. M. H. Cullis P.R. Lipid nanoparticle delivery of siRNA to to silencing of and of in PubMed Scopus Google Scholar and tumor Tam Tam Y.K. Cullis P.R. Lipid nanoparticle siRNA systems for silencing the receptor in cancer in J. 2012; PubMed Scopus Google Scholar The for immune cells is that they accumulate LNP systems in the and tumor tissues may be to preferentially accumulate LNP to EPR for the to gene silencing in and as well as tumor cells are at of than for gene leading to therapeutic that do not clinical It is that significant in LNP siRNA potency will be doses can be As Tam Chen S. Cullis P.R. a for in lipid nanoparticle Chem. B. 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Mui Tam Y.K. et of mRNA from PubMed Scopus Google Scholar The ability to use the as a in up new therapeutic to enabling the rapid of from the provide a to rapid following to infections such as or In to the the are of in that is in is the potential of LNP mRNA systems in for The or administration of LNP systems containing mRNA for protein with diseases are immune in in from in and as for M.J. H. H. K.A. R. et by a single mRNA PubMed Scopus Google Scholar Given the ability of the LNP delivery to enable mRNA protein in in tissue, it will be to applications in the of gene the gene-editing rely vectors to into of vectors is the of that a problem that has not been for LNP systems D. J. J. S. et and of for Engl. J. 2013; PubMed Scopus Google Scholar As with the of in we of in the gene-editing will be in the LNPs containing plasmid DNA have in The ability of the loading in combination with lipids, to efficient encapsulation of plasmids as large as has been Chen S. Tam Cullis P.R. of lipid nanoparticles for in and in delivery of plasmid Scopus Google Scholar Further, these systems can be highly for a of cells in tissue of these systems into has in robust of a protein in tissues with therapeutic applications are LNP systems have the potential to enable the of gene therapy. significant the immune to LNP of RNA and DNA polymers and the for LNP systems with that siRNA, mRNA, and therapies can be to It is that the flexibility of LNP technology to the the dominant of immune to LNP containing and genetic drugs are the of to that up the cells from A.G. Unterholzner L. Viral evasion and subversion of pattern-recognition receptor signalling.Nat. Rev. Immunol. 2008; 8: 911-922Crossref PubMed Scopus (520) Google immune can be reduced by D. the of for therapeutic RNA Drug Discov. Google Scholar however, it is to the potential for significant immune in J. a in by and Immunol. PubMed Scopus Google Scholar As a the clinical administration of LNP siRNA, for is by with such as J. a in by and Immunol. PubMed Scopus Google Scholar This the clinical development of LNP of genetic drugs. the ability of LNP systems to as delivery systems can be used to has for that of as as to of a of into LNP systems can the to in Chen and can be used to the potency of LNP systems containing genetic drugs in tissues than the The potential for is clear in that of LNP siRNA is into the J. G. S. M. et of lipid siRNA and 2013; PubMed Scopus Google Scholar to is to the G. S. Chen D. R. et of siRNA delivery by lipid nanoparticles is by 2013; PubMed Scopus Google Scholar and of the LNP siRNA to such as in of the or to can the of siRNA and genetic drugs to into the it has been that of the protein by the small molecule in LNP siRNA in cells in leading to H. Tam Chen S. J. R. Cullis P.R. The gene silencing potency of lipid nanoparticles containing PubMed Scopus Google Scholar of of and of leading to may be be to improve potency It is that the of following first small molecule drugs and will be to of biological and to by highly LNP systems containing genetic drugs are to a in remarkable is that the development of LNP systems capable of the size of siRNA in is leading to LNP systems that can larger mRNA and plasmid DNA many of gene therapies to be It is that these advances are by of such as in hepatocytes or the of nanoparticles to immune than the of Further, we that LNP systems contain a of in to the genetic drug that a to potency and and to and and to the many and have in and has in The to over many years with based at and the of for has been through the for and the for the has been by the and and