key: cord-1053548-0oh40b6r
authors: Witzigmann, Dominik; Kulkarni, Jayesh A.; Leung, Jerry; Chen, Sam; Cullis, Pieter R.; van der Meel, Roy
title: Lipid nanoparticle technology for therapeutic gene regulation in the liver
date: 2020-07-02
journal: Adv Drug Deliv Rev
DOI: 10.1016/j.addr.2020.06.026
sha: c4c60433230b002fa5ee0ab481e210a5e3c37748
doc_id: 1053548
cord_uid: 0oh40b6r

Hereditary genetic disorders, cancer, and infectious diseases of the liver affect millions of people around the globe and are a major public health burden. Most contemporary treatments offer limited relief as they generally aim to alleviate disease symptoms. Targeting the root cause of diseases originating in the liver by regulating malfunctioning genes with nucleic acid-based drugs s holds great promise as a therapeutic approach. However, employing nucleic acid therapeutics in vivo is challenging due to their unfavorable characteristics. Lipid nanoparticle (LNP) delivery technology is a revolutionary development that has enabled clinical translation of gene (editing) therapies. LNPs can deliver siRNA, mRNA, DNA, or gene-editing complexes, providing opportunities to treat hepatic diseases by silencing pathogenic genes, expressing therapeutic proteins, or correcting genetic defects. Here we discuss the state-of-the-art LNP technology for hepatic gene therapy including formulation design parameters, production methods, preclinical development and clinical translation.

"Survival rates have improved for almost every disease of every organ in the last few decades, with one notable exception: liver disease" [1] . This statement by The Lancet Commission clearly illustrates the global burden of liver disorders and the need for more effective therapeutic strategies [2] . The most frequently occurring liver diseases include hepatitis, liver cancer, alcoholic liver disease, fatty liver disease, and hereditary diseases. In addition to direct harmful effects, these diseases can significantly affect the liver's carbohydrate, fat, and protein metabolism. The increase in lifestyle-related incidence rates and the limited therapeutic efficacy of currently available treatments have resulted in substantial drug development efforts targeting the liver [2] . Our ability to treat (hepatic) diseases by targeting their genetic background is increasingly becoming a clinical reality owing to the development of nucleic acidbased therapeutics. In contrast to small molecule drugs and biologics which target gene products (i.e. proteins), nucleic acid therapeutics have the potential to therapeutically regulate essentially any gene of interest at the DNA or RNA level. Their versatility in treating inherited or acquired disorders originating in the liver stems from the ability to induce efficient gene silencing (inhibiting pathological/mutant protein production), gene expression (producing therapeutic proteins) or gene editing (correcting dysfunctional/mutated genes). Several nucleic acid therapeutics have been approved by the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) with many more in various stages of clinical evaluation. These therapeutics include antisense oligonucleotides (ASO) [3] , small interfering RNA (siRNA) [4, 5] , plasmid DNA (pDNA) [6, 7] , messenger RNA (mRNA) [8, 9] , and complexes containing guide RNA (gRNA) as part of gene editing approaches [10, 11] .

Using nucleic acids therapeutically in vivo is challenging because of their unfavorable physicochemical characteristics, such as negative charge and relatively large size, which prevents their efficient uptake into cells [12] . In addition, nucleic acids are susceptible to degradation by nucleases in the circulation, suffer from rapid renal clearance, and induce immunostimulatory effects via pattern recognition receptors, resulting in adverse effects [13] . Therefore, the clinical translation of nucleic acid therapeutics has been dependent on chemical modifications and advanced (nanocarrier) delivery technologies to improve nucleic acids' stability, promote their target tissue accumulation, enable their Kupffer cells > hepatocytes. Higher LNP doses corresponding to 1 mg/kg mRNA shifted expression slightly towards hepatocytes while keeping the same pattern. Transfection of all major liver cell types with equal potency was recently demonstrated for LNP-mRNA systems (composed of branched-tail 306O i10 ) at a dose of 2 mg RNA/kg by Hajj et al. [51] .

Indeed, targeting the right cell type with the right dose is crucial to developing effective therapeutics. It should be noted that the LNP compositions described in these preclinical studies deviate from those used in the clinic (except for MC3-based LNPs). Systematic studies are therefore needed to improve our fundamental understanding of LNPs' in vivo behavior. Rigorous control of physicochemical LNP characteristics such as size distribution, zeta potential, and entrapment will be crucial to assess the intrahepatic distribution of a single LNP composition with different payloads.

Following intravenous injection, liver-resident macrophages, i.e. Kupffer cells, are the first hepatic cells to interact with LNPs (Figure 1) . These phagocytic cells are part of the mononuclear phagocyte system (MPS), also known as the reticuloendothelial system (RES). They comprise 80% of the entire macrophage population within the body, illustrating their importance in host defense and LNP elimination [52] . [54] . (C) Kupffer cell (KC) located within the hepatic sinusoid in close proximity to endothelial cells. Adapted with permission from UCSF Office of Medical Education [55] .

Three major elimination pathways have been described [56] . First, negatively charged LNP systems are recognized by class A scavenger receptors (SR-A) expressed primarily on Kupffer cells resulting in rapid clearance [41, 57, 58] . Second, mannose-and fucose-type receptors can be leveraged to selectively target LNP systems to Kupffer cells. Third, LNP opsonization by serum proteins results in MPS sequestration. Complement factors (e.g. C3b or C1q) and serum opsonins such as fibrinogen can coat LNPs with unfavourable characteristics including large size, high surface charge, or lack of PEGylation J o u r n a l P r e -p r o o f [59] [60] [61] . Several research groups have explored strategies to prevent Kupffer cell clearance in order to redirect LNPs to hepatocytes. Transient Kupffer cell depletion using clodronate-loaded liposomes or by knocking out the endocytic Caveolin1 gene are efficient methods in a research setting [41, 43] . However, the clinical utility of such approaches is limited.

LSECs are located in close proximity to Kupffer cells and play important roles in sequestering LNPs and restricting access to hepatocytes [62] . Many structural and functional features have been elucidated by Braet and Wisse [54, [63] [64] [65] [66] [67] . LSECs line the hepatic sinusoids and form pores, so-called fenestrations, that are clustered in sieve plates (Figure 2) . Endothelial fenestrae range from 50 to 200 nm in diameter and differ between species (Table 1) . Therefore, liver fenestrae physically restrict circulating LNPs' access to the perisinusoidal space and thus limit cellular interactions with hepatocytes according to size.

Several research groups have investigated using pore-opening substances to modulate fenestrae size with limited success [64] . In addition to their structural characteristics, LSECs have high endocytic activity.

A number of scavenger receptors, including stabilin-2, can efficiently sequester anionic nanoparticles [68] . 

Hepatocytes, comprising 70-80% of the total liver cell population, are the most relevant hepatic target cell type for nucleic acid therapeutics (Figure 1 ). Owing to their broad range of functions, hepatocytes play a key pathogenic role in many disorders ( Table 2) . Hepatocytes are highly differentiated with a sinusoidal (basolateral) membrane towards the blood circulation and an apical membrane towards bile canaliculi.

The sinusoidal membrane with its microvilli exhibits surface receptors important for LNP recognition. The most important receptors for LNP-nucleic acid are the low-density lipoprotein receptor (LDLR) and asialoglycoprotein receptor (ASGPR) [76] . Within a healthy liver, hepatocytes are postmitotic (i.e. nondividing cells) with an average life span of up to 6 months. 

Many factors can alter LNP accumulation and clearance. The following sections detail important (patho)physiological factors affecting intrahepatic LNP distribution.

An often-overlooked challenge in hepatic gene therapy is metabolic and cellular liver zonation, a phenomenon that separates various pathways along the porto-central axis of a liver lobule. First, some genetic disorders manifest in periportal or perivenous hepatocytes [110, 111] . Second, metabolic zonation can vary among species and during development (infant versus adult). Third, different non-parenchymal cell subtypes within the liver microenvironment can affect LNP clearance [45] . All these factors impact LNP development and gene therapy outcomes. Figure 1 details the liver microarchitecture and its major metabolic pathways. Metabolic liver zonation for glucose homeostasis, urea synthesis, carbohydrates, bile acids, or lipid metabolism has been discussed in several excellent reviews [110] [111] [112] [113] . Advancements in omics and single-cell techniques are continuously elucidating new cell subtypes and improve our understanding of liver zonation [45, 110] [118] . These results demonstrate that whole-tissue (entire liver) analysis should be replaced by dissociated single cell-based techniques considering the metabolic liver zonation.

Liver disease progression results in pathological remodelling including microanatomical or target receptor alterations that could affect nanoparticle delivery and sequestration. Firstly, liver infections or metabolic disorders can lead to chronic cell damage and cell activation. This can result in liver fenestrae rearrangement or fibrotic material deposition by activated stellate cells within the perisinusoidal space.

Thus, LNP transport to hepatocytes is inhibited, as is access to the key target cell for most gene therapies [119] . Hepatic inflammatory processes can also enhance hepatic nanoparticle sequestration by Kupffer cell activation [116] . Secondly, downregulation of surface receptors crucial for LNP binding decreases gene delivery efficiency. For example, two independent studies have demonstrated lower ASGPR expression with increasing stage of HCC (according to the Barcelona Clinic Liver Cancer staging) [120, 121] . This has serious implications for liver cancer interventions using ASGPR-targeting approaches.

Thirdly, variations in serum proteins, such as apolipoprotein E (ApoE), are known to mediate specific LNP binding and might affect efficacy. A recent study investigated the effect of ApoE polymorphisms in therapeutic outcomes. Diagnostic tools to stratify patients for LNP-based gene therapy therefore offer interesting possibilities [123] .

The fundamental LNP design parameters for nucleic acid delivery are based on those established for small molecule liposomal formulations. These parameters include appropriate particle size (for efficient terminal sterile filtration and hepatic delivery), long-term stability in storage, optimized payload release rates to produce a therapeutic effect, robust and scalable manufacturing processes, and efficient entrapment. In applying these requisites to nucleic acid delivery systems, it became obvious that additional lipid components and functionalities were required beyond those used to compose smallmolecule carriers. The very first nucleic acid formulations, containing only phosphatidylcholine and cholesterol, demonstrated that nucleic acid entrapment within a particle was feasible, but the entrapment efficiency was poor [124, 125] . Subsequent development of the cationic lipid 1,2-dioleoyl-3trimethylammonium-propane (DOTAP) showed that ionic interactions between the lipids and payload can dramatically increase entrapment efficiencies and intracellular delivery. Toxicity issues, resulting from the cationic lipids' permanent positive charge and non-biodegradable nature, plagued these initial lipoplexlike formulations [126] . Through additional formulation development, and manufacturing process optimization, it was determined that LNP systems required four components: ionizable cationic lipids, phospholipids (typically phosphatidylcholine), cholesterol, and PEG-lipids. The role of each component, the evolution of the composition, and the manufacturing processes are discussed in the following sections.

To date, a vast number of ionizable cationic lipids covering a wide range of structures have been developed (Figure 3 ), yet they all share a few aspects: (1) The headgroups contain tertiary amines that become protonated under acidic pH and typically are uncharged (or zwitterionic) at neutral pH; (2) The lipid tails contribute to making the molecule sufficiently hydrophobic to promote incorporation into a nanoparticle during formation; and (3) The protonated lipids generate structures that help elevate propensity for membrane fusion in acidified endosomes following internalization by the target cell. In addition to these similarities, the various lipids' performed functions are essentially identical. As the pH of the environment dictates the headgroup protonation, LNP are prepared in an acidic aqueous buffer (e.g. pH 4) that promotes the charge interaction between the ionizable cationic lipid and the anionic nucleic acid. Subsequent buffer exchange into isotonic and pH-neutral buffer generates the final LNP suspension with a near net-neutral surface charge. This uncharged state is critical to preventing immune responses upon intravenous administration and facilitates delivery to hepatocytes [126] . The next function is to maintain a positive charge in the acidified endosome and promote membrane fusion to allow cytosolic delivery of the nucleic acid. This fine balance of positive charge at acidic pH and neutral charge at J o u r n a l P r e -p r o o f physiological pH is the result of substantial efforts towards optimizing the ionizable lipid for in vivo nucleic acid delivery.

One of the first tested ionizable lipids, known as dioleyl-dimethylaminopropane (DODMA), contained oleyl lipid tails (C18:1) conjugated to the dimethylamino-propyl headgroup through ether linkers. Using the molecular shape hypothesis as a guiding principle [127] , the three components of these lipids (headgroup, linker, and tails) were systematically studied to determine optimal characteristics for each.

The molecular shape hypothesis describes the macrostructure obtained upon hydration of a lipid with specific geometries. More specifically, lipids containing tails with larger cross-sectional areas than the lipid headgroups result in H II phases or inverted micelles; comparatively, when the cross-sectional area of the tails is similar to that of the head group (resulting in a cylindrical geometry), the lipids tend to from bilayers. Comparing different lipid tail-unsaturation suggested that the linoleyl chains (DLinDMA) provide optimal particle internalization and potential to generate membrane-destabilizing H II phases [128] .

DLinDMA with ester bonds resulted in a lipid, DLin-DAP, with substantially reduced potency [30] . Further studies suggested that degrading the ester bond within the acidified endosome contributed to efficacy loss [129] . Simultaneously, a series of headgroup modified lipids were tested, and DLin-KC2-DMA was designed with vastly higher potency than DLinDAP and DLinDMA (Figure 3 ) [30] . Further modifications and screening led to the development of DLin-MC3-DMA (Figure 3 ) [31] , used in the clinical formulation, Onpattro ® , and now considered the gold-standard for ionizable cationic lipids.

Although several screening methods for ionizable lipids have been devised, the critical potency test for hepatic targets was the in vivo model for hepatic gene knocking down; the Factor VII (FVII) model provided a modestly high-throughput approach [29] . FVII is a serum protein produced by hepatocytes in the liver and secreted into the blood circulation. Its short half-life enables gene silencing assessment on the protein level within a short timeframe. It is important to stress that FVII-knockdown screens specifically identify LNPs that target hepatocytes and ignore all other hepatic cell types. LNP containing siRNA against murine FVII were intravenously administered over a dose range of 0.001-10 mg siRNA per kg body weight and circulating FVII levels were determined by ELISA 24 hours later. The metric used to compare formulations was the effective dose required to achieve 50% gene silencing (ED 50 ), and DLin-MC3-DMA (MC3) was determined to be the most potent ionizable cationic lipid for LNP-based gene silencing. The potency improvements cover the range of DLinDAP with an ED 50 of ~20 mg/kg, while that for MC3 was 0.005 mg/kg in mice [30, 31] .

Further developments focused on lipid biodegradability to reduce potential toxicity, immunogenicity, and other adverse effects [130] . The design parameters for these lipids included high in vivo transfection efficiency, increased ability to be metabolized, and no generation or accumulation of toxic metabolites.

One approach incorporates an ester linkage, which can be easily hydrolyzed by intracellular esterases or DLinDMA [133] , DLin-KC2-DMA [30] , and DLin-MC3-DMA [31] ; (ii) lipidoids like cKK-E12 [134] and C12-200 [29] ; and (iii) next-generation lipids including the biodegradable molecules L319 [130] , TT3 [135] , and ssPalmE [136] as well as lipids from proprietary libraries belonging to Acuitas (A9) [137] and Moderna (L5) [138] .

Two LNP componentsphospholipids and cholesterolhave generally been seen to promote formulation stability [139] . Although that evidence is largely anecdotal in the LNP context, phospholipids such as DSPC, with strong bilayer-forming properties and high phase transition temperatures, help increasing membrane rigidity and reducing membrane permeability. While the role of cholesterol remains J o u r n a l P r e -p r o o f largely unclear in the context of nucleic acid delivery systems, cholesterol-deficient particles can sequester cholesterol while in circulation, leading to potentially destabilizing effects [140] . This sequestration process is largely driven by the exchange of cholesterol away from the plasma membrane of peripheral tissues into lipoproteins in circulation followed by equilibration into circulating liposomes.

Recently, Harashima and colleagues studied cholesterol-free LNP-siRNA systems (only composed of the ionizable cationic lipid CL15H6, phospholipid, and PEG-lipid) and they observed decreased potencies in the presence of serum likely due to particle instability as a result of cholesterol accumulation [141] .

Two studies suggested that the cholesterol amount typically formulated into an LNP is larger than what can be stably retained in LNPs. More recently, it was determined that ~30-40 mol% helper lipid is required to efficiently entrap siRNA within LNPs, providing additional insight into the role of these helper lipids [27] .

The helper lipids serve as a mechanism for spacing out ionizable lipids to achieve a membrane surface charge of approximately +1 per nm 2 (siRNA has a surface charge of approximately -1 per nm 2 ).

Limited information is available on the role of helper lipids for LNP activity. However, some evidence has suggested that the replacement of DSPC with DOPE in lipidoid-based LNPs improves mRNA delivery in vivo [142] . For LNP-pDNA formulations, certain unsaturated phosphatidylcholines (i.e., SOPC and DOPC) improved the LNP activity over DSPC in the presence of FBS in vitro [28] . DOPE-containing LNP-pDNA systems showed best activity in murine serum suggesting a potential role of helper lipids in modifying the LNP surface affinity to distinct apolipoprotein subtypes. An additional role of cholesterol in LNP systems was recently investigated [143] . Incorporating oxidized cholesterols such as 20α-OH redirected LNP-mRNA systems from hepatocytes to hepatic endothelial cells and Kupffer cells. Although the mechanism of modifying LNP tropism remained elusive, formation of different protein coronas and/or recognition by scavenger receptors expressed on hepatic RES (such as scavenger receptor class B type I as binding site for oxidized LDL) might have resulted in redirection of LNPs [144, 145] .

The final LNP component, the PEG-lipid, is engineered to perform two specific functions. First, PEG-lipids incorporate into the emerging nanoparticle during LNP formation. As LNP systems do not contain an aqueous core, PEG-lipids reside almost exclusively on the LNP surface, and their concentrations control particle size [146] . Both the PEG molecular weight as well as the molar percentage of PEG-lipid affect the characteristics of lipid-based particles [147] [148] [149] . Specifically, as the PEG-lipid is increased from 0.25 mol% to 5 mol%, a reduction in LNP size is observed from ~120 nm to 25 nm, but further increases to PEG-content do not modify particle size [147, 24] . Second, they improve the shelf-stability by creating a steric barrier that extends away from the surface of the LNP, thereby preventing particle aggregation and improving in vivo circulation lifetimes. However, for transfection purposes, PEG-lipids have an established inhibitory effect [150, 151] . Based on the hypothesized mechanism of LNP function, the nanoparticle requires an intricate balance between stability in storage and circulation, and instability within the cell to support intracellular delivery [152] . Diffusible PEG-lipids helped stabilize particles while enabling J o u r n a l P r e -p r o o f Journal Pre-proof intracellular delivery [25, 151] . These lipids are composed of acyl chains that are 14-carbons in length and can dissociate rapidly from the LNP in the circulation [153] . Two hours post administration, only 20% of the injected PEG-lipid is associated with the LNP. In contrast, PEG-lipids with 18-carbon acyl chains, incorporate into the LNP and do not dissociate from the particle in the circulation. At high concentrations, these PEG-lipids can contribute to extending circulation half-life (from < 30 minutes for diffusible PEGlipid to > 2 hours) [23, 153] . However, LNPs designed to target hepatic disorders do not require a prolonged circulation lifetime due to the liver's natural ability to sequester nanoparticles. Therefore, diffusible PEG-lipids are ideal for such applications.

LNP production methods have evolved over time with certain processes gaining prominence. Rapidmixing methods have gained favor for their decreased labour requirements as they combine nanoparticle formation and nucleic acid entrapment into a single step [154] , and provide more homogenous nanoparticles. The first report of rapid-mixing was by Batzri and Korn, where an ethanolic lipid solution was rapidly injected into an aqueous solution to form liposomes [155] . Applying this method to nucleic acids involved combining pre-formed cationic liposomes with nucleic acids to produce lipoplexes [156] .

More recently, a T-junction mixing chamber was used for two separate mixing steps [154] . The first mixing step brought together an ethanolic lipid stream with an acidic aqueous buffer containing nucleic acid at an equal flow rate (1:1 v/v mixing). This created metastable particles that were combined with aqueous buffer in a second mixing step (through the T-mixer) to dilute the ethanol content and stabilize the nanoparticles. To simplify this process into a single step, the mixing ratio was modified to 1 part ethanol and 3 parts aqueous. These rapid-mixing methods produce homogenous nanoparticles with entrapment efficiencies > 90% and, importantly, have been proven to be fully scalable [33, 146, 157, 158] . A key advance during the development of Onpattro ® for hepatocyte gene silencing was identifying an optimized ionizable cationic lipid with an apparent pKa between 6.2 and 6.5 [17, 31] . Further increasing the pKa value to 7.15, resulted in improved gene silencing in LSECs [159, 160] . Incorporating ionizable cationic lipids exhibiting higher pKa values increased accumulation in the MPS, most likely due to scavenger receptor recognition [159] .

The lipid sensitivity to phospholipase is another important factor modulating intrahepatic LNP distribution and activity. Three different lipases have been described including the lipoprotein and endothelial lipase in LSECs and the hepatic lipase in hepatocytes [161] . LNP-siRNA systems that incorporate ionizable cationic lipids that are sensitive to endothelial lipase (e.g. ester linkages between head and tail functions) have enhanced gene silencing in hepatocytes but exhibit significantly reduced activity in LSECs [159] . Co-treatment with lipase inhibitors or incorporating lipase-resistant ionizable cationic lipids can recover gene silencing in LSECs [159] .

Based on micro-anatomical, subcellular, and (patho)physiological considerations, an ideal LNP for gene regulation in hepatocytes must satisfy the following design criteria: nanoparticle size < 80 nm to efficiently pass through liver fenestrae and improve LNP stability, apparent ionizable cationic lipid pKa value around 6.4, near neutral surface charge to prevent sequestration by the MPS, and lack of immune stimulation and toxic effects. Achieving these and other criteria facilitating efficient nucleic acid entrapment and LNP formulation are detailed in the following section.

It is important to mention that upon intravenous administration LNPs adsorb serum proteins on their surface. Many, if not all, of the abovementioned physiochemical characteristics impart distinct properties to the LNPs which ultimately influence protein adsorption. This "biomolecular corona" covering nanoparticles significantly impacts systemic circulation and nano-bio interactions [162] [163] [164] . Efficient targeting and gene regulation in hepatocytes stems from the presence of ApoE in the corona of LNPs and enabled the success of Onpattro ® [33, 76] . A recent publication suggested that the ionizable lipid composition plays a major role in the corona formed [165] . How the biomolecular corona can be leveraged to optimize targeting of different cell types within the liver microenvironment needs to be investigated.

Research in the late 1980s focusing on in vivo pDNA delivery showed that in the absence of a delivery system, naked nucleic acid injected into the circulation rapidly broke down and the products accumulated in hepatic tissue [166] . As interest towards ASOs and siRNA grew, LNP compositions and production methods simply translated from plasmids to these shorter nucleic acids [167] . More recently, formulations have become sufficiently potent to support mRNA therapeutics' discovery and translation [168] . Figure 4 illustrates the different LNP-based treatments for hepatic diseases by silencing pathogenic genes, J o u r n a l P r e -p r o o f expressing therapeutic proteins, or correcting genetic defects. Table 3 highlights preclinical LNP-based hepatic gene therapy approaches.

Refining lipid-DNA complexes to more advanced formulations required additional lipids, and such nanoparticles were termed stabilized plasmid lipid particles (SPLP) [169, 170] . The composition of these formulations largely drew from those used for small molecules therapeutics and included about 6-8 mol% The ability to transfect dividing (liver cancer) cells was most recently highlighted in a study where LNP systems optimized for pDNA were found to yield potent transfection [28] . While most DNA delivery applications have focused on gene therapy, a highly interesting application is employing DNA as barcodes for diagnostic and screening purposes [148] . Utilized as short fragments and each with a unique sequence, DNA barcodes allow for high-throughput, multiplexed in vivo screening to determine the biodistribution, uptake, and functional activity within the liver microenvironment (as outlined in section 2.2. and 3.5) [50, 143] . Notably, as a diagnostic tool, DNA barcodes have also been used for developing personalized cancer nanomedicines by co-loading them together with anticancer drugs into lipid nanocarriers. Utilizing this strategy, multiple anticancer medicines can be administered at sub-therapeutic doses and the most effective drug can subsequently be identified in the biopsies according to their barcode [175] . As only limited therapies are available for liver cancer, this methodology could well be used towards identifying effective and novel treatments for liver cancer. Although lipid calcium phosphate nanoparticles (LCPs) are beyond the scope of this review, in the context of liver cancer, it is relevant to note the work by Leaf Huang and colleagues demonstrating that LCP-based DNA delivery enables mitigation of liver metastasis [176] [177] [178] .

All procedures and compositions developed for DNA delivery readily translated into effective delivery systems for other nucleic acids [167] . siRNA only requires cytoplasmic delivery as all RNA-induced silencing complex (RISC)-related machinery is located in the cytosol. This quick translation resulted in demonstrating the first robust gene silencing in non-human primates (NHP) using nanoparticles known as stable nucleic-acid lipid particles (SNALPs) containing siRNA against apolipoprotein B (ApoB) [133] . Only twelve years later, Onpattro ® was approved by the FDA for treating ATTRv [179] .

In the early 2000s, the concept of modifying nucleic acids was largely applied to improving their cytoplasmic persistence to enable long term knockdown (decreased siRNA turnover). As such, modified siRNAs were entrapped into LNP systems with the rationale that a delivery system was specifically required to increase siRNA's liver accumulation and intracellular quantity. This had to be achieved in a manner where the cost of raw materials and processing was offset by a potent formulation, i.e. a drastic reduction in material requirement made the formulation commercially viable. With LNP formulations containing DLin-MC3-DMA, murine data suggested that as a little as 0.005 mg siRNA/kg body weight was required to achieve 50% gene silencing, with no observable toxicities. While alternative technologies such as siRNA-conjugates are also gaining prominence [4] , the applicability of LNP technology for hepatic targets is quite clear. It should be noted that siRNA-conjugates require substantially higher doses (~1 mg/kg, weekly subcutaneous administration) in order to achieve gene knockdown [180] . LNP-siRNA systems have shown utility in decreasing viral loads and virulence, various applications in hepatic oncology, and in metabolic liver disease treatment.

RNAi finds strong support in anti-viral applications where strict adherence to treatment regimens is critical to success. LNP-siRNA treatments can provide sustained knockdown for months leading to longterm viral gene suppression with a potential to eliminate certain viruses. One example is using LNP-siRNA as a therapeutic intervention for the Ebola outbreak in 2013, which resulted in almost 28,000 cases and 11,300 deaths [181] . LNP-siRNA formulations could be rapidly adapted to provide siRNA complementarity to the specific strain and showed that a combination of three siRNAs against the viral RNA synthesis genes suppressed the infection in non-human primates (NHP) [181] . Similarly, LNP-siRNA modification with GalNAc-conjugated PEG-lipids to specifically accumulate in hepatocytes (of chimeric mice with humanized livers) reduced Hepatitis B Virus (HBV) genomic DNA and antigens [182] . Other anti-viral LNP system examples include those for hepatitis delta virus (co-infected with HBV) and hepatitis C virus [183, 184] .

LNP-mediated siRNA delivery for hepatic oncology applications has largely focused on downregulating genes critical for cell cycle regulation, thereby inducing apoptosis. One example is LNP-siRNA against polo-like-kinase 1 (PLK1), which regulates multiple cell cycle progression stages. PLK1 is over expressed in multiple tumors including liver cancer and down-regulation has been shown to perform well as an intervention [185] . Similarly, simultaneous vascular endothelial growth factor (VEGF) and kinesin spindle protein (KSP) knockdown has been shown to inhibit proliferation in hepatocellular carcinoma and induce apoptosis [186] . Zhou et al. demonstrated that delivery of the small RNA let-7g J o u r n a l P r e -p r o o f inhibited tumor growth and dramatically extended survival in a MYC-driven genetic liver cancer tumor model [187] .

Examples of LNP-siRNA delivery for liver-related metabolic disorders are plentiful. An interesting clinical observation was that loss-of-function mutations in proprotein convertase subtilisin/kexin type 9

(PCSK9) resulted in low cholesterol levels in circulation. This finding prompted the investigation into using siRNA to downregulate PCSK9 as a treatment for hypercholesterolemia. Murine and NHP studies showed that specific PCSK9 transcript lowering resulted in reversible and durable knockdown of PCSK9, apolipoprotein B (ApoB), and low-density lipoprotein associated cholesterol [188] . Similarly, in the first demonstration of RNAi in higher-order mammals, ApoB knockdown resulted in reduction of ApoB levels, serum cholesterol content, and LDL particle concentration in NHPs [133] . Other lipid-trafficking related targets include apolipoprotein C3 knockdown for hyperlipidemia [189] , and angiopoietin-like 3 protein inhibition for hypertriglyceridemia [190] .

Lastly, we discuss the specific case of Onpattro ® (patisiran), an LNP-siRNA formulation targeting the ttr gene. TTR is a homotetrameric serum protein that is synthesized in hepatocytes and secreted into the systemic circulation (note similarity to FVII) [191] . When mutated, TTR deposits as amyloid fibrils in cardiac or peripheral nervous tissue resulting in multi-system failure including ocular, cardiovascular, nephropathy, gastrointestinal, and neuropathy (autonomic and peripheral sensorimotor) manifestations.

TTR downregulation with LNP-siRNA is a powerful approach to treat this disease. Murine data suggested that at doses of 0.1 mg/kg siRNA, > 85% liver ttr mRNA knockdown and TTR protein serum concentrations could be achieved [192] . Further testing in NHPs showed that an intravenous dose of 0.3 mg/kg every 4 weeks resulted in rapid and reversible knockdown although serum levels increased two weeks after each administration. Increasing the dosing frequency to once every three weeks resulted in sustained and robust knockdown (> 90%) following the third dose.

Introducing exogenous mRNA to induce a therapeutic effect has great potential for a variety of applications. The true benefits of LNP technology for gene regulation in the liver are best highlighted with mRNA. Specifically, mRNA requires a delivery system as modifications to the nucleotides alone have not proven successful in meeting the potency requirements for clinical translation. In addition to this, the exorbitant costs of mRNA production imply that lower doses and less frequent dosing regimens are more likely to gain favourable reception. As such, dramatic advances are seen for mRNA formulations as vaccines, in protein replacement therapies, and gene editing. reader is referred to several recent articles [193] [194] [195] [196] [197] [198] [199] [200] . However, several recent studies have used the "liver as a bioreactor" to produce relevant neutralizing antibodies.

Pardi et al. showed that intravenous delivery of LNP-mRNA encoding a broadly neutralizing antibody against HIV-1 resulted in sufficient expression to protect from HIV-1 challenge [201] . Similarly, another study showed that an LNP-mRNA system as prophylactic and therapeutic anti-rabies intervention protected mice from a Rabies virus challenge [202] . and elevated hematocrit in porcine and non-human primate (NHP) models [203] . Similarly, delivering mRNA encoding human clotting factor IX (FIX) to FIX-knockout mice displayed a reduction in hematocrit loss following injury, indicating FIX expression can rescue hemophilia B phenotypes [204] .

Gene editing is the next major application of mRNA therapeutics. Various approaches have been explored including CRISPR/Cas9 and Zinc-finger nucleases (ZFN). An initial gene editing demonstration used a combination of viral delivery (sgRNA and repair template) combined with LNP-mRNA encoding Cas9 to correct a mutation in the fumarylacetoacetate hydrolase gene [205] . The study showed approximately 6% of hepatocytes were edited and it is assumed that the limitation was the viral delivery.

Comparatively, Finn et al. used LNP-mRNA formulations encoding for Cas9 protein, co-delivered with sgRNA targeting ttr. They showed sustained 12-month circulating TTR knockdown (97%) following a single administration of 3 mg/kg RNA body weight in a murine model with ~70% editing in the liver (~70% liver cells are hepatocytes) [206] . Similarly, LNP-mediated delivery of mRNA encoding zinc-finger nucleases (ZFN) targeting ttr and pcsk9 resulted in > 90% knockout at mRNA doses 10-fold lower than reported previously [137] . In the same study, co-delivery of LNP-mRNA encoding ZFN targeting the albumin gene and a viral vector for templates of promotor-less human IDS or FIX resulted in integration of those templates at the albumin locus and generated therapeutically relevant levels of those proteins in murine models. In addition to continuous efforts in optimizing ionizable cationic lipids for enhanced genome editing in the liver, a recent study by Cheng et al. demonstrated that bioengineering LNP formulations with additional lipids, so-called selective organ targeting (SORT) molecules, can tune the J o u r n a l P r e -p r o o f

The rapid translation from lab bench to patients was primarily driven by a holistic design of LNP composition and processes to support scalability while maintaining potency. Onpattro ® paved the way for the next generation of lipid-based therapeutics and its success in phase 2 trials spurred development of mRNA therapeutics. Gene therapies enabled by LNPs are under clinical development for a broad range of applications (Table 4 ) [211] . In this section we discuss the clinical data for Onpattro ® and some mRNA therapeutics currently under development.

The Onpattro ® story, while heavily reviewed in literature, makes for a compelling case to support the development of other LNP nucleic acid formulations [33] . Initial efforts laid the foundations to support further clinical development, although it was clear that improved potency was required. DLinDMA-based LNP-siRNA against ttr (ALN-TTR01) was administered once to 24 healthy subjects at doses ranging from 0.01 to 1.0 mg siRNA per kg body weight, with another eight subjects receiving placebo [212, 213] . Over the period of 30 days, 38% serum TTR reduction was observed with persistent reduction for approximately one week. While the knockdown was arguably insufficient for therapeutic efficacy at the highest dose, the study validated the RNAi approach in humans. Subsequent clinical development used MC3-based LNP, named ALN-TTR02 or Onpattro ® (patisiran). Another phase 1 study included 13 healthy subjects receiving Onpattro ® , four subjects receiving placebo, and another six receiving a control siRNA [214] . The Onpattro ® doses ranged from 0.01 to 0.5 mg/kg siRNA and TTR serum levels were measured over 70 days. At siRNA doses of 0.3 mg/kg, rapid and robust ttr knockdown was observed; this was sustained over two weeks for a period of 21 days following administration. At these doses and with promising results, further development was warranted.

In a subsequent phase 2 study, the dosing regimen for Onpattro ® was established [86] . ATTRv patients received two Onpattro ® infusions at doses 0.01-0.3 mg/kg every four weeks or 0.3 mg/kg every three weeks. The Q3W dosing regimen resulted in a mean 85% knockdown after the second dose. Only few mild-to-moderate infusion-related reactions were observed and one patient reported three serious adverse events. The similarity to preclinical data is quite astonishing; in NHP studies, increasing dosing frequency to Q3W (from Q4W) resulted in 96% maximal knockdown, and ~85% mean knockdown following the initial dose [192] .

In the phase 3 APOLLO study, 148 patients received Onpattro ® at a dose of 0.3 mg/kg once every three weeks, with 77 patients receiving placebo [32] . The primary endpoint was the modified neuropathy impairment score + 7 (mNIS + 7), which is used to measure the level polyneuropathy in ATTRv patients.

The test uses highly standardized, quantitative methods to measure muscle weakness, muscle stretch reflexes, sensory loss, and autonomic impairment with higher scores corresponding to disease worsening [215] . Over a period of 18 months, ATTRv patients on placebo showed a linear increase in their mNIS+7 from 0 to 28.0. Onpattro ® , with an mNIS+7 of -6.0, is the only ATTRv treatment that has been able to halt and even reverse disease progression in patients [32] . In addition to this, Onpattro ® also met all secondary endpoints. This led to EMA and FDA approval in August 2018 [33] .

With LNP technology validated as a safe approach for gene modulation in the liver, a wide range of applications have emerged. A substantial effort is focusing on vaccine applications without necessarily transfecting the liver. However, the potential for treating liver diseases is also clear. Translate Bio was developing a formulation for treating OTC deficiency, however disappointing preclinical toxicology data resulted in the termination of the program [216] . Moving forward, they chose to focus on developing their cystic fibrosis mRNA therapeutic. Moderna Therapeutics is advancing an LNP candidate formulation for treating methylmalonic acidemia [217] . The focus of this review is on the hepatic applications of LNP formulations, and indications for extrahepatic targets have been summarized elsewhere [218] . Highlighted LNP-based nucleic acid therapeutics in the clinic are summarized in Table 4 .

Therapeutic development, and gene therapy in particular, requires concerted efforts from formulation developers, process developers, and clinical sponsors to allow for successful clinical translation.

Specifically, the therapeutic has to be safe and effective, be producible at a large scale, and meet all regulatory requirements for the corresponding drug class. Onpattro ® has shown that this is possible for systemic nucleic acid therapeutics, as it overcame barriers that typically halt the clinical translation of such nanocarrier-based therapeutics.

Intravenous administration of nanoparticulate formulations can potentially result in infusion-related reactions such as hypersensitivity manifesting as mild flu-like symptoms, or more severe cardiac anaphylaxis [219] . Both complement activation as well as complement-independent phagocytosis are involved in such reactions. The reader is referred to excellent articles on complement activation-related pseudoallergy (CARPA) and complement independent pseudoallergy (CIPA) [220, 219] . Several physiochemical properties such as lamellarity, surface charge, and cholesterol content may influence hypersensitivity reactions [221] . Infusion-related reactions can be managed by pre-dosing patients with a combination of anti-histamines (H1/H2 blockers), corticosteroid immunosuppressants (e.g., dexamethasone), and oral acetaminophen in addition to reducing the rate of infusion [222] . Onpattro ® 's phase 3 trial suggested that the most frequent reactions included flushing, backpain, abdominal pain, and nausea described as mild-to-moderate. The severity and frequency of these reactions decreased with repeated administration and exposure of Onpattro ® . It should be noted that ASOs and GalNAc-siRNA conjugates do not require pre-medication and can be administered subcutaneously (by healthcare professionals), but the doses required to achieve equivalent gene silencing are a few orders of magnitude higher than required for LNPs and can only be limited to gene silencing applications [192] .

Another substantial barrier to clinical translation is producing formulations at commercial scales. As Following this, the next processing steps introduce shear as buffer exchange is not done by dialysis, but rather by tangential flow filtration. Given the inherent instability of these formulations, particle size increases are observed during this step. These processes use terminal, redundant sterile filtration rather than complete aseptic processing. The impact that buffer exchange has on particle size also affects the ability to sterile filter the formulation and the yield of material. Robust process design is critical for successful and timely clinical translation of such formulations. 

Developing LNP delivery technology has enabled the clinical translation and approval of the first siRNA drug for inhibiting pathogenic protein production in hepatocytes [32, 33] . Importantly, Onpattro ® provides a valuable treatment for ATTRv amyloidosis patients, whose options were previously limited to TTR stabilizers or a liver transplant [34] . At the same time, LNP-siRNA development has yielded fundamental insights into optimally designing formulations for hepatocyte gene silencing, (large scale) production methods, in vivo behaviour, immunostimulatory effects, and cost-effectiveness. As these criteria and parameters are now firmly established, it is anticipated that other hepatocyte-targeted LNP-siRNA treatments will be developed, such as to knockdown proprotein convertase subtilisin/kexin type 9 for hypercholesteremia treatment [234] .

While these advances in LNP development are ground-breaking, other liver-targeted nucleic acid therapeutics, such as ASOs [3] and GalNAc-siRNA conjugates [4] , are also gaining momentum. For example, the ASO Tegsedi ® (inotersen) was recently approved for the same indication as Onpattro ® [235] .

With both Onpattro ® and Tegsedi ® set at the same list price ($450,000 per year), it remains to be seen which treatment will prove to be most beneficial and cost-effective. Tegsedi's ® major advantage is its subcutaneous administration (versus Onpattro's ® intravenous infusion), this advantage could be outweighed by its less favorable toxicity profile; patients require monitoring of platelet count, renal and hepatic impairment. Subcutaneous administration (and a less complex production process) is also the J o u r n a l P r e -p r o o f main advantage of GalNAc-siRNA conjugates although currently approved conjugates have to be administered by healthcare professionals. Most recently, the GalNAc-siRNA conjugate Givlaari™ (givosiran, $575,000 per year) was approved for treating acute hepatic porphyria [236, 237] , while New Drug Applications were filed for lumasiran for treating primary hyperoxaluria type 1, [238, 239] and inclisiran for treating hypercholesteremia [240] [241] [242] . Vutrisiran, a GalNAc-siRNA conjugate for treating ATTRv amyloidosis, is currently undergoing phase 3 trials and has been granted Orphan Drug designation in the U.S. and the European Union [243] . Although there is preclinical evidence that LNP-siRNA can induce hepatic gene silencing following subcutaneous administration, the dose needed for effective gene silencing is considerably higher than for intravenously administered formulations [147] . Of note, while LNP-siRNA systems have been optimized for hepatic gene silencing, preclinical studies have also demonstrated their ability to induce effective gene silencing in extrahepatic target sites including the bone [244] and tumors [245, 246] . A major area of interest is applying LNP-siRNA for immunotherapy, by silencing target genes in lymphocytes following intravenous administration for immunotherapy [247] [248] [249] [250] [251] (covered by Peer et al. in this issue [252] ).

As mentioned before, LNP technology's true benefits are currently proving to be of significant value for gene regulation approaches using large nucleic acid-based therapeutics, such as mRNA and gene editing complexes, which cannot be accomplished by nucleic acid modification or GalNAc conjugation.

Intravenously administered LNP-mRNA effectively transfect hepatocytes and induce protein expression in the liver, providing opportunities for protein replacement therapy without affecting the genome. For example, An et al. demonstrated that treatment with LNP containing mRNA encoding human methylmalonyl-CoA mutase (hMUT) had sustained functional benefits in mouse models of methylmalonic acidemia, a rare, inherited, pediatric metabolic disorder [217] . Other examples include using the liver to produce coagulation factors [253] , or therapeutic antibodies against HIV [201] and chikungunya virus [254] . Although this review focuses on gene therapy for diseases originating in the liver, it is worth mentioning that as with LNP-siRNA, intravenously injecting LNP-mRNA to induce protein expression in immune cells is gaining considerable traction [255, 256] , especially for developing (personalized) cancer immunotherapies [257, 258] . In addition, LNP-mRNA systems have revealed their potential for ex vivo CAR T cell engineering [259] .

Moreover, LNP-mRNA-based vaccinations following subcutaneous, intradermal, or intramuscular administration have demonstrated to effectively protect from viral challenge [260] [261] [262] [263] [264] [265] . Given mRNA's relatively short optimization time from target identification to therapeutic, several companies including Moderna, BioNTech, and CureVac as well as universities around the globe have initiated LNP-mRNA vaccine programs to combat the recent SARS-CoV-2 pandemic [266] [267] [268] [269] . Typical vaccine production relies on isolation and large-scale virus propagation with subsequent processing to purify material (e.g.

inactive virus or specific surface protein) that raises a response against a specific viral antigen. With mRNA delivery, these timelines can be dramatically reduced, and the breadth of immune coverage expanded. Additionally, mRNA vaccines leverage several aspects of LNP technology: (1) LNP systems J o u r n a l P r e -p r o o f are not completely immune-silent and can act as adjuvants [270, 271] , (2) few doses are required (i.e. prime and booster), and (3) the mRNA dosage is relatively low (compared to protein replacement therapies).

LNP technology, and ionizable cationic lipid development in particular, have been instrumental for translating therapeutic gene regulation in hepatocytes from bench to bedside. As LNPs are a multicomponent and modular platform, they represent a versatile toolbox with many opportunities to develop future gene therapies with more potent therapeutic effects and improved toxicity profiles. For example, incorporating lipophilic prodrugs in LNP systems has shown to be an attractive approach for reducing nucleic acid therapeutics' immunostimulatory effects [272] or for designing combination therapies with additive therapeutic effects [273] . Generating more potent (ionizable cationic) lipids and improved understanding of nano-bio interactions in vivo are continuously fueling the optimization of LNP systems for delivering nucleic acid therapeutics. Deciphering intracellular trafficking pathways and mechanism(s) of endosomal escape will facilitate efforts to boost LNP potency [274] [275] [276] [277] . Elucidating the nature and dynamic of the biomolecular corona formed on LNPs (following intravenous injection) and understanding its implications for biodistribution will be crucial to develop gene therapies beyond the liver [162] [163] [164] 278, 279] . Therefore, we expect that LNP-based gene therapies will be developed for indications beyond (ultra) rare diseases in the near future and increasingly become integrated in mainstream medicine. 

PRC is a co-founder of Acuitas Therapeutics and Precision Nanosystems; and Scientific Director and CEO of the NMIN.

DW, JAK, JL, SC, PRC and RvdM conceived and co-wrote the manuscript. The final manuscript was approved by all authors.

Addressing liver disease in the UK: a blueprint for attaining excellence in health care and reducing premature mortality from lifestyle issues of excess consumption of alcohol, obesity, and viral hepatitis

Burden of liver diseases in the world

RNA-Targeted Therapeutics

GalNAc-siRNA Conjugates: Leading the Way for Delivery of RNAi Therapeutics

Lipid Nanoparticle Technology for Clinical Translation of siRNA Therapeutics

Gene therapy comes of age

Adeno-associated virus vector as a platform for gene therapy delivery

mRNA vaccines-a new era in vaccinology

Personalized vaccines for cancer immunotherapy

Rational designs of in vivo CRISPR-Cas delivery systems

Delivery Aspects of CRISPR/Cas for in Vivo Genome Editing

Overcoming cellular barriers for RNA therapeutics

Safety profile of RNAi nanomedicines

Knocking down disease: a progress report on siRNA therapeutics

Delivery of oligonucleotides with lipid nanoparticles

Liposomal drug delivery systems: from concept to clinical applications

Lipid Nanoparticle Systems for Enabling Gene Therapies

Lipid Nanoparticles Enabling Gene Therapies: From Concepts to Clinical Utility

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans

Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells

Strategies, design, and chemistry in siRNA delivery systems

Chemical and structural modifications of RNAi therapeutics

Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNA Lipid Nanoparticles

Shielding of Lipid Nanoparticles for siRNA Delivery: Impact on Physicochemical Properties, Cytokine Induction, and Efficacy

Hypersensitivity and Loss of Disease Site Targeting Caused by Antibody Responses to PEGylated Liposomes

Synthesis and characterization of novel poly(ethylene glycol)-lipid conjugates suitable for use in drug delivery

On the role of helper lipids in lipid nanoparticle formulations of siRNA

Design of lipid nanoparticles for in vitro and in vivo delivery of plasmid DNA

A combinatorial library of lipid-like materials for delivery of RNAi therapeutics

Rational design of cationic lipids for siRNA delivery

Maximizing the potency of siRNA lipid nanoparticles for hepatic gene silencing in vivo

Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis

The Onpattro® story and the clinical translation of nanomedicines containing nucleic acid-based drugs

Hereditary transthyretin amyloidosis: a model of medical progress for a fatal disease

Advances in microfluidics for lipid nanoparticles and extracellular vesicles and applications in drug delivery systems

State-of-the-Art Design and Rapid-Mixing Production Techniques of Lipid Nanoparticles for Nucleic Acid Delivery

The liver

The Global Burden of Liver Disease: The Major Impact of China

Structural and functional hepatocyte polarity and liver disease

Hepatocytes: a key cell type for innate immunity

Nanoparticle-liver interactions: Cellular uptake and hepatobiliary elimination

Elimination Pathways of Nanoparticles

Modifying a Commonly Expressed Endocytic Receptor Retargets Nanoparticles in Vivo

Lipidoid Nanoparticles Containing PD-L1 siRNA Delivered In Vivo Enter Kupffer Cells and Enhance NK and CD8(+) T Cellmediated Hepatic Antiviral Immunity

Single cell RNA sequencing of human liver reveals distinct intrahepatic macrophage populations

A human liver cell atlas reveals heterogeneity and epithelial progenitors

The journey of a drug-carrier in the body: An anatomo-physiological perspective

Mosby's Medical Dictionary

Biodistribution of small interfering RNA at the organ and cellular levels after lipid nanoparticle-mediated delivery

Cell Subtypes Within the Liver Microenvironment Differentially Interact with Lipid Nanoparticles

A Potent Branched-Tail Lipid Nanoparticle Enables Multiplexed mRNA Delivery and Gene Editing In Vivo

Role of Kupffer cells in host defense and liver disease

The Hepatic Microcirculation: Mechanistic Contributions and Therapeutic Targets in Liver Injury and Repair

The size of endothelial fenestrae in human liver sinusoids: implications for hepatocyte-directed gene transfer

Liver endocytosis and Kupffer cells

A Consensus Definitive Classification of Scavenger Receptors and Their Roles in Health and Disease

The function of scavenger receptorsexpressed by macrophages and their rolein the regulation of inflammation

Preclinical Studies To Understand Nanoparticle Interaction with the Immune System and Its Potential Effects on Nanoparticle Biodistribution

Nanoparticle interaction with plasma proteins as it relates to particle biodistribution, biocompatibility and therapeutic efficacy

The role of surface chemistry in serum protein coronamediated cellular delivery and gene silencing with lipid nanoparticles

Liver sinusoidal endothelial cells: Physiology and role in liver diseases

An electron microscopic study of the fenestrated endothelial lining of rat liver sinusoids

Structural and functional aspects of liver sinusoidal endothelial cell fenestrae: a review

The liver sieve: Considerations concerning the structure and function of endothelial fenestrae, the sinusoidal wall and the space of disse

Thirty-five years of liver sinusoidal cells: Eddie wisse in retirement

Three-dimensional organization of fenestrae labyrinths in liver sinusoidal endothelial cells

Directing Nanoparticle Biodistribution through Evasion and Exploitation of Stab2-Dependent Nanoparticle Uptake

Influence of acute alcohol administration on endothelial fenestrae of rat livers: an in vivo and in vitro scanning electron microscopic study

Species differences in transgene DNA uptake in hepatocytes after adenoviral transfer correlate with the size of endothelial fenestrae

Perisinusoidal stellate cells of the liver: important roles in retinol metabolism and fibrosis

Liver inflammation and fibrosis

Mechanisms of hepatic stellate cell activation

Pathogenesis of Liver Fibrosis

Models of liver fibrosis: exploring the dynamic nature of inflammation and repair in a solid organ

Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms

Hepatocyte-specific drug delivery using active targeted nanomedicines -evaluation of targeting strategies in vitro and in vivo

State-of-the-art gene-based therapies: the road ahead

Gene therapy for haemophilia: a long and winding road

Monogenic diseases that can be cured by liver transplantation

Late-Onset Ornithine Transcarbamylase Deficiency: Treatment and Outcome of Hyperammonemic Crisis

Homozygous familial hypercholesterolemia: Current perspectives on diagnosis and treatment

PCSK9: A Key Modulator of Cardiovascular Health

Evolving landscape in the management of transthyretin amyloidosis

Efficacy and safety of patisiran for familial amyloidotic polyneuropathy: a phase II multi-dose study

Protein C and protein S deficiencies: similarities and differences between two brothers playing in the same game

Prevalence of inherited antithrombin, protein C, and protein S deficiencies in portal vein system thrombosis and Budd-Chiari syndrome: A systematic review and meta-analysis of observational studies

An update on primary hyperoxaluria

Inherited disorders of bilirubin metabolism

Gene replacement therapy for genetic hepatocellular jaundice

Gene therapy for alpha-1 antitrypsin deficiency

A Review of alpha(1)-Antitrypsin Deficiency

Pathogenesis and Clinical Considerations in Diagnosis and Treatment

The treatment of Wilson's disease, a rare genetic disorder of copper metabolism

Tyrosinemia Type 1: Metastatic Hepatoblastoma With a Favorable Outcome

Hereditary Hemochromatosis: Pathogenesis, Diagnosis, and Treatment

The Molecular Pathogenesis of Hereditary Hemochromatosis

Glycogen storage diseases: new perspectives

Pompe disease gene therapy

Hepatocellular carcinoma: epidemiology and molecular carcinogenesis

Hepatocellular carcinoma

Hepatitis B and C virus hepatocarcinogenesis: lessons learned and future challenges

Pathogenesis of hepatitis B virus infection

Hepatitis B virus infection

Hepatitis B virus infection

Evolving epidemiology of hepatitis C virus

Liver zonation: Novel aspects of its regulation and its impact on homeostasis

Metabolic zonation of the liver: The oxygen gradient revisited

Functional specialization of different hepatocyte populations

Metabolic zonation of the liver: Regulation and implications for liver function

Inverse zonation of hepatocyte transduction with AAV vectors between mice and non-human primates

Gene therapy for monogenic liver diseases: clinical successes, current challenges and future prospects

Phenotype Determines Nanoparticle Uptake by Human Macrophages from Liver and Blood

Liver Macrophages: Old Dogmas and New Insights

Relation between localization and function of rat liver Kupffer cells

Therapeutic targeting of liver inflammation and fibrosis by nanomedicine

Expression of asialoglycoprotein receptor 1 in human hepatocellular carcinoma

Variable asialoglycoprotein receptor 1 expression in liver disease: Implications for therapeutic intervention

APOE polymorphism in ATTR amyloidosis patients treated with lipid nanoparticle siRNA

Entrapment of a bacterial plasmid in phospholipid vesicles: potential for gene transfer

Introduction of liposome-encapsulated SV40 DNA into cells

Transfection reagent Lipofectamine triggers type I interferon signaling activation in macrophages

The Molecular Basis of Nonbilayer Phases

Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids

Influence of cationic lipid composition on uptake and intracellular processing of lipid nanoparticle formulations J o u r n a l P r e -p r o o f Journal Pre-proof of siRNA

Biodegradable Lipids Enabling Rapidly Eliminated Lipid Nanoparticles for Systemic Delivery of RNAi Therapeutics

Synthesis and Characterization of Degradable Multivalent Cationic Lipids with Disulfide-Bond Spacers for Gene Delivery

Molecular Tuning of a Vitamin E-Scaffold pH-Sensitive and Reductive Cleavable Lipid-like Material for Accelerated in Vivo Hepatic siRNA Delivery

RNAi-mediated gene silencing in non-human primates

Lipopeptide nanoparticles for potent and selective siRNA delivery in rodents and nonhuman primates

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

A neutral lipid envelope-type nanoparticle composed of a pH-activated and vitamin E-scaffold lipid-like material as a platform for a gene carrier targeting renal cell carcinoma

Nonviral Delivery of Zinc Finger Nuclease mRNA Enables Highly Efficient In Vivo Genome Editing of Multiple Therapeutic Gene Targets

A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Nonhuman Primates

The role of helper lipids in lipid nanoparticles (LNPs) designed for oligonucleotide delivery

Transbilayer Movement and Net Flux of Cholesterol and Cholesterol Sulfate between Liposomal Membranes

Hydrophobic scaffolds of pH-sensitive cationic lipids contribute to miscibility with phospholipids and improve the efficiency of delivering short interfering RNA by small-sized lipid nanoparticles

Optimization of Lipid Nanoparticle Formulations for mRNA Delivery in Vivo with Fractional Factorial and Definitive Screening Designs

Nanoparticles Containing Oxidized Cholesterol Deliver mRNA to the Liver Microenvironment at Clinically Relevant Doses

Binding characteristics of scavenger receptors on liver endothelial and Kupffer cells for modified low-density lipoproteins

Scavenger receptor class B type I as a receptor for oxidized low density lipoprotein

Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA

Development of lipid nanoparticle formulations of siRNA for hepatocyte gene silencing following subcutaneous administration

Barcoded nanoparticles for high throughput in vivo discovery of targeted therapeutics

Zebrafish as a predictive screening model to assess macrophage clearance of liposomes in vivo

Poly(ethylene glycol)--lipid conjugates regulate the calcium-induced fusion of liposomes composed of phosphatidylethanolamine and phosphatidylserine

Use of poly(ethylene glycol)-lipid conjugates to regulate the surface attributes and transfection activity of lipid-DNA particles

On the mechanism whereby cationic lipids promote intracellular delivery of polynucleic acids

Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA

A scalable, extrusion-free method for efficient liposomal encapsulation of plasmid DNA

Single bilayer liposomes prepared without sonication

Simple mixing device to reproducibly prepare cationic lipid-DNA complexes (lipoplexes)

On the Formation and Morphology of Lipid Nanoparticles Containing Ionizable Cationic Lipids and siRNA

Rapid synthesis of lipid nanoparticles containing hydrophobic inorganic nanoparticles

Relationship Between the Physicochemical Properties of Lipid Nanoparticles and the Quality of siRNA Delivery to Liver Cells

Mixing lipids to manipulate the ionization status of lipid nanoparticles for specific tissue targeting

Endothelial lipase is synthesized by hepatic and aorta endothelial cells and its expression is altered in apoE-deficient mice

Biomolecular coronas provide the biological identity of nanosized materials

Corona Composition Can Affect the Mechanisms Cells Use to Internalize Nanoparticles

Secreted Biomolecules Alter the Biological Identity and Cellular Interactions of Nanoparticles

Synergistic lipid compositions for albumin receptor mediated delivery of mRNA to the liver

The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake

Spontaneous entrapment of polynucleotides upon electrostatic interaction with ethanoldestabilized cationic liposomes

Lipid-Based DNA Therapeutics: Hallmarks of Non-Viral Gene Delivery

Stabilized plasmid-lipid particles: construction and characterization

Stabilized plasmid-lipid particles for systemic gene therapy

Nuclear localization signal peptides enhance cationic liposomemediated gene therapy

Synergism between a cell penetrating peptide and a pH-sensitive cationic lipid in efficient gene delivery based on double-coated nanoparticles

Improving in vivo hepatic transfection activity by controlling intracellular trafficking: the function of GALA and maltotriose

Theranostic barcoded nanoparticles for personalized cancer medicine

Delivery of oligonucleotides with lipid nanoparticles

Liver specific gene immunotherapies resolve immune suppressive ectopic lymphoid structures of liver metastases and prolong survival

Relaxin gene delivery mitigates liver metastasis and synergizes with check point therapy

FDA approves first-of-its kind targeted RNA-based therapy to treat a rare disease

Advanced siRNA Designs Further Improve In Vivo Performance of GalNAc-siRNA Conjugates

Lipid nanoparticle siRNA treatment of Ebolavirus-Makona-infected nonhuman primates

Highly specific delivery of siRNA to hepatocytes circumvents endothelial cell-mediated lipid nanoparticleassociated toxicity leading to the safe and efficacious decrease in the hepatitis B virus

Hepatitis B Virus Therapeutic Agent ARB-1740 Has Inhibitory Effect on Hepatitis Delta Virus in a New Dually-Infected Humanized Mouse Model

Inhibition of hepatitis C virus in mouse models by lipidoid nanoparticle-mediated systemic delivery of siRNA against PRK2

Abstract 2829: Preclinical characterization of TKM-080301, a lipid nanoparticle formulation of a small interfering RNA directed against polo-like kinase 1

Simultaneous silencing of VEGF and KSP by siRNA cocktail inhibits proliferation and induces apoptosis of hepatocellular carcinoma Hep3B cells

Modular degradable dendrimers enable small RNAs to extend survival in an aggressive liver cancer model

Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates

Loss-of-function mutations in APOC3 and risk of ischemic vascular disease

Silencing of ANGPTL 3 (angiopoietin-like protein 3) in human hepatocytes results in decreased expression of gluconeogenic genes and reduced triacylglycerol-rich VLDL secretion upon insulin stimulation

Transthyretin and familial amyloidotic polyneuropathy. Recent progress in understanding the molecular mechanism of neurodegeneration

Preclinical evaluation of RNAi as a treatment for transthyretin-mediated amyloidosis

Characterization of HIV-1 Nucleoside-Modified mRNA Vaccines in Rabbits and Rhesus Macaques

mRNA vaccines-a new era in vaccinology

Nucleoside Modified mRNA Vaccines for Infectious Diseases

Nucleoside-modified mRNA immunization elicits influenza virus hemagglutinin stalk-specific antibodies

Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses

Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination

Optimization of Lipid Nanoparticles for Intramuscular Administration of mRNA Vaccines

Blankschtein, mRNA vaccine delivery using lipid nanoparticles

Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1 challenge

Schlake, mRNA mediates passive vaccination against infectious agents, toxins, and tumors

Sequenceengineered mRNA Without Chemical Nucleoside Modifications Enables an Effective Protein Therapy in Large Animals

Therapeutic efficacy in a hemophilia B model using a biosynthetic mRNA liver depot system

Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype

A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing

Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing

Nanotechnology for organ-tunable gene editing

Monitoring Translation Activity of mRNA-Loaded Nanoparticles in Mice

Fast and Efficient CRISPR/Cas9 Genome Editing In Vivo Enabled by Bioreducible Lipid and Messenger RNA Nanoparticles

Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery

Trial to Evaluate Safety and Tolerability of ALN-TTR01 in Transthyretin (TTR) Amyloidosis -Full Text View -ClinicalTrials.gov

Safety and efficacy of RNAi therapy for transthyretin amyloidosis

Trial to Evaluate Safety, Tolerability, and Parmacokinetics of ALN-TTR02 in Healthy Volunteer Subjects -Full Text View -ClinicalTrials.gov

Development of measures of polyneuropathy impairment in hATTR amyloidosis: From NIS to mNIS + 7

Translate Bio Announces FDA Clearance to Proceed with a Single-ascending Dose (SAD) Phase 1/2 Clinical Trial for Ornithine Transcarbamylase (OTC) Deficiency, GlobeNewswire News Room

Long-term efficacy and safety of mRNA therapy in two murine models of methylmalonic acidemia

Current issues of RNAi therapeutics delivery and development

Mechanism of nanoparticle-induced hypersensitivity in pigs: complement or not complement?

Activation of complement by therapeutic liposomes and other lipid excipient-based therapeutic products: Prediction and prevention

Liposome-induced complement activation and related cardiopulmonary distress in pigs: factors promoting reactogenicity of Doxil and AmBisome

Trial design and rationale for APOLLO, a Phase 3, placebo-controlled study of patisiran in patients with hereditary ATTR amyloidosis with polyneuropathy

Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis

3rd, First-in-humans trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement

Pharmacokinetics and exploratory exposure-response of siRNAs administered monthly as ARB-001467 (ARB-1467) in a Phase 2a study in HBeAg positive and negative virally suppressed subjects with chronic hepatitis B

A phase I/II study of TKM-080301, a PLK1-targeted RNAi in patients with adrenocortical cancer (ACC)

First-in-human phase I study of the liposomal RNA interference therapeutic Atu027 in patients with advanced solid tumors

Safety, tolerability, and pharmacokinetics of BMS-986263/ND-L02-s0201, a novel targeted lipid nanoparticle delivering HSP47 siRNA, in healthy participants: A randomised, placebo-controlled, double-blind, phase 1 study

Preclinical mammalian safety studies of EPHARNA (DOPC Nanoliposomal EphA2-Targeted siRNA)

Abstract CT156: A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles encoding shared tumor antigens for immunotherapy of malignant melanoma

Safety and efficacy in advanced solid tumors of a targeted nanocomplex carrying the p53 gene used in combination with docetaxel: A phase 1b study

455PDFirst-in-human, first-in-class phase I study of MTL-CEBPA, a RNA oligonucleotide targeting the myeloid cell master regulator C/EBP-α, in patients with advanced hepatocellular cancer (HCC)

Intellia Therapeutics Announces Second Quarter 2019 Financial Results and Company Update, Intellia Ther

Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled

Inotersen Treatment for Patients with Hereditary Transthyretin Amyloidosis

Phase 1 Trial of an RNA Interference Therapy for Acute Intermittent Porphyria

GS-14-ENVISION, a phase 3 study to evaluate efficacy and safety of givosiran, an investigational RNAi therapeutic targeting aminolevulinic acid synthase 1, in acute hepatic porphyria patients

SUN-325 SAFETY AND EFFICACY OF LUMASIRAN, AN INVESTIGATIONAL RNA INTERFERENCE (RNAi) THERAPEUTIC, IN ADULT AND PEDIATRIC PATIENTS WITH PRIMARY HYPEROXALURIA TYPE 1

Mp12-14 safety and efficacy study of lumasiran, an investigational rna interference (rnai) therapeutic, in adult and pediatric patients with primary hyperoxaluria type 1 (ph1)

Inclisiran: UK to roll out new cholesterol lowering drug from next year

Inclisiran in Patients at High Cardiovascular Risk with Elevated LDL Cholesterol

Effects of Renal Impairment on the Pharmacokinetics, Efficacy, and Safety of Inclisiran: An Analysis of the ORION-7 and ORION-1 Studies

Phase 1 study of ALN-TTRsc02, a subcutaneously administered investigational RNAi therapeutic for the treatment of transthyretin-mediated amyloidosis

Lipid Nanoparticle Delivery of siRNA to Osteocytes Leads to Effective Silencing of SOST and Inhibition of Sclerostin In Vivo

A Glu-urea-Lys Ligand-conjugated Lipid Nanoparticle/siRNA System Inhibits Androgen Receptor Expression In Vivo

siRNA Lipid Nanoparticle Potently Silences Clusterin and Delays Progression When Combined with Androgen Receptor Cotargeting in Enzalutamide-Resistant Prostate Cancer

Systemic Gene Silencing in Primary T Lymphocytes Using Targeted Lipid Nanoparticles

Harnessing RNAi-based nanomedicines for therapeutic gene silencing in B-cell malignancies

A modular platform for targeted RNAi therapeutics

Leukocyte-specific siRNA delivery revealing IRF8 as a potential anti-inflammatory target

Systemic RNAi-mediated Gene Silencing in Nonhuman Primate and Rodent Myeloid Cells

Targeted Lipid Nanoparticles for RNA therapeutics and immunomodulation in Leukocytes

Systemic delivery of factor IX messenger RNA for protein replacement therapy

A lipidencapsulated mRNA encoding a potently neutralizing human monoclonal antibody protects against chikungunya infection

Cell specific delivery of modified mRNA expressing therapeutic proteins to leukocytes

Synthesis and Biological Evaluation of Ionizable Lipid Materials for the In Vivo Delivery of Messenger RNA to B Lymphocytes

Lipid Nanoparticle Assisted mRNA Delivery for Potent Cancer Immunotherapy

Systemic RNA delivery to dendritic cells exploits antiviral defence for cancer immunotherapy

Ionizable Lipid Nanoparticle Mediated mRNA Delivery for Human CAR T Cell Engineering

Characterization of HIV-1 Nucleoside-Modified mRNA Vaccines in Rabbits and Rhesus Macaques

Nucleoside-modified mRNA immunization elicits influenza virus hemagglutinin stalk-specific antibodies

Nucleoside-modified mRNA vaccines induce potent T follicular helper and germinal center B cell responses

Zika virus protection by a single low-dose nucleoside-modified mRNA vaccination

Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes

Ciaramella, mRNA vaccines against H10N8 and H7N9 influenza viruses of pandemic potential are immunogenic and well tolerated in healthy adults in phase 1 randomized clinical trials

Self-amplifying RNA SARS-CoV-2 lipid nanoparticle vaccine induces equivalent preclinical antibody titers and viral neutralization to recovered COVID-19 patients

Work on a COVID-19 Vaccine Candidate | Moderna

BioNTech and Pfizer announce completion of dosing for first cohort of Phase 1/2 trial of COVID-19 vaccine candidates in Germany

CureVac´s Optimized mRNA Platform Provides Positive Pre-Clinical Results at Low Dose for Coronavirus Vaccine Candidate | English

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Unmodified mRNA in LNPs constitutes a competitive technology for prophylactic vaccines

Lipid-derived nanoparticles for immunostimulatory RNA adjuvant delivery

Dexamethasone prodrugs as potent suppressors of the immunostimulatory effects of lipid nanoparticle formulations of nucleic acids

Modular lipid nanoparticle platform technology for siRNA and lipophilic prodrug delivery | bioRxiv

Efficiency of siRNA delivery by lipid nanoparticles is limited by endocytic recycling

Brief update on endocytosis of nanomedicines

Boosting Intracellular Delivery of Lipid Nanoparticle-Encapsulated mRNA

Endocytic Profiling of Cancer Cell Models Reveals Critical Factors Influencing LNP-Mediated mRNA Delivery and Protein Expression

Isolation methods for particle protein corona complexes from protein-rich matrices

An Analysis of the Binding Function and Structural Organization of the Protein Corona