Modulating intracellular pathways to improve non-viral delivery of RNA therapeutics

https://doi.org/10.1016/j.addr.2021.114041Get rights and content

Abstract

RNA therapeutics (e.g. siRNA, oligonucleotides, mRNA, etc.) show great potential for the treatment of a myriad of diseases. However, to reach their site of action in the cytosol or nucleus of target cells, multiple intra- and extracellular barriers have to be surmounted. Several non-viral delivery systems, such as nanoparticles and conjugates, have been successfully developed to meet this requirement. Unfortunately, despite these clear advances, state-of-the-art delivery agents still suffer from relatively low intracellular delivery efficiencies. Notably, our current understanding of the intracellular delivery process is largely oversimplified. Gaining mechanistic insight into how RNA formulations are processed by cells will fuel rational design of the next generation of delivery carriers. In addition, identifying which intracellular pathways contribute to productive RNA delivery could provide opportunities to boost the delivery performance of existing nanoformulations. In this review, we discuss both established as well as emerging techniques that can be used to assess the impact of different intracellular barriers on RNA transfection performance. Next, we highlight how several modulators, including small molecules but also genetic perturbation technologies, can boost RNA delivery by intervening at differing stages of the intracellular delivery process, such as cellular uptake, intracellular trafficking, endosomal escape, autophagy and exocytosis.

Introduction

Ribonucleic acid (RNA) therapeutics have gathered a lot of attention the past three decades as promising tools to modulate gene expression levels, both by academia and pharmaceutical companies [1], [2], [3], [4], [5]. From a functional point of view, these therapeutics can be divided into three main categories: those targeting cellular RNA or DNA (e.g. antisense oligonucleotides and small interfering RNAs), those encoding for proteins (mRNA) and, to a lesser extent, those directly targeting proteins (aptamers) [6], [7].

As the majority (∼80%) of the human genome is transcribed into RNA and virtually any coding or non-coding RNA transcript can be considered as a therapeutic target, the introduction of exogenous RNA-targeting small nucleic acids into cells has the potential to treat a myriad of disorders (e.g. cancer, viral infections, autoimmune diseases, cardiovascular disorders). Notably, also gene products that were previously thought to be ‘undruggable’ by conventional small molecules or proteins can be targeted with specific nucleic acids [1], [4], [8], [9], [10]. For example, antisense oligonucleotides (ASOs) and nucleic acids triggering the RNA interference (RNAi) pathway, such as small interfering (si)RNA, short hairpin (sh)RNA and micro (mi)RNA mimics, can induce sequence-specific silencing of disease-promoting genes (e.g. silencing of growth factors in cancer cells or cytokines in immune cells) via mRNA degradation and/or translational repression [11], [12], [13], [14]. ASOs can also act as antagonists of endogenous miRNAs (antagomiRs), thereby controlling expression of miRNA-regulated genes [15], [16]. In turn, ASOs applied to modulate the splicing of pre-mRNA in the nucleus (i.e. correcting aberrant splicing or inducing alternative splicing) are named splice switching oligonucleotides (SSOs) and can be used to repair protein function [16], [17]. In addition, modulation of nonsense-mediated mRNA decay [18] or mRNA translation [19], [20], [21] by specifically designed ASOs has shown an ability to increase gene expression, demonstrating their versatility. Short activating RNA (saRNA), which has a similar structure to siRNA, can also boost gene expression, although this involves binding to promoter regions of genes in the nucleus to enhance transcription [3], [22]. Finally, short single-stranded RNA aptamers can also selectively and directly block protein function [3], [16].

Next to the small non-coding nucleic acids discussed above, also in vitro transcribed (IVT) messenger (m)RNA can be therapeutically exploited to transiently induce/restore protein expression, providing a powerful alternative to conventional DNA-based gene therapy, vaccination (against cancer [23], [24], [25], [26] or infectious diseases [26], [27], [28], [29]) or traditional protein replacement strategies (e.g. mRNA expressing an effective chloride channel in cystic fibrosis or mRNA encoding for growth factors in tissue engineering) [30], [31], [32], [33], [34]. In contrast to plasmid DNA (pDNA), mRNA does not require nuclear translocation and is therefore effective in transfecting both dividing and non-dividing cells [35]. Moreover, mRNA therapeutics do not hold the risk of insertional mutagenesis and genotoxicity. In addition, mRNA can be used in the field of regenerative medicine (e.g. cell reprogramming [36]) or genome editing [37]). Indeed, the components of the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) nuclease system can be co-delivered as Cas9-encoding mRNA and single-guide RNA (sgRNA) [38], [39], [40], [41], where the sgRNA recognizes specific target genomic loci, while the mRNA produces the Cas9 nuclease that cleaves the sgRNA-bound DNA sequence [42]. Here, transient Cas9 expression by mRNA allows improved control over (the duration of) intracellular nuclease activity, thus reducing the risk of off-target mutations. Alternatively, the sgRNA can also be delivered as a ribonucleoprotein complex with the Cas9 protein [41], [43]. Self-amplifying RNA (SAM) is a special type of mRNA construct that, in addition to the protein of interest, also encodes a viral enzyme complex for self-amplification (i.e. replicase polyprotein) [44]. Hence, upon transfection, the replicase is co-translated and via the generation of RNA intermediates multiple copies of the protein-encoding subgenomic mRNA are synthesized, producing very high levels of the encoded protein [44], [45], [46]. Consequently, compared to conventional IVT mRNA, substantially lower amounts of SAM need to be delivered intracellularly to enable effective protein production [44].

Locally administered (i.e. intravitreal injection in the eye) ASO (Fomivirsen, 1998) or aptamer (Pegaptanib, 2004) therapies were the earliest small non-coding nucleic acid drugs to obtain FDA-approval, of which Fomivirsen is no longer marketed [47], [48]. Next, a subcutaneously injected ASO (Mipomersen, 2013) [49], a systemically applied SSO (Eteplirsen, 2016) [50] and a locally administered (via intrathecal injection) SSO (Nusinersen, 2016) [51] were approved by the FDA and/or EMA and marketed with varying degrees of success [47]. In 2018, the clinical approval of Patisiran, the first siRNA-based therapeutic, represented a crucial milestone for the field of nucleic acid-based drugs [52], [53], [54]. This initial success was shortly followed by the approval of Inotersen, an ASO developed for the same target and disease (i.e. transthyretin amyloidosis) [55]. Since then, also Givosiran (siRNA) [56], Volanesorsen (ASO) [57], Golodirsen (SSO) [58], Lumasiran (siRNA) [59] and Inclisiran (siRNA) [60] have obtained approval by the FDA and/or EMA [61]. Very recently, also two COVID-19 mRNA vaccines, that encompass mRNAs encoding the spike protein of SARS-CoV-2, have been FDA and EMA approved [62], [63], [64], [65], [66], [67], [68], [69]. Currently, many pharmaceutical companies have several RNA therapeutics in their pipelines [2], [70], [71], including (additional) mRNA vaccines [72], [73], [74], [75], [76], [77], [78], [79], [80], [81], [82] and siRNA therapeutics [83], [84], [85] to support the fight against the COVID-19 pandemic. In the latter case, siRNAs targeting the SARS-CoV-2 genome [86] are being actively explored. For an extensive overview of the RNA therapeutics currently approved or in clinical trials, we would like to refer the reader to other recent reviews on this topic [3], [16], [61], [70], [71], [87], [88], [89].

Despite recent progress, the clinical translation of nucleic acid drugs is still relatively modest [4], [90]. This can largely be attributed to the numerous biological barriers encountered by nucleic acid therapeutics, both extra- and intracellular, which hinder their safe and effective delivery to the interior of target cells (as schematically shown in Fig. 1) [1], [91], [92], [93]. Indeed, despite their distinct pharmacological effects, all classes of nucleic acid drugs share the particular physicochemical characteristic of being relatively unstable, negatively charged, macromolecular and hydrophilic, altogether complicating in vitro and in vivo delivery [8], [94].

In general, several extracellular barriers should be surmounted to distribute nucleic acid drugs to the tissue and cells of interest, while minimizing delivery to off-target tissues. The specific barriers to which nucleic acids are exposed will in part depend on the administration route. For naked (and/or unmodified) nucleic acids that are administered systemically, these include degradation by serum nucleases, unwanted activation of the innate immune response, unfavorable interactions with blood components and, especially for small nucleic acids, rapid renal clearance [1], [93], [94], [95]. Chemically modifying the nucleic acid strands (e.g. by introducing phosphorothioate (PS) backbones or other modified nucleosides) could confer nuclease resistance [47], [95], reduce the immunostimulatory effects [96], [97], [98] and, in the case of PS-containing single-stranded oligonucleotides, prolong the circulation half-life due to enhanced plasma protein binding [47], [95], [99], [100]. Hence, PS-containing ASOs (PS-ASOs) can be delivered in vivo to some tissues (mainly the liver) after formulation in simple saline solutions [47], [101], [102], [103]. However, double-stranded siRNA molecules have a much lower propensity for protein binding than single-stranded oligonucleotides, resulting in rapid elimination by the kidneys (half-life of < 5 min for unformulated siRNA) [104], [105], [106], [107]. Therefore, siRNAs need appropriate in vivo delivery systems to allow sufficient circulation, extravasation to target tissues and eventually cellular internalization [1], [16], [93], [108], [109], [110]. For these same reasons, carefully designed delivery systems are also critical for the larger mRNA therapeutics. Exceptions to this rule are, for example, naked mRNA constructs that can be efficiently internalized by immature dendritic cells in the case of local (e.g. intradermal, intranodal) mRNA vaccine applications [5], [70]. While both viral [111], [112] and non-viral [1] vectors are being evaluated for gene delivery, RNA therapeutics are predominantly formulated in non-viral delivery systems. Such synthetic vectors are typically composed of polymeric [113], [114], lipidic [52], [115], [116], [117] and/or inorganic [118] materials that complex/encapsulate the negatively charged nucleic acids to form nanosized assemblies. Multiple types of nanoparticles have now been developed for nucleic acid delivery [108], with lipid nanoparticles (LNPs) being state-of-the-art [117], [119], [120] and culminating in the first approval of both a siRNA therapeutic (Patisiran) [52] and the first mRNA vaccines (for COVID-19) [65], [66]. Another well-known approach to enhance the tissue-specific accumulation and uptake of siRNAs and oligonucleotides is the use of receptor-targeted ligands that are covalently linked to the nucleic acid strands (e.g. N-acetylgalactosamine (GalNAc)-siRNA conjugates, such as Givosiran, which actively target the highly expressed asialoglycoprotein receptor (ASGPR) on hepatocytes) [8], [47], [121], [122], [123], [124], [125], [126]. Advances in the chemical stability of the siRNA duplex allowed these novel GalNAc-siRNA conjugates to show impressive pharmacodynamic properties in clinical trials, including sustained months-long knockdown of e.g. the proprotein convertase subtilisin/kexin type 9 (PCSK9) target [127], [128], [129]. Next to GalNAc conjugates, there has also been a considerable interest in conjugation of small non-coding nucleic acids with other components, including lipophilic ligands [130], [131], [132], [133], [134], [135] (e.g. cholesterol that enhances association with blood lipoproteins and subsequent uptake by lipoprotein receptor-enriched tissues [136], [137], [138]), cell-penetrating peptides (CPPs) [139], antibodies, antibody-peptide fragments [140] (e.g. nanobodies [141], [142], [143], [144]), aptamers, small molecules [145] or combinations of different conjugations (e.g. dynamic polyconjugates [146], [147]). Ideally, targeting moieties bind cell-specific surface receptors that are rapidly internalized through receptor-mediated endocytosis, followed by rapid release of the ligand from the receptor and receptor recycling to the cell surface [145].

Nevertheless, despite the recent success for siRNA delivery in the liver and spleen [52], [56], [148], state-of-the-art delivery systems still face several extra- and intracellular obstacles [8], [91], [92], [93], [149], [150]. First, opsonization of plasma proteins on the nanoparticle surface creates a protein corona, which strongly influences their in vivo fate [91]. For example, macrophages of the mononuclear phagocyte system (MPS) quickly sequester and remove the nanoparticles from the circulation via specific recognition of adsorbed proteins [91]. In addition, protein corona deposition has been shown to shield active targeting ligands on the nanoparticle surface and reduce, for example, tumor-specific delivery [151]. Next, unless circulating cells (e.g. leukocytes [152], [153], [154], [155]) or endothelial cells are targeted, the endothelial lining of the blood vessels imposes a second extracellular barrier that circulating nanoparticles need to overcome to reach their target tissue. Liver and spleen sinusoidal capillaries have large endothelial fenestrae (i.e. pores between adjacent endothelial cells) [91], [156], with liver sinusoids also having a slower blood flow [156], which promotes passive nanoparticle accumulation. Such passive targeting has also been demonstrated, albeit to a lesser extent, to other tissues that likewise show discontinuous endothelium (e.g. inflamed tissues and certain solid tumors) [157], [158]. Thirdly, if a nanoparticle successfully reaches its target site, it should be distributed throughout the tissue, which might be complicated by a high interstitial pressure, high cell density and a dense extracellular matrix (ECM) [91].

Multiple strategies have been developed to overcome the different extracellular barriers and to improve the biodistribution of systemic (nucleic acid-loaded) nanoparticles. These include (a) the functionalization of nanoparticle surfaces or nucleic acid strands with polyethylene glycol (PEG) to hinder protein adsorption and MPS clearance or (b) the use of targeting agents. Nevertheless, despite these technological advances, the currently approved siRNA drugs still mainly target the liver upon systemic administration, rationalizing their use for the treatment of liver-related diseases [71], [88], while the only approved mRNA therapeutics are COVID-19 vaccines which do not need to distribute throughout the body. Still, it is also evident that RNA therapeutics could have a greatly extended impact if extrahepatic delivery or non-vaccine applications become a clinical reality for systemically administrated RNA formulations [87]. Consequently, the improvement of biodistribution and extra-hepatic delivery of systemically applied nucleic acid drugs remains an active area of research [159], [160]. Notably, careful modulation of the nanoparticle’s (physico)chemical properties, such as nanoparticle size or surface charge [41], [148] by e.g. changing the lipid composition [161], [162], [163], [164], can have a profound impact on its biodistribution. In addition, pre-treatment with decoy nanoparticles or chloroquine (CLQ) can prevent nanoparticle clearance by the MPS by saturating or actively blocking the phagocytic response [165], [166], [167]. Interestingly, increasing the nanoparticle dose above a certain threshold (1 trillion nanoparticles in mice) could strongly improve passive extravasation of nanoparticles into mice tumors due to Kupffer cell saturation [160], [168]. Note however that the abovementioned studies do not necessarily investigate the biodistribution of RNA therapeutics, but they altogether highlight the fact that systemic administration of (nucleic acid-loaded) nanoparticles generally still results in a high non-specific accumulation in the spleen and liver [165], [169]. To reach other tissues, local/topical administration (e.g. pulmonary [114], ocular [170], [171], skin, oral, intrathecal [172] routes) is also intensively explored, albeit these routes encompass their own specific extracellular barriers (e.g. enzymatic degradation, immobilization in mucus, etc.) [92].

Upon arrival to the target cells, RNA therapeutics additionally have to overcome multiple intracellular barriers. Following interaction with the plasma membrane, nucleic acids and their carriers are typically internalized by endocytosis. Endocytic uptake localizes the cargo in early endosomes, which further mature via late endosomes into endolysosomes, where both the cargo and the carrier face degradation due to the acidic pH and the presence of lysosomal hydrolases. However, as nucleic acids exert their function in the cytosol or nucleus, the cargo should escape this endosomal confinement [173]. Consequently, numerous groups have focused on methods that could enhance endosomal escape, including, but not limited to, (a) cationic polymers with endosomal buffering capacity that cause osmotic swelling and eventual rupture of the vesicles (the so-called proton-sponge effect [174]), (b) pH-sensitive fusogenic peptides/lipids/lipid-like materials that interact with anionic endosomal membranes and induce membrane fusion or (c) materials that induce pore formation such as certain CPPs. For a detailed description of each of these techniques, we refer the reader to the numerous comprehensive reviews on this topic [173], [175], [176], [177], [178]. Unfortunately, despite years of extensive research, endosomal escape remains a major bottleneck as it has been shown that only a minor fraction of the confined siRNA or mRNA molecules are able to escape into the cytosol when delivered via state-of-the-art delivery systems (i.e. 1–2% in case of siRNA-[179], [180] or mRNA-[181] loaded LNPs and an estimated < 0.01% in case of GalNAc-siRNA conjugates [94], [126], [182]), albeit that recent improvements in ionizable lipid design demonstrated higher intracellular delivery efficiencies for mRNA-loaded LNPs [120], [183]. Furthermore, multiple additional intracellular barriers exist such as (a) the cellular excretion of nucleic acids/nanoparticles via exocytosis or recycling, (b) the degree and kinetics of vector unpacking and nucleic acid decomplexation [184], [185], (c) the clearance of damaged endosomes or cytosolic nucleic acids/nanoparticles via autophagy and (d) the degradation of released nucleic acids by cytosolic nucleases [92]. Finally, for those nucleic acids that require delivery into the nucleus, the nuclear envelope can be an important barrier as well [1], [92]. The various intracellular barriers and novel approaches to overcome them will be discussed in more detail in this review (see Table 1). Importantly, overcoming those intracellular barriers more efficiently might overall enhance the efficacy of the next-generation of RNA therapeutics, certainly in those tissues and cell types where only a limited amount of nucleic acid drug accumulates [87], [94].

Numerous studies in the field of nucleic acid therapy have focused on the development of innovative delivery systems to overcome the abovementioned intra- and extracellular hurdles, which coincidently led to an exponential growth of novel nanomaterials and increasingly sophisticated nanocarrier designs [187], [188]. Indeed, using the available technologies, it is possible to formulate billions of chemically distinct nucleic acid delivery vehicles [189]. Although such complex delivery technologies significantly impacted advanced drug delivery, they also encompass major challenges from a manufacturing and regulatory point of view, which lowers their potential for clinical translation [188]. In addition, successful delivery strategies mainly rely on simple, but robust, physicochemical or biological principles [188]. However, in the case of therapeutics with intracellular targets, the exact cellular biological pathways (and molecular regulators) that drive productive delivery are largely unknown and our current view on this process is usually oversimplified [190]. Indeed, even for existing (and approved) nanomedicines, the most fundamental interactions within the body seem to be much more complex than previously anticipated [187]. Likewise, even the mechanisms by which nucleic acid-loaded LNPs are formed, were recently shown to be different than hitherto assumed [191]. Hence, expanding our fundamental knowledge of the physicochemistry of existing nucleic acid-loaded nanoparticles, the biology of the target tissues and cells, and the interaction between both, might fuel the rational development of less sophisticated, but more effective delivery technologies [187], [188], [190], [192], [193]. Intriguingly, important clues could be found in the way viruses and bacterial toxins exploit host trafficking machinery to gain access to intracellular targets, which are mechanisms that have been optimized by Mother Nature for millions of years [190]. Mimicking these virus-derived strategies by manipulation of key cellular processes or proteins may be a highly interesting approach to boost non-viral nucleic acid delivery as well.

In this review, we provide a critical discussion of the established as well as emerging techniques that can be used to investigate the influence of different intracellular barriers on nucleic acid transfection efficiency. Next, we consider each intracellular barrier in more detail and provide an overview on how several molecular agents (small molecules, lipids, siRNAs, etc.) can boost transfection upon manipulation of the intracellular delivery process (see Table 1). Although it is still poorly understood which specific (set of) genes or cellular pathways eventually regulate the transfection process of RNA therapeutics, we describe a number of general processes that proved to be essential for successful delivery. Increasing delivery efficiency of nanoformulations by modulating intracellular barriers could lower the required dose of RNA drugs to achieve a therapeutic response and strongly reduce the risk of off-target effects. Moreover, gaining additional insight into the intracellular behavior of RNA formulations is expected to fuel the design of next-generation RNA therapies for improved treatment of a plethora of diseases.

Section snippets

Small molecules as non-genetic tools

Small molecules have been extensively used, both as chemical probes to dissect complex biological processes and as drugs to treat human disease [194]. Hence, low molecular weight compounds were likewise applied as tools to elucidate endocytic uptake/intracellular processing of nanoparticles [195], [196] or as adjuvants to improve nucleic acid delivery through modulation of intracellular pathways [145].

The use of small molecules, either as single compounds or part of a compound library screen,

Cellular targeting and intracellular uptake

Upon arrival at their target site, both ‘naked’ nucleic acids as well as nucleic acid-conjugates and nucleic acid-loaded nanoparticles need to traverse the plasma membrane to reach their site-of-action. Apart from some approaches that reported direct cytosolic delivery of the nucleic acid cargo [285], [286], macromolecules and their nanocarriers are typically internalized by endocytosis and subsequently traffic through distinct membrane-bound vesicles [95], [236]. Endocytosis generally can be

Conclusion and perspectives

A key obstacle to the development of more potent, more widely-applicable and safer RNA therapeutics is the limited mechanistic understanding of the interaction of nucleic acid molecules and their delivery systems, both extra- and intracellular, with target cells. Although multiple well-conducted studies, using e.g. state-of-the-art microscopic technologies, have indisputably contributed to our current understanding of intracellular nucleic acid delivery, this process is generally presented in

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

T. Van de Vyver is a doctoral fellow of the Research Foundation-Flanders (grant 1198719N, FWO, Belgium). K. Raemdonck acknowledges the FWO for a postdoctoral Research Grant (grant 1517516N) and the Ghent University Special Research Fund for a Starting Grant (01N03618). Funding by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No 101002571) is acknowledged with gratitude. The authors additionally thank R. Guagliardo

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