Protein downregulation—siRNA, ASOs, and microRNA

In simplistic terms, disease-relevant proteins can be altered in one of two ways: upregulated or downregulated. The use of RNAs to selectively downregulate proteins experienced a paradigm shift following the discovery of siRNA by Fire and colleagues [56]. Short interfering RNAs are typically 21–23 base-pairs in length and can selectively bind and degrade complementary mRNA through the RISC (Fig. 2) [57]. After almost two decades of research, siRNA-based therapies represent one of the more clinically advanced platforms for RNA drugs. Alnylam Pharmaceuticals, in particular, has several siRNA drugs in clinical trials. Their most advanced drug, also one of the most advanced siRNA therapeutics, patisiran, is a LNP containing siRNA against mutant transthyretin for the treatment of transthyretin amyloidosis [58]. Patisiran is currently in phase III of clinical trials [59], having shown significant dose-dependent knockdown, with minimal adverse events, in phase II trials [60], and other companies have also invested in the use of lipoplex-based siRNA drugs (Table 2). Increasingly, however, Alnylam and others have reported significant progress with the GalNAc conjugate technology (Table 2). Despite Alnylam’s recent decision to discontinue development of revusiran, a GalNAc–siRNA conjugate drug that also treats transthyretin amyloidosis [61], the company has several more GalNAc conjugates in its pipeline that utilize a newer ‘enhanced stabilization chemistry’ [62] that could address the issues that led to the removal of revusiran from clinical trials [61]. Surprisingly, some of the current clinical trials utilize naked, albeit chemically modified, siRNAs. Almost all of these naked siRNAs are delivered locally (Table 2), reducing the risk of RNA degradation and systemic immune activation compared with that associated with systemic delivery. An intriguing use of naked siRNA is Silenseed’s siG12D LODER, which encapsulates siRNA targeted against the KRAS oncoprotein in an implantable and degradable polymeric matrix for the treatment of pancreatic cancer [63, 64]. However, there is concern that the positive effects of such treatments might in some cases be mediated by the induction of non-specific and immunological mechanisms such as siRNA binding to toll-like receptors [65].

Despite its significant presence in clinical trials, siRNA is not the only, or even the first, RNA drug to be investigated for protein knockdown at the clinical stage. The first RNA drugs widely used in clinical trials were anti-sense oligonucleotides (ASOs). Like siRNA, ASOs are designed to block protein translation through Watson–Crick base-pairing with the target mRNA [66] and can be modified to improve stability [67]. The ASOs, however, inhibit protein production through a variety of mechanisms, such as sterically blocking ribosome attachment or eliciting RNase-H activation [68]. They can also promote exon skipping (a form of RNA splicing which leaves out faulty exons), which allows for the deletion of faulty sequences within proteins [69], and, in some cases, can even lead to protein upregulation, which could be used therapeutically in diseases where certain genes are repressed [70]. An additional utility of ASOs is their ability to enter cells without the use of a transfection reagent, although this uptake does not always lead to therapeutic action [71]. Four ASOs have been clinically approved, all of which are chemically modified and used without a delivery vehicle, representing the only RNA drugs for protein modulation to be cleared by the FDA so far. The most recent, Spinraza (nusinersen), is injected intrathecally to treat spinal muscular atrophy [72]. It joined Exondys 51 (eteplirsen), an intravenously infused ASO for treatment of Duchenne muscular dystrophy [73], Vitravene (fomivirsen), an intravitreally injected ASO indicated for the treatment of ocular cytomegalovirus [74], and Kynamro (mipomersen), which is injected subcutaneously and targets mRNA encoding apolipoprotein B for the treatment of hypercholesterolemia [75, 76]. There are still several ASOs in clinical trials, the majority of which are delivered without a vehicle (Table 2). Of particular interest are studies by Ionis Pharmaceuticals utilizing a GalNAc–ASO conjugate similar to that developed by Alnylam to deliver siRNA. Optimism from such approvals and clinical studies has also led researchers to continue investigation of ASOs to treat diseases such as amyotrophic lateral sclerosis (ALS) [77] and spinocerebellar ataxia [78].

An emerging, albeit less clinically advanced, RNA-based platform for protein knockdown is microRNA (miRNA). Endogenous microRNAs are non-coding RNAs that act as key regulators for a variety of cellular pathways, and are often downregulated in diseases [79]. Thus, exogenous microRNAs, or microRNA mimics, delivered therapeutically could be used to knockdown several proteins simultaneously, which is particularly useful in diseases such as cancer where having a single disease-relevant target is rare [80]. It is also worth noting that a rare subset of microRNAs is thought to enhance protein production, and that targeting of gene-suppressing microRNAs using ASOs could also be used to increase protein production [81]. The majority of current clinical trials involving microRNA are screens to investigate microRNA involvement in certain diseases, although there are several ongoing animal studies utilizing microRNA delivery. Examples include the use of LNPs to treat a mouse model of colorectal cancer [82], and polymeric nanoparticles to deliver microRNA to the heart to treat fibrosis [83]. The first microRNA mimic therapy to enter clinical trials was MRX-34—a liposomal-encapsulated microRNA mimic from Mirna Therapeutics meant to treat a variety of cancers [84]. However, the company terminated the study earlier in 2017 after reports of several immune-related severe adverse events [85]. The fact that the adverse events were immunological in character further highlights the importance of RNA modification for clinical applications, as such modifications remain one of the most important means of evading immune detection for RNA drugs. Chemical modification of miRNA mimics in particular, however, might prove challenging owing to the complex nature of miRNA-induced gene regulation [86].

Protein overexpression—mRNA

Expression of disease-relevant proteins can be achieved by intracellular delivery of plasmid DNA (pDNA) or messenger RNA (mRNA). Application of DNA or mRNA as protein intermediate enables expression of virtually any desired protein inside the host cells and tissues. This approach can address formulation and delivery challenges encountered with protein-based drugs, especially those aimed at intracellular targets [87]. mRNA-based therapeutics in particular offer several advantages over pDNA, including rapid and transient protein production, no risk of insertional mutagenesis, and greater efficacy of non-viral delivery by virtue of mRNA cytoplasmic activity (Fig. 2) [88]. Since the first pre-clinical studies in the 1990s, mRNA technology has greatly developed and now holds the potential to revolutionize vaccination, protein-replacement therapies, and treatment of genetic diseases, consequently gaining a considerable level of interest among the scientific community and biotech industry [53].

The delivery of mRNA therapeutics has been facilitated by significant progress in maximizing the translation and stability of mRNA, preventing its immune-stimulatory activity and the development of in vivo delivery technologies, some of which are discussed below. The 5′ cap and 3′ poly(A) tail are the main contributors to efficient translation and prolonged half-life of mature eukaryotic mRNAs. Incorporation of cap analogs such as ARCA (anti-reverse cap analogs) and poly(A) tail of 120–150 bp into in vitro transcribed (IVT) mRNAs has markedly improved expression of the encoded proteins and mRNA stability [89, 90]. New types of cap analogs, such as 1,2-dithiodiphosphate-modified caps, with resistance against RNA decapping complex, can further improve the efficiency of RNA translation [91]. Replacing rare codons within mRNA protein-coding sequences with synonymous frequently occurring codons, so-called codon optimization, also facilitates better efficacy of protein synthesis and limits mRNA destabilization by rare codons, thus preventing accelerated degradation of the transcript [92, 93]. Similarly, engineering 3′ and 5′ untranslated regions (UTRs), which contain sequences responsible for recruiting RNA-binding proteins (RBPs) and miRNAs, can enhance the level of protein product [53, 94]. Interestingly, UTRs can be deliberately modified to encode regulatory elements (e.g., K-turn motifs and miRNA binding sites), providing a means to control RNA expression in a cell-specific manner [95]. Some of the previously discussed RNA base modifications such as N1-methyl-pseudouridine have not only been instrumental in masking mRNA immune-stimulatory activity but have also been shown to increase mRNA translation by enhancing translation initiation [96, 97]. In addition to their observed effects on protein translation, base modifications and codon optimization affect the secondary structure of mRNA, which in turn influences its translation [98]. Understanding the importance of, and the ability to predict, the folding structure of mRNA could aid engineering of mRNA therapeutics—however, the accuracy of available prediction tools is currently limited. Despite the plethora of carriers studied for other types of RNA drugs, mRNA molecules are significantly larger (600–10,000 kDa) than the previously discussed siRNAs (~14 kDa) and ASOs (4–10 kDa), which poses an additional challenge for delivery of mRNA therapeutics [99]. Accommodation of large and charged mRNAs into nanoparticles and their effective intracellular release has been shown to require fine-tuning of existing formulations and the development of a new-generation of biomaterials with higher potency [36, 37].

Therapeutic applications of mRNA that are currently being explored are vaccinations against cancer and infectious disease, protein-replacement therapy, and gene editing. A comprehensive list of ongoing clinical trials involving mRNA can be found in Table 2. mRNA vaccines are in the most-advanced stages of clinical development, following in the footsteps of competing DNA and protein-based technologies. Synthetic mRNA vaccines allow simultaneous delivery of a wide variety of antigens and are both faster and easier to manufacture at low cost in comparison with other systems, enabling a more-rapid response towards emerging pathogens [100]. Additionally, immune responses generated by naked mRNA can be beneficial for vaccination purposes [101, 102]. Immunization against infectious diseases using ex vivo mRNA-transfected dendritic cells (DCs) is now being pursued in clinical trials and has demonstrated good safety profiles and ability to induce antigen-specific T-cell responses [103].

Another RNA vaccination approach is the use of self-amplifying mRNA replicons that have been developed to extend the duration and magnitude of antigen expression as well as boost the immune response [104, 105]. In a recent study, replicon vaccines formulated into nanoparticles comprising repeatedly branched dendrimer (tree-like) molecules have generated protective immunity against a broad spectrum of lethal pathogens, including Zika, Ebola and influenza viruses [106]. Conventional, modified mRNAs are also being explored for vaccination [105]. Lipid-nanoparticle-encapsulated mRNAs encoding pre-membrane and envelope glycoproteins of Zika virus have recently been reported to elicit potent and durable neutralizing antibody responses in mice and non-human primates against the virus after intradermal administration [107]. Moreover, expression of modified mRNA encoding broadly neutralizing antibody in the liver, after systemic administration of mRNA–LNPs, has protected humanized mice against HIV-1 challenge [108]. Cancer mRNA vaccines have experienced accelerated development and clinical translation driven by the success of cancer immunotherapy. The majority of approaches tested in clinical trials employ adoptive-transfer of DCs transfected with mRNAs coding for tumor-specific antigens (TSAs) and immunomodulation of T cells with mRNAs expressing chimeric antigen receptors (CARs) or TSAs [109]. In addition, direct intradermal and systemic administration of LNP-formulated mRNAs coding for tumor-specific antigens is currently being investigated in the clinic for induction of T-cell immune responses [100, 110, 111].

By contrast, most mRNA-based protein replacement therapies are still in the preclinical stages of development and involve supplementation of deficient or aberrant proteins as well as modulation of cell behavior by expression of exogenous proteins. The in vivo efficacy of RNA–protein therapy has been demonstrated for a number of diseases. The majority of the studies preferentially target the liver owing to the well-established and efficient methods for RNA delivery into liver tissue. Therapeutically relevant amounts of human FIX (hFIX) protein were reached and sustained physiological activity for 4–9 days upon a single intravenous dose of hFIX mRNA-loaded LNPs in mice with hemophilia B [112, 113]. Similarly, LNPs formulated with mRNA encoding erythropoietin (Epo) have been shown to elicit a systemic physiological response in large animals, including pigs and nonhuman primates [93]. Therapeutic effects of mRNA have also been demonstrated in other tissues. Lung delivery of surfactant protein B (SP-B) mRNA protected mice from respiratory failure [114], whereas myocardial injection of RNAiMAX-formulated mRNA, encoding human vascular endothelial growth factor A (VEGF-A), improved heart regeneration after myocardial infarction in mice [115]. Based on this notion, Astra Zeneca partnered by Moderna has launched a phase I clinical trial for local delivery of VEGF mRNA, starting January 2017 [116]. Pre-clinical studies have demonstrated the translational potential of mRNA-based protein therapy for both secreted and intracellular protein targets. However, treatment of chronic diseases might carry an elevated risk of toxicity, associated with the repeated mRNA–LNP administrations required to sustain therapeutic levels of protein. Using mRNA for delivery of gene editing tools could address this challenge and is discussed below.