An Overview of RNA Delivery: Challenges and Innovation
Abstract
Historically, RNA has been used to uncover the genetic basis and details of specific biological mechanisms. During the last three decades, the potential for RNA to serve as a therapeutic modality has been recognized and early successes achieved. There are challenges to the delivery of RNA, however. Various approaches have been developed for both in vitro and in vivo applications. Lipid-based solutions have been used in approved products, but issues with specificity and stability remain that continue to be the focus on innovative research.
RNA Brings Greater Versatility
Ribonucleic acid (RNA) in its various forms helps translate information encoded in deoxyribonucleic acid (DNA) into cellular functions via the production of various proteins and enzymes. Messenger RNA (mRNA), transfer RNA (tRNA) and ribosomal RNA (rRNA) comprise the three major classes.1
Unlike DNA, RNA is relatively short-lived due to degradation by RNase enzymes. On the positive side, RNA does not need to enter the nucleus to initiate protein production. For this reason, RNA – both mRNA and anti-sense RNA (RNAi), which binds to specific RNA molecules to inhibit their activity – has been extensively used by researchers to uncover the genetic basis and details of specific biological mechanisms, information that has increased understanding of many diseases and their causes.
RNA interference was first discovered in 1990 when scientists observed that particular RNA strands had the ability to suppress gene expression in plants.2 This discovery catalyzed extensive research focused on understanding RNAi mechanisms of action, leading to the identification of different types of RNA, including small interfering RNA (siRNA), a small non-coding, double-stranded RNA that is generally complementary to the coding region of a target mRNA; microRNA (miRNA), small non-coding RNA molecules that regulate the expression of multiple mRNAs by blocking translation or promoting degradation; and short/small hairpin RNA (shRNA), synthetic RNA strands with tight hairpin turns that can be used for gene silencing.
The blockage of post-transcriptional translation by small pieces of RNA to silence particular genes or sets of genes is a natural process in normal cell regulation. By targeting specific RNA sequences, researchers can determine which genes or sets of genes control the expression of specific proteins. This knowledge has been applied to the development of RNA-based therapeutics.1,3
RNAi therapies, several of which have been approved by the US Food and Drug Administration (FDA), are based on short single-stranded oligonucleotides (antisense oligonucleotides, or ASOs) that are complementary to a certain region of a target RNA (such as an mRNA).4 A couple of FDA-approved RNA treatments are based on siRNA, such as Patisiran (marketed as Onpattro) and Givosiran (marketed as Givlaari)5. One RNA aptamer drug – a short single-stranded nucleic acid that can bind a specific protein, peptide, carbohydrate, or other types of molecules depending on its tertiary structure – has also been approved by FDA, under the name of Macugen (pegaptanib).
Increasing numbers of RNA interference therapies and vaccines are also progressing rapidly through clinical development, including those based on ASOs, siRNAs, and miRNAs, among others.5,6
In addition to RNA technologies that directly interfere with expressed mRNA or mRNA expression, blocking protein production, there are those that cause the expression of target proteins whose deficiencies can lead to disease.
Exogenous delivery of coding synthetic mRNA has of course been leveraged for COVID-19 vaccines and is being developed for many other antiviral and anticancer vaccines and cancer immunotherapies progressing through clinical studies.7-14 Newer candidates leverage self-amplifying mRNA to enable reduced doses and dosing frequencies.15 RNA is also being explored in the fields of regenerative medicine, in vivo delivery of gene-editing agents, gene therapy, and for the reprogramming of cells for research and therapeutic applications.4,16-18 Compared to plasmid DNA, RNA therapeutics and vaccines provide transient expression in the cytoplasm. There is no requirement for delivery into the nucleus and no risk of insertional mutagenesis.4,8 mRNA vaccines, meanwhile, can be rapidly designed and manufactured using platform processes.20
In addition, unlike small-molecules drugs, which are limited to interacting with the small percentage of proteins that have active binding sites (known as “druggable targets”)2, RNA as an active therapeutic or immunological agent modulate protein production by leveraging existing cellular biochemistry3. Similarly, biologic drugs such as recombinant proteins and antibodies need to interact with proteins that have already been expressed, and also have the correct structure, folding properties, and post-translational modifications to do so.
Furthermore, to produce RNA therapeutics and vaccines only requires the target nucleotide sequence, whether that is a malfunctioning mRNA or a viral protein.3 The active drug substance or vaccine active can then be quickly synthesized. There is no need for the development of the specialized cell lines and cell culture/fermentation processes required to produce recombinant proteins or establishment of the complicated synthetic routes necessary to manufacture structurally complex chemical APIs, both of which are time- and cost-intensive.
Market estimates for RNA therapies and vaccines are generally presented separately for RNA antisense/interference and mRNA products. Global revenue for the former in 2021 is reported to be nearly $5.88 billion and expanding at a compound annually growth rate (CAGR) of 18.68% through 2032.21 The value of the market is projected to surpass $6.99 billion by the end of that year.22 Drivers include the applicability to treat both common and rare diseases, the specificity and safety of RNA-based medicines, growing use of RNA molecules for gene-editing applications and the expansion of many DNA-focused companies into the RNA sector.
The value of the global market for mRNA therapeutics and vaccines is estimated to reach $128.14 billion by 2030.23 Not surprisingly, by application, the infectious diseases segment accounts for the highest revenue currently, as does the prophylactic vaccines segment by type. In addition to the success of the COVID-19 mRNA vaccines, other growth drivers include the rising prevalence of long-lasting infectious diseases, advances in stabilization and delivery technologies for mRNA, and the ability to use mRNA therapeutics as personalized medicines. According to one market research firm, as of late October 2022, more than 35 companies were evaluating over 195 mRNA therapeutic and vaccine candidates at different stages of development for a myriad of disease indications.24
Delivery Challenges
The delivery of RNA to cells both in vitro and in vivo is essential to enabling fundamental research and the development of effective RNA-based therapies, vaccines, and regenerative medicines. Whether for smaller RNAi forms or larger mRNA strands, safe and efficient methods of delivery without causing cellular damage or RNA degradation are needed. For instance,controlling mRNA activity once it has entered the desired cellscan be problematic (4).Most critically, RNA is inherently unstable with respect toenvironmental conditions (temperature, pH) and readilydegraded by RNase, a ubiquitous enzyme both outside andinside the body. As such, unprotected RNA strands rapidlydegrade in the bloodstream, making delivery to target cells andachievement of long-term inhibitory or expression effectschallenging (4). In addition, due to their size and charge, nakedRNA molecules also have difficulty passing through cellularmembranes. The need for delivery of naked RNA is generally limited toresearch applications performed in vitro/ex vivo. In this context,physical methods including electroporation, microinjection,and gene guns are typically used. These technologies result indisruption of the cell membrane.(8) Electroporation isassociated with high cell death rates and requires serum-freetransfection conditions and large quantities of RNA.The use oflipid Nanoparticle (LNP)-mediated mRNA delivery to human Tcells has also been reported in the literature to inducefunctional protein expression, suggesting that LNPs hold agreat potential to enhance mRNA-based CAR T cellengineering methods. High mRNA transfection efficiency data in primary human T-cells from peripheral blood and in primaryHuman CD34+ HSC from placental blood have also been presented in the webinar “BioInsights - How are cationic lipids offering new possibilities in the delivery of RNA therapeutics?"citing the use of Sartorius Polyplus® lipid-based jetMESSENGER®, invivo-jetRNA®+ and cationic LNPs.When working with in vivo RNA delivery methods, ensuring both safety and efficacy requires efficient and targeted delivery with minimal production of unwanted side products and off-target effects. Physical methods are generally not suitable because they result in the degradation and clearance of foreign materials during circulation (10).
Exploring Delivery Solutions
Chemical methods are more attractive for in vivo delivery of RNA. Polymers, lipid-based solutions, polypeptides, dendrimers, gold nanoparticles and hybrid systems are all under investigation (Figure 1). The use of conjugates, such as N-acetylgalactosamine (GalNAc), that bind to specific receptors on the surfaces of cells is also being explored as a delivery mechanism for RNA (1). The most widely employed solution is the encapsulation of the RNA in lipid nanoparticles (LNPs).(25) This solution was used for both FDA-approved mRNA-based COVID-19 vaccines and other marketed RNA therapeutics. Many RNA-based drug candidates currently in the clinic also leverage LNP delivery technology (25,26). Extracellular vesicles and patient-derived dendritic or mesenchymal stem cells can also be used to deliver RNA to many different types of cells, even those in the brain (30) Chemical transfection reagents such as polyethyleneimine (PEI) if appropriately formulated can also be used for both in vitro and in vivo RNA transfection. For example, in vivo- jetPEI® has been recently used in the frame of a saRNA vaccine study against HIV, described in the publication by Aldon et al (31). Although viral vectors are widely employed for the delivery of DNA-based gene therapies, recombinant viruses do not play an important role as delivery systems for RNA (8). RNA molecules are typically larger than plasmid DNA, particularly mRNA, and generally too large for most viral vectors. It is worth noting, however, that mRNA packing virus-like particles have been investigated for the therapeutic in vivo delivery of gene- editing agents (16).
The use of LNPs as delivery vehicles for RNA-based therapeutics and vaccines can in part be attributed to the successful approval of both RNAi and mRNA products formulated as LNPs. The first RNAi therapeutic approved by the FDA in 2018 (Onpattro®, patisiran, Alnylam Pharmaceuticals) (32) and the COVID-19 vaccines (33) from Pfizer-BioNTech (BNT162b2, also known as Comirnaty® and Moderna (mRNA-1273, also known as Spikevax®) have demonstrated the value and rapid translational potential of LNPs. Furthermore, in preclinical studies, LNPs have been used to deliver nucleic acids beyond siRNA and mRNA, such as ASOs (34), microRNA (35), and DNA (36). Several clinical trials are currently assessing LNP delivery of a broad range of payloads, including the first in vivo CRISPR/Cas9 treatment delivered intravenously to treat patients with ATTR (37).
Innovative Technologies as Solutions
Significant advances have been made with respect to the development and delivery of RNA-based therapeutics and vaccines. Even so, there are still challenges that must be overcome (38). Targeting of certain types of tissues and cells will be necessary before RNA therapeutics can be expanded to a broader set of diseases. Reduction of toxicity continues to bean issue as well. The thermal instability of RNA molecules mustalso be addressed to enable products that can be stored andshipped at reasonable temperatures. Approaches to these problems include chemical modificationof both the RNA structure and the lipids used to form theencapsulating LNPs (39). Antibodies, nanobodies, aptamers,peptides, small molecules, and other options are beinginvestigated as means for providing targeted tissue delivery.Modifications to various positions on the RNA backbone toincrease RNA stability are also being explored.
Figure 1: Polyplus alternative approches to improve in vivo nucleic acid delivery challengers: cationic polymer, lipids and lipid nanoparticles
An alternative approach involves the development of novel ionizable and cationic lipids—key components of LNP formulations—and potentially additional types of lipids that can increase selectivity for certain tissues (40). Attachment of mAbs or other molecules that can bind to specific cell receptors to the LNP lipid components is also being investigated. Sartorius Polyplus® has contributed over the years to tackle challenges of in vitro and in vivo gene delivery. The cationic polymer- based in vivo -jetPEI® formulation for DNA/siRNA/miRNA/oligonucleotides in vivo delivery has been successfully used over the years in multiple studies, and cited in over 600 peer-reviewed publications. Reagent’s efficiency, safety and GMP grade availability has also made it the delivery vector of choice for several clinical trials in different Phases (Preclinical, I, II and III). As an alternative to cationic polymers, classical cationic lipids used for LNPs (DOTMA and DOTAP) have been recognized to elicit toxicity due to their tri-methyl ammonium part. The use of ionisable lipids (Dlin-MC3-DMA, SM-102 or ALC-0315) has been a valid alternative so far, however, more efforts are needed to overcome their restrictive biodistribution and stability.
For this purpose, Sartorius Polyplus® has recently developed a library of cationic lipids LipidBrick® (European Patent EP3646854 B1),which represents a remarkable innovation in comparison to the classical cationic lipids. The polar head which is an imidazolium, and the presence of a R1 alkyl group “masking” the positive charge, both contribute to a reduced toxicity. As LNP electric charge is known to control in vivo distribution and expression of mRNA (40), the use of Sartorius Polyplus® cationic lipid in LNPs formulations broadens their current applications spectrum interms of potency and targeting. Available soon to be used as an active component for LNPs manufacturing, our proprietary cationic lipid (LipidBrick® IM21.7c) is already included in the formulation of in vivo-jetRNA®+, our liposome-based product for in vivo mRNA delivery. This product shows an amazing capacity to encapsulate 100% of the mRNA available, leading to comparable LNP delivery results, while remaining an off-the-shelf, user friendly and easy to use reagent for mRNA delivery in vivo.
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