Christian Stackhouse

This is the first article of a series on the state of emerging therapeutic modalities and related technologies. In this installment, we review the RNA therapeutics scientific and clinical development landscape with emphasis on oncology, immunology, neurology, & diabetes applications. I will start each of these installments with operational definitions & essential background of the therapeutic modality used to frame the overview. Then I will provide a brief history on the field from its first appearance through the present day. We will then explore the present state of the field including current opportunities & applications in the disease areas of interest. Finally, each installment will end with a look at future prospects & potential developments in the field as they relate to the clinical development landscape. Background & Definitions: Ribonucleic acid (RNA) is an integral part of the central dogma of biology where genetic information is encoded in the deoxyribonucleic acid (DNA) sequence of a cell which is then transcribed into RNA, some of which are then translated into proteins which are the functional (or not) molecules for cellular processes. Traditional medicine has focused on targeting proteins which are uniquely associated with a diseased cell or which may be malfunctioning to cause a disease state. RNA therapeutics broadly can refer to the treatment or prevention of disease utilizing RNA-based agents or by targeting RNAs whose expression is related to the disease state. For this article, we will use this definition when referring to RNA therapeutics as a field. History: Nucleic acids (the base units of DNA & RNA) were discovered by Friedrich Miescher in the last half of the 19th century. It wasn’t until the 1930s that biologists began to recognize a relationship of nucleic acids to cellular growth and development. In 1947, Andre Boivin and Roger Vendrely published the hypothesis that DNA governed the assembly of RNA which in turn controlled the production of cytoplasmic enzymes. In the 1950’s, work by Rosalind Franklin, Francis Crick, and James Watson revealed the double helical structure of DNA followed by the postulation of Crick for the existence of messenger RNA (mRNA) that serves as a template for protein production. In 1961, Watson and colleagues published in Nature that they had isolated these mRNA intermediates. Over the next three decades, scientists continued to discover new forms and functions of RNAs until a breakthrough experiment by Jon Wolff demonstrated that in vivo transcribed mRNA or plasmid DNA directly injected into the muscle tissue of mice were able to enter the cells and were translated into proteins in the cytoplasm (1). This experiment and the subsequent discovery of the RNA interference (RNAi) process 8 years later by Andre Fire & Craig Mello sparked a surge in RNA research giving rise to the field of RNA therapeutics. Mechanisms of RNA Therapeutics: The direct introduction of synthesized mRNAs can be used for protein or peptide replacement or introduction. Once mRNAs reach the cytoplasm of target cells, they take advantage of the cell’s translational machinery (ribosomes & tRNA) to produce a peptide or protein of interest with therapeutic benefit. These mRNAs can be chemically modified are designed to increase their stability in the cell, but ultimately, liner mRNA transcripts are degraded fairly rapidly in most cells by nucleases making this type of therapy more transient than other methods. One potential solution for improved stability is being leveraged by Orna Therapeutics in the form of engineered circular RNAs (oRNA) that are inherently less suitable to nuclease degradation than linear RNAs. Merck (MSD) recently announced a collaboration with Orna based on the promise of the improved in vivo stability of the oRNA platform verses linear mRNA. RNAi refers to the delivery of RNAs (or RNA/DNA hybrids) such as antisense oligonucleotides (ASOs or AONs) or short interfering RNAs (siRNAs) designed to bind nucleic acid targets to degrade, sequester, or block active domains of the target. ASOs can also be chemically modified with added labels or tags to identify subcellular localizations of targets or to isolate targeted transcripts. This has applications in drug design, basic research, diagnostics, and for measuring a patient’s response to treatment or disease progression when paired with medical imaging modalities such as positron emission tomography (PET or PET-CT) (2). An additional application involves chemically modified ASO-mediated control of RNA splicing which can be directed to block the incorporation of mutated exons (exon skipping) to rescue expression of near-full length, functional proteins such as in Duchenne Muscular Dystrophy (DMD) (3). ASO-mediated steric hindrance can also affect gene regulation by interfering with epigenetic modification & chromatin state mediated by long non-coding RNAs (lncRNAs) (4). In March of this year, NextRNA Therapeutics launched with $9.3M from seed financing and $46.8M from Series A on a platform aimed to disrupt the binding activity of non-coding RNAs, particularly lncRNAs, with small molecules. Development of RNA vaccines are also increasing for applications beyond infectious disease including cancer vaccines. The premise of mRNA vaccines is that delivery of a synthetic mRNA encoding cancerspecific epitopes found on the surface of malignant cells are expressed in antigen-presenting cells (APCs) which facilitate activation of the innate & adaptive immune system. In this way, the patients own immune system is trained to recognize cancer cells and eliminate them. In contrast to the immune stimulatory effect for most vaccines, a recent publication in Science describes the development of a 1 methylpseudouridine-modified mRNA vaccine that may reduce the damaging autoimmune response for disorders such as multiple sclerosis (MS) (5). They find that maintenance of this therapeutic effect is dependent on expression of immune checkpoint signaling including PD-1 & CTLA-4 signaling. Treatment with this modified mRNA vaccine reduced proinflammatory cytokine release in animal models of MS suggesting a possible application for CRS management experienced with certain cell therapies. Gene editing traditionally is mediated by DNA plasmids encoding the gene-editing enzyme machinery and guide RNA (gRNA) sequences to accomplish targeted edits within cells. We will discuss gene editing in greater detail as the subject for the next installment. These plasmids are often delivered using transfection reagents or viral vectors which have variable efficacy based on cell type & significant safety risks. A drawback of DNA plasmid transfection for in vivo editing applications is that the persistent expression of gene editing components can increase the risk of off-target effects and of altering cellular phenotypes in unpredictable ways. Researchers at Tufts University recently described lipid nanoparticle (LNP) delivery of CRISPR-Cas9 ribonucleoprotein (RNP) co-packaged with a guide RNA (gRNA) or LNP delivery of CRISPR-Cas9 mRNA resulting in efficient, transient gene editing with non-integrating Cas9 expression (6). This represents a significant opportunity for the future development of in vivo gene editing therapeutic approaches leveraging the systems and lessons from RNA therapeutics to combine these therapeutic modalities. RNA aptamers are sometimes referred to as chemical antibodies. These molecules are short nucleic acid sequences (DNA or RNA) that bind to specific targets (proteins, DNA, or RNA) similar to traditional protein antibodies, but they are much more versatile than traditional antibodies and have distinct properties that differentiate them from other RNA-based therapies. Aptamers, like ASOs, can be chemically modified to reduce immunogenicity, resist degradation, enhance potency, or attach labels. There are also peptide-based aptamers with broad therapeutic applications. I will discuss aptamers as a therapeutic modality in the 3rd installment of this series. Challenges – Delivery/stability, structure/chemical synthesis, & off-target effects/immunogenicity: As mentioned previously, cells in the body including malignant cells have a host of mechanisms for degrading and recycling RNAs in the cytoplasm and nucleus. Additionally, RNA is inherently unstable, especially in linear form. Your skin and the surfaces around you have nuclease enzymes that rapidly degrade RNA meaning the study, production, storage, and administration of RNA-based therapeutics require special processes & conditions to protect the integrity of these molecules. Many ASOs employ a “gapmer” design strategy with 3 or 5 modified RNA bases on each end of the oligo separated by 8 to 12 oligodeoxynucleotide gap. This design along with the base modifications enhance the binding of the ASO to the target. Adding phosphorothioate (PS) modified linkages to the backbone of ASOs further improves their stability and activity by protecting the oligo from nuclease degradation while promoting RNase H1-mediated degradation of the ASO target. PS modifications also introduce chiral centers into the oligonucleotide, which can be controlled using stereoselective synthesis methods which can further control the binding affinity, stability, and other properties of ASOs (7). Furthermore, PS gapmer ASOs acting through RNase H1 degradation are not cleaved in the process meaning the activity of these ASOs can persist for long periods of time and high levels of activity can be achieved with fewer molecules in the cell. Still other modifications can produce an ASO which binds but does not degrade its target serving to disrupt activity by steric hindrance or even can be linked to a tag or marker to label the target molecule. RNA therapies can sometimes be delivered “naked” in suspension by direct injection or administration to the targeted site. These RNAs can then enter the cell via a process called gymnotic uptake (gymnosis) by which transmembrane transporters bring the extracellular RNAs into the cell (8). RNAs can be chemically modified to increase the efficiency of uptake and can be designed to enter the cell nucleus or to remain in the cytoplasm depending on the subcellular location of the intended target. RNA therapies have also commonly utilized lipid nanoparticle (LNP) packaging and delivery for systemic administration. There are advantages and disadvantages to this approach, the largest drawback being accumulation in the liver and spleen (unless those are the target organs). Kumthekar et.al. have described the use of spherical nucleic acids (SNAs) that consist of a gold nanoparticle core with covalently linked oligonucleotides for RNAi applications in the CNS (9). There are numerous alternative nanoparticle delivery systems in development including self-assembled RNA nanoparticles like the phi29 pRNA hexameric ring formation which can be modified for in vivo delivery applications (10,11). Delivery using nanoparticles is a complex bioengineering problem. Patisiran (Alnylam Pharmaceuticals siRNA for polyneuropathy FDA approved in 2018), Tozinameran (Pfizer-BioNTech COVID-19 vaccine approved in 2021), and Elasomeran (Moderna’s conditionally approved COVID-19 vaccine) all deliver RNA payloads via LNPs. Two large differentiation points across these products demonstrate the complexity of LNP-RNA design. While the two COVID vaccines can be administered intramuscularly in seconds, Patisiran is administered by IV with over 70min infusion time (12). Patisiran must be stored between 2 & 8 degrees C and will lose efficacy if frozen. The Pfizer/BioNTech vaccine must be stored between -60 & -90 degrees C while the Moderna vaccine can be stored from -15 to -50 degrees C. These differences are in part because of the stability of siRNA vs mRNA, but also due to the composition & design of the LNP (12). Off-target and undesired host immune activation are additional challenges for RNA therapy design. Base constrained ASO modifications such as locked nucleic acids (LNA), 2’-O-methoxyethyl (2’-MOE) oligonucleotides, and 2’,4’-constrained ethyl (cEt) modifications can further enhance binding affinity, target specificity, and resistance to nucleases. However, LNA ASOs have been found to have neurotoxic and hepatotoxic effects. Base modifications within CpG motifs of oligonucleotides can reduce potentially toxic immunogenicity. AUM Biotech’s proprietary FANA technology has the advantages of self-delivery, reduced off-target effects, and improved stability over other RNA modalities. AUM Biosciences is the clinical stage development arm leveraging modified ASOs for oncology applications. Applications of RNA Therapeutics: RNA therapies in the U.S. marketplace are relatively young. The first RNA-based FDA approval was the ASO Fomivirsen for cytomegalovirus retinitis (CMV) delivered via intraocular injection in 1998. It was developed by Ionis Pharmaceuticals (formerly Isis Pharmaceuticals) and licensed to Novartis to address the high incidence of CMV in HIV/AIDs patients. Ionis also secured FDA approval for the first ASO, Eteplirsen, to affect RNA-splicing by steric binding to promote exon skipping and restore mostly functioning dystrophin protein to DMD patients. Alnylam Pharmaceuticals secured the first FDA approval for Patisiran, the first siRNA used to treat polyneuropathy caused by a form of hereditary amyloidosis (13). BNT162b2, now Comirnaty, was the first mRNA vaccine granted Emergency Use Authorization (EUA) and then the first FDA-approved mRNA vaccine having been developed by BioNTech and Pfizer. There are several disease-specific applications in development described below: NeurologyThere are 10 FDA-approved RNA-based therapeutics for the treatment of neurological diseases including 8 ASOs from 1998 (fomivirsen) to 2021(Casimersen), 1 siRNA (patisiran 2018), and 1 RNA aptamer (pegaptanib 2004) (14). Four of the approved ASOs are for DMD. These are examples of splice-switching ASOs (ssASOs). A host of neurological and neurodegenerative diseases are related to alternative splicing or mutations in exon-encoding regions that produce malignant or non-functional proteins. Preclinical studies suggest ssASOs may have therapeutic applications in dementia-causing diseases and “tauopathies” by reducing or preventing plaque formations (15,16). A challenge for all drugs is delivery to the brain and crossing the blood-brain barrier (BBB). Intrathecal (IT) administration is a common CNS delivery route introducing drugs into the subarachnoid space and cerebrospinal fluid (CSF) for efficient distribution in the CNS compartment. Intraventricular injections are another route entering CSF circulation and intravitreal or intraocular injections are also common routes of administration. A class of arginine-rich cell-penetrating peptides conjugated to ASOs have shone to cross the BBB in animal models (17). Exosomes and nanoparticles have also been decorated with brain-targeting peptides for enhanced delivery to the brain. OncologyImmunotherapeutic approaches in oncology are a rapidly growing market segment across the globe. RNA therapies are crossing into this space through RNA cancer vaccines which are targeted to APCs to train the host immune cells to recognize and eliminate cancer cells (18). A PD-L1 targeted siRNA conjugated to a magnetic nanocarrier has demonstrated robust pancreatic cancer tumor reduction preclinically that thanks to the magnetic conjugate, can also be used to monitor therapy response by MRI (19). Some RNA therapies are delivered with engineered viral vectors because of their payload capacities and long history of use as vectors in biotechnology. Inorganic gold or iron-oxide nanoparticle delivery system have theragnostic potential both in targeted delivery of the drug, but also enabling noninvasive visualization to identify distal metastases or gauge tumor mass and invasion into normal parenchyma (20). Photothermal and photodynamic therapies are being combined with RNA therapies for additive or synergistic anti-tumor effects including highly-specific RNAi targeting where focused electromagnetic or x-ray wavelengths trigger release of the RNAi payload only in the specific targeted site (21). RNA therapies could represent a revolution in the treatment of cancer and other diseases by opening new therapeutic targets to pathways or malignant programs once thought “un-druggable.” It is possible the species of RNAs such as lncRNAs or circRNAs can serve as master regulators of upstream disease-programs by affecting cellular programs at the epigenetic level (22). Enfuego Therapeutics founded by Chad Pecot at UNC-CH received STTR Phase 1 funding in 2020 to design ASOs to elusive oncology targets previously thought “undruggable.” ImmunologyClearly, mRNA vaccines against COVID-19 have left their mark on the RNA and immunology landscapes for protection against infectious diseases. As mentioned in the previous section, RNA therapies including RNA cancer vaccines are bridging the two modalities and demonstrating the versatility of RNAimmunotherapy combinations. Traditional vaccines are based on live attenuated infectious peptides or whole killed pathogens to stimulate immune response and memory. RNA-based vaccines can be used to express one or more specific epitopes of an infectious organism on APCs producing safe, long-lasting immune responses and have the potential for rapid development and production (23). Another potential advantage for mRNA vaccines is described in a 2017 manuscript by Stitz et.al. where they developed a thermostable mRNA vaccine against rabies (24). Most vaccines require special storage conditions including cryopreservation from production until administration to the patient. The thermostable mRNA vaccine described here represents opportunities for greater access to life-saving prophylactic vaccines even in remote areas that lack modern refrigeration. DiabetesType 1 diabetes is a lifelong disease that could potentially be cured with xenotransplantation or allotransplantation of pancreatic tissue, but these procedures are highly invasive with significant mortality and morbidity (25). Recent efforts to engineer pancreatic islet cells for transplantation are now leveraging gene editing and RNAi methods to potentiate immune responses, reduce risk of acute or chronic rejection, and improve outcomes for transplant patients (26). In 2006, researchers at the University of Bracelona, demonstrated that transient RNAi against Phosphoenolpyruvate carboxykinase (PEPCK) lowered blood glucose and improved glucose tolerance in diabetic animal models suggesting an alternative treatment mechanism for diabetes (27). Diabetic patients often suffer from chronic wounds related to the disease that can be life-threatening. Two recent articles have described improved diabetic wound healing using engineered miR-31 exosomes or engineered hydrogels containing siRNAs which reduce inflammatory signaling and promote wound healing programs (28,29). This again demonstrates the breath of therapeutic applications and opportunities for RNA-based therapies. Conclusions & Future Directions: The RNA world is vast, diverse, and our understanding of RNA biology is constantly evolving. A class of circular RNAs (circRNAs) which were discovered more than 4 decades ago have become attractive therapeutic targets and engineered synthetic circRNAs are gaining commercial interest as an RNA-based therapeutic platform. Similar to NextRNA Therapeutics previously mentioned, some companies like Ribometrix are developing small molecules targeting malignant RNAs based on their unique 3D structure. The global pandemic has attracted more interest than ever before in the field. Scientific knowledge and commercial interest in RNA-based therapeutics are on the rise. As of August 18, 2022, 1,376 Scientific publications of RNA therapies appear in NCBI PubMed records since the early 1990’s (PubMed). Nearly 81% of these were published in the past decade and more than 40% since 2020. This represents a 22.56% CAGR of publications from 2015 to 2021. There are 407 U.S. patent filings for “RNA vaccines” with nearly 72% filed since 2015 and over 31% of all patents filed since 2020 (USPTO). However, there are still opportunities for advancement and discovery in RNA therapy field. Less than 5% of oncology/cancer-related U.S. patents include RNAi applications and only around 0.1% of oncology patents mention lncRNAs. Targeted in vivo delivery remains challenging for many disease applications. Methods for discovering disease-related targets and subsequent design of RNA therapies with high efficacy, specificity, and favorable safety profiles are rapidly evolving. High-throughput screening approaches for candidate selection and testing can be costly. Numerous in silico-based approaches for rational design of RNA drugs are under development including a commercial digital platform (Benchling) that has garnered attention from numerous pharmaceutical & biotechnology companies. Staying on top of the RNA scientific and clinical development landscape can be challenging, especially given the rapid growth of new companies and the pace of new discoveries in this space. There are a multitude of promising preclinical and clinical stage RNA-based or RNA targeting products and platforms. For pharmaceutical, CRO, and biotechnology companies in or planning to enter this space, opportunities abound, but having the right partners with the knowledge and resources to navigate the RNA therapeutic/biotechnology landscape can help jumpstart your program(s) while avoiding pitfalls or setbacks. Want to learn more? Abbreviations: RNA – ribonucleic acid DNA – deoxyribonucleic acid mRNA – messenger RNA ncRNA – non-coding RNA lncRNA – long non-coding RNA miRNA – microRNA rRNA – ribosomal RNA RNAi – RNA interference siRNA – silencing or short/small interfering RNA LNP – lipid nanoparticles circRNA – circular RNA cET - 2’,4’-constrained ethyl base modification ASO/ANO – antisense oligonucleotides About the author: Christian T. Stackhouse, Ph.D. Christian has more than a decade of primary scientific research experience in the Life Sciences with more than 8years of experience in translational oncology research. He received his doctorate in Neuroscience from the University of Alabama at Birmingham working principally in the areas of neurooncology and RNA biology. Before joining Sedulo as a Client Engagement Manager, he completed a postdoctoral fellowship at Duke University in the hematology-oncology division of the Department of Pediatrics. Christian has numerous peer-reviewed scientific publications, abstracts, and has presented at national & international conferences for basic science, epidemiology, oncology, and computer science. His most recent article: “An in vivo model of glioblastoma radiation resistance identifies long non-coding RNAs and targetable kinases” was published in JCI Insight in August 2022. References: 1) Wolff, J. A., Malone, R. W., Williams, P., Chong, W., Acsadi, G., Jani, A., & Felgner, P. L. (1990). Direct gene transfer into mouse muscle in vivo. Science (New York, N.Y.),- Pt 1),-. https://doi.org/10.1126/science-) Saleem A, Matthews JC, Ranson M, Callies S, André V, Lahn M, Dickinson C, Prenant C, Brown G, McMahon A, Talbot DC, Jones T, Price PM. Molecular Imaging and Pharmacokinetic Analysis of Carbon-11 Labeled Antisense Oligonucleotide LY- in Cancer Patients. Theranostics. 2011;1:290-301. doi: 10.7150/thno/v01p0290. Epub 2011 Jun 1. PMID:-; PMCID: PMC-) Matsuo M. 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