Can RNA Simplify Gene Therapy Development?

Published on: 
Pharmaceutical Technology, Pharmaceutical Technology-07-02-2020, Volume 44, Issue 7
Pages: 22–25, 64

RNA is easier to manipulate than DNA but challenging to deliver to the right cells.

Originally it was thought that the only role of ribonucleic acid (RNA) was to translate genetic information encoded in DNA into protein sequences that perform critical biological functions. While messenger RNA (mRNA) is crucially important, it is just one of many types of RNA involved in a range of activities that affect the transmission of genetic information and the functioning-or dysfunctioning-of cells. Appropriately designed RNA-based therapies can thus modulate genetic information in a controlled and targeted manner, effectively acting as gene therapies. They also offer several advantages over DNA plasmid-based therapies.

Strands of RNA by themselves, however, are unstable and degrade rapidly in the bloodstream. Delivery to specific targeted cells is one of the main challenges that developers of RNA-based gene therapies are tackling today.

RNA technologies fall into two main categories: those that directly interfere with expressed mRNA or mRNA expression, blocking protein production, and those that cause the expression of target proteins whose deficiencies can lead to disease.

The first type includes RNA interference (RNAi) and antisense therapies, which in effect silence the expression of specific genes that are drivers of disease mechanisms. One example is small interfering RNAs (siRNAs), which according to David Evans, chief scientific officer at Sirnaomics, are useful as therapeutics where the gene(s) to be targeted is (are) overexpressed in the diseased state and where silencing these genes may produce a therapeutic benefit.

Indeed, Doug Fambrough, president and CEO of Dicerna Pharmaceuticals, believes that while supporting protein production is useful in a few contexts, such as treating rare genetic diseases or making vaccines, there are far more uses for silencing genes. “For example, silencing can be used to treat viral diseases, cardiovascular conditions, cancer, inflammatory diseases, fibrotic diseases, select rare diseases, and generally any condition where biological processes are inappropriately activated,” he asserts.

Sirnaomics and Dicerna are both focusing on RNA interference therapies. Belgium-based biotech company eTheRNA immunotherapies, on the other hand, has elected to focus on mRNA-based technologies that lead to the expression of proteins the body is unable to produce on its own, according to Wim Tiest, a special advisor at the company.

Several advantages over DNA

Unlike DNA-based gene therapies that must be delivered into the nucleus and create permanent changes, RNA therapies only need to reach the cytoplasm to perform their post-transcriptional activity and are not permanent, thus treatment can be modified or halted if necessary. They are also smaller, more easily manipulated, and do not present the risk of unintentional genetic effects.

“Messenger RNA and other RNA-based therapies have the advantage of being versatile (they can code for a broad range of targets), easy to produce (basically the production process is transposable between mRNA designs), and safe (due to the transient expression of the target and absence of risk for integration into the patients genome),” asserts Tiest. “Additionally,” he says, “mRNA is compatible with a range of systemic and local delivery modalities and can be designed to be nearly invisible to the host’s immune system, thus avoiding any vector-directed immune responses.”

RNAi technologies such as siRNAs also provide high target specificity by using gene sequences that match with targeted genes and rely on naturally occurring cellular enzymes to mediate gene silencing, according to Fambrough. Due to this high specificity-and reversibility-they offer the potential of fewer side effects. “Unlike gene therapy or gene editing, which can be permanent and could have unknown, unwanted, and irreversible consequences, RNAi therapies can offer targeted, effective treatment that does not permanently edit or change DNA. An RNAi therapy’s effect is targeted, precise, and can be stopped at any time by discontinuing treatment,” he explains.

More druggable targets

With the rapid progress made in sequencing the human genome, there is now a wealth of information on sequences of genes, some of which have been identified as playing a direct role in the etiology of diseases, according to Evans. “Many of these therapeutic targets, however, are undruggable by traditional small molecules or antibodies but are upregulated in disease states like cancer and fibrosis,” he notes. “siRNAs are readily designed against these gene sequences and can silence expression of the proteins that are driving disease, producing therapeutic benefit to patients with disease,” Evans says.

In addition, RNAi therapies offer a highly versatile approach that can benefit patients with an array of diseases, Fambrough points out. “Today, we can create siRNAs to match almost any messenger RNA. In effect, the RNAi approach to drug development can silence virtually any gene,” he notes.

Another benefit of RNA therapies compared to many conventional small-molecule and biologic drugs is the possibility of reducing the treatment burden for patients, according to Fambrough. “With a longer duration of effect, RNAi therapies can be administered with infrequent subcutaneous injections, and depending on the disease modality, can be self-administered at home,” he observes.



Stabilization and delivery challenges

RNA by its nature is rapidly degraded once injected. As such, delivery to the targeted cells is the main hurdle at this time. “We need to ensure that the siRNAs are able to reach their target tissue and cell types,” Evans states. In cancer, for example, that means accumulation of nanoparticles, for instance, at the tumor site with delivery of the RNA into the tumor cells specifically. Stabilization of RNA by modification of the chemical backbone is possible, and this approach can also help to make the RNA less immunogenic, Tiest notes.

Similarly, the inhibitory or expression effect wanes as the RNA degrades inside the cell. Self-amplifying RNA can to some extent prolong this active period, according to Tiest. The need for regular, repeated dosing and maintenance of a sufficient active dose is therefore also a major bottleneck. He does note that local administration helps to overcome some of this limitation.

Overall, careful selection of targets that require short interventions and can benefit from the increased safety profile of RNA is required to fully profit from RNA-based therapy development, Tiest concludes.


Multiple delivery solutions

Like most DNA plasmid-based gene therapies, RNA therapies can also be delivered into cells using viral vectors, but the majority of these therapies under development today rely on nonviral methods, such as organic (lipid) or inorganic nanoparticle-encapsulated complexes, extracellular vesicles, and patient-derived dendritic or mesenchymal stem cells, allowing delivery even across the blood-brain barrier (1).

Delivery technologies for intravenous, subcutaneous, and intramuscular administration are largely focused on preventing RNA-based therapies from degrading in the blood stream. For mRNAs administered in this manner, lipid nanoparticle solutions appear to be most common. Other delivery technologies are designed to enable local administration of mRNAs to the target site or organ, according to Tiest. “For cancers,” he explains, “intratumoral injection of mRNA offers advantages in safety (very local effect), dosage (no need to saturate circulation and reach the target), and easier combination with other existing therapies.”

Lipid-based nanoparticles have also been used for siRNA-based drugs, including some that have been approved by regulatory authorities. N-Acetylgalactosamine (GalNAc)-mediated delivery of chemically modified siRNAs has been demonstrated to result in significant delivery and silencing of genes within the hepatocytes of the liver and has also resulted in an approved product.

Dicerna’s GalXC platform is a leading example. “This approach uses a unique, proprietary, and rationally designed tetraloop configuration of double-stranded RNA molecules that interfaces effectively with the RNAi process and allows us to easily modify a compound’s chemical structure to maximize stability and potency,” Fambrough explains. He adds that it offers the potential for more convenient administration, infrequent dosing, high specificity, a high therapeutic index, and scalable, straightforward manufacturing. So far, the company has focused on treating diseases of the liver, but is now exploring the use of its GalXC platform across different organs, systems, and rare and common diseases.

Sirnaomics uses a polypeptide nanoparticle consisting of a histidine-lysine branched-chain polypeptide (HKP). HKP mixed with siRNAs spontaneously forms nanoparticles due to the charge-charge interaction between the negative phosphates on the siRNAs and the positively charged lysines, Evans notes. An advantage of this technology over some other delivery mechanisms, he says, is its ability to deliver multiple siRNAs to a cell at the same time, allowing synergistic effects of two siRNAs to be utilized as a therapeutic.

HKP-based nanoparticles can also accumulate near tumors, possibly mediated by the enhanced permeability and retention (EPR) effect, according to Evans. They further benefit from the presence of histidines, because these moieties have a pKa of approximately 6.3 and become protonated during acidification of the endosomes that are intermediary in the uptake of siRNAs by the nanoparticles. “This protonation helps release the siRNA payload into the cytoplasm where the siRNAs function to silence targeted genes,” he observes.

Controlling mRNA activity

A third challenge has been regulation of mRNA activity once it is delivered to the right cells, which is needed to ensure the right dose of the targeted therapeutic protein is expressed. A research team at the Massachusetts Institute of Technology developed one possible solution: incorporation of additional genes for RNA-binding proteins (2). The interaction between the mRNA and RNA-binding proteins is controlled using a small-molecule drug, and thus dosing of the small-molecule drug regulates the mRNA activity.



Breakthrough approvals

Inhibitory RNA approaches are most advanced, while mRNA therapies are at earlier stages, with some vaccines in Phase II trials and candidates using mRNA for in-vivo expression of antibodies that target a specific therapeutic effect, according to Tiest.

The two RNAi therapies that have received approval to date were both developed by Alnylam Pharmaceuticals. Onpattro (patisiran) was granted approval by FDA in August 2018 for the treatment of the polyneuropathy of hereditary transthyretin-mediated amyloidosis (3). It has received marketing authorization from the European Medicines Agency. This drug halts the production of faulty transrethrytin.

In November 2019, Alnylam received FDA approval for Givlaari (givosiran) for the treatment of adults with the rare genetic disease acute hepatic porphyria (4). This RNAi therapy targets aminolevulinic acid synthase 1.

Numerous products in development

The three companies interviewed for this article alone have several RNA-based therapies in development, but they represent only a small portion of candidates moving through clinical development.

eTheRNA is mainly active in oncology, with both vaccines against tumor targets and intratumoral injection of mRNA expressing targets involved in cell death and immune stimulation in its portfolio that leverage propriety LNP and immune stimulation (Trimix) technologies. Currently, the company has an ongoing Phase I/II program in melanoma and breast cancer. It also recently entered the field of infectious diseases and is working on a cross-protective coronavirus vaccine based on T-cell response that is administered intranasally, according to Tiest.

Sirnaomics has developed a combination of two siRNAs targeting TGFbeta 1 and Cox2 within an HKP nanoparticle that it has formulated for intratumoral delivery (STP705 in Phase IIs in non-melanoma skin cancer) and intravenous administration (STP707 for delivery to key liver cells for fibrosis treatment; it also has demonstrated in animal models to mitigate hepatocellular carcinoma [HCC]). STP707 has further been shown to increase the activity and improve the efficacy of anti-PDL1 antibodies against HCC, according to Evans.

The company is also working on chimeric siRNAs that combine a small-molecule therapeutic into the structure of the siRNA. “Once inside the tumor cell, the small molecule is released from the sense strand where it is designed to augment the activity of the siRNA inhibitor and produce a synergistic effect,” Evans says. The activity of these constructs has been demonstrated against a number of tumor types including pancreatic tumors, colon cancers, bladder cancer, and head and neck cancers.

Dicerna is leveraging its GalXC technology on its own and through several collaborations. The company’s lead candidate, nedosiran, is in development to treat primary hyperoxaluria (PH), an ultra-rare and devastating condition that causes calcium oxalate crystals to form in the kidneys and often results in kidney failure. Nedosiran silences the lactate dehydrogenase enzyme, which is implicated in all three types of PH. It is currently being evaluated in the PHYOX clinical development program, which includes an ongoing registrational trial.

Two other top candidates include an siRNA therapy to treat alpha-1 antitrypsin-associated liver disease (Phase I/II) and RG6346 for the treatment of hepatitis B virus infection (Phase I), which is being pursued in collaboration with Roche. “Between Dicerna and our collaborative partners, we currently have more than 20 active discovery, preclinical, or clinical programs focused on rare, cardiovascular, cardiometabolic, viral, chronic liver, and complement-mediated diseases, as well as neurodegeneration and pain,” Fambrough notes.

Broadening applications

Both mRNA and siRNA therapeutics and vaccines are advancing through the clinic, with a steady stream of readouts expected in the next few years. “Apart from clinical data, these results should validate a number of delivery and chemical modification platforms that can then open up the field into an increased development of RNA-based therapeutics,” Tiest states.

Over the next year, Evans expects an influx of additional siRNAs targeting liver diseases and being targeted to the hepatocytes using GalNAc conjugates because this approach has already demonstrated success. “Efforts will also be focused on additional chemically modified siRNAs with different targeting ligands that enable delivery to tissues and cells outside the liver, with the biggest obstacle being avoidance of uptake by the liver for any and all systemically delivered siRNAs,” observes Evans.

Fambrough agrees that the next frontier for RNAi therapies will be non-hepatic tissues. “There is significant interest and persistent unmet need across a range of disorders,” he says.

Moving new therapies on to the main stage

Going forward, the key advantages of RNA-based therapies compared to DNA-based gene therapies will continue to drive greater interest in all types of RNA treatments. “RNA combines the advantages and versatility of genetic-based therapy modalities with the lack of (perceived) risks associated with DNA-based gene therapy. Technology has now reached a level of maturity, particularly around stabilization and delivery of RNA, and the upcoming flow of clinical data could enable the break-through of this technology onto the main stage of drug development,” comments Tiest.

Looking specifically at RNAi therapies, Fambrough notes that while the technology is a newer approach to treating disease, it has experienced a “coming of age” in the past couple of years. “We have seen not only the first approvals in the space, but also a surge of interest among big pharma and big biotech companies seeking an entry into RNAi,” he says.


1. X. Tang, et. al. Front. Oncol., 9:1208 (2019).
2. MIT, “This RNA-Based Technique Could Make Gene Therapy More Effective,” Press Release, Oct. 16, 2018.
3. Alnylam Pharmaceuticals, “Alnylam Announces First-Ever FDA Approval of an RNAi Therapeutic, ONPATTRO (patisiran) for the Treatment of the Polyneuropathy of Hereditary Transthyretin-Mediated Amyloidosis in Adults,” Press Release, Aug. 10, 2018.
4. FDA, “FDA Approves Givosiran for Acute Hepatic Porphyria,” Press Release, Nov. 20, 2019.

Article Details

Pharmaceutical Technology
Vol. 44, No. 7
July 2020
Pages: 22–25, 64


When referring to this article, please cite it as C. Challener, “Can RNA Simplify Gene Therapy Development?" Pharmaceutical Technology 44 (7) 2020.