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The recent success of mRNA vaccines has opened the door for further and future development of nucleic acid-based therapies.
Nucleic acid-based therapeutics have been stepping up into the spotlight following the deployment of messenger RNA (mRNA) COVID-19 vaccines in 2021 and the rise in clinical development programs for gene therapies. These areas of development may just be the surface of the types of therapeutic applications where nucleic acid-based therapeutics can come into play. Tackling the development challenges of these therapeutics can potentially benefit more expansive areas of unmet medical need, particularly in difficult-to-treat patient populations.
Current immune therapies in development may often fail or not work for everyone. The result of these failures, in part, is what may be driving the R&D engine for alternative modalities, including nucleic acid-based therapeutics.
“We do not yet fully understand why current immune therapies are highly efficacious in some patients while other patients fail to respond, but research scientists are exploring several hypotheses. With cancer immunotherapies, it is important to remember that tumors are highly heterogeneous, in respect to the expression of tumor antigens and their effects on the immune system that enable tumor cells to escape immune surveillance,” explains John Lewis, CEO of Entos Pharmaceuticals.
Lewis points out that some cancers may not respond to current immuno-oncology (IO) drugs because these cancers do not express the target protein, or because they suppress immune surveillance and inhibit T-cell responses. Mutational burden, which is the number of mutations present in a cancer cell, may also play a role in response to IO therapies, especially checkpoint inhibitors, he adds.
“Checkpoint pathways evolved to regulate the T-cell response to cancer cells. Some tumors suppress T-cell response, so checkpoint inhibitors can overcome this suppression. While this allows normal T-cell anti-tumor activity to develop, this activity appears more effective in cells with a higher mutational burden,” says Lewis.
Another reason for failure is that current immune therapies often have safety issues or lack efficacy, or a combination of both, points out Gerrit Dispersyn, CEO, Phio Pharmaceuticals. Regardless of background, he explains, there is a need for more refined therapies to reprogram immune cells and tumor cells. Therefore, there is a need to target the internal cell machinery that can be achieved with oligonucleotide (i.e., nucleic acid)-based therapeutics.
Currently available commercial IO products mainly fall into two categories: immune checkpoint antibodies and chimeric antigen receptor T cell (CAR-T) therapy, Dispersyn notes. He explains that immune checkpoint antibodies “remove the brakes” of all T cells, including the auto-reactive ones, which leads to dose-limiting toxicity. Meanwhile, CAR-T therapies have limited access to solid tumors. Thus, the use of oligonucleotides has the potential to improve efficacy against solid tumors.
“Of the different platform technologies, it is becoming more and more clear that the shortcomings of the currently used platforms—whether it’s antibodies, cell-based therapeutics, or gene therapy—are being realized. Therefore, new approaches to fine tuning or helping overcome these challenges is partially explaining the turn to RNA-based therapeutics,” says Dispersyn.
“Triggering immune responses to fight disease is incredibly challenging, and we do not fully understand the complexities behind it,” adds Matthew Scholz, CEO of Oisin Biotechnologies, a US-based late preclinical stage company specializing in therapeutics for age-related diseases, and OncoSenX, a US-based late-stage pre-clinical cancer company. Scholz notes that there is significant variability in the population as well as heterogeneity within tumors. A therapeutic that targets a surface protein or requires functional T-cell infiltration can therefore be rendered ineffective in many patients. Targeting disease at the cellular level using RNA and DNA to “code” for a desired response is a more specific and nuanced approach, he asserts.
“With nucleic acid-based technologies, only the code for proteins is delivered. With DNA as a payload, tissue-specific expression of the cargo can be achieved through rational promoter selection as well,” says Scholz. “Think of it as a software approach to mobilizing the immune system: nucleic acids provide the program, and the immune system interprets the program and responds accordingly. The caveat of this approach is the nucleic acids must be delivered to the right cells in the body for the program to run effectively, in this case T cells for immune modulation and tumor cells themselves to eliminate tumors.”
For oncology indications, some immunotherapies are powerful at activating immune responses to attack and destroy tumor cells, specifies Jake Becraft, PhD, co-founder and CEO of Strand Therapeutics, an emerging biopharmaceutical company. However, one of the major shortcomings of these therapeutic agents is unexpected, off-target effects that can cause serious adverse reactions and life-threatening toxicities, he adds. The major advantage of nucleic acid-based therapeutics lies in the fact that they can be used to accurately target a tumor or tissue, then have a specific therapeutic protein, biologic, or immune engager expressed only at the site of interest. “This localization dramatically decreases the risks of off-target effects and improves safety and efficacy,” Becraft states.
While nucleic acid-based therapies may not overcome all their challenges, they enable more precise cellular targeting and extended activity than small-molecule or protein-based therapies, adds Lewis. According to Lewis, preclinical studies demonstrate that therapeutic gene expression can be targeted to specific tissue types with tissue-specific promoters, which can potentially improve efficacy while reducing side effects that result from off-target activity. Furthermore, cancer cells have high turnover rates, and DNA-based therapies provide a longer duration of activity than protein and small-molecule therapies, Lewis adds.
Much of the focus today is on RNA and RNA-related technology for developing “next-generation” biotherapeutics. The benefits of RNA technology, according to Dispersyn, is that it does not require modification of the genome. For instance, Phio is using an RNA interference (RNAi) approach to developing therapeutics because RNAi is transient and not permanent. In comparison, gene-editing approaches may result in permanent issues or toxicity.
“In cases where permanent modification is not required, we have the option to use RNA-based therapeutics. This is because DNA is not directly linked to proteins. RNA is a required intermediary step between genetic material and the protein. It is the proteins that make a disease present or absent. Since every translation of the genome goes to RNA, theoretically, almost everything can be solved using RNA therapies. This may be a more helpful solution than editing DNA, which is permanent and the long-term effects of which are largely unknown at this time,” Dispersyn says.
With much of the focus on RNA-based approaches for developing nucleic acid-based therapeutics, where does DNA fit in?
Scholz states that, while DNA is harder to deliver than RNA, DNA does offer many advantages over RNA, such as durability of expression, reduced manufacturing costs, increased stability, and contextual control of expression. “DNA-based vaccines are in clinical trials now, and Oisin Biotechnologies has a pipeline of candidates targeting indications including chronic kidney disease, metabolic diseases, and sarcopenia, to name a few,” says Scholz. “As these technologies are currently being evaluated in human clinical trials, we expect it will not be long before pharmaceutical companies broadly adopt this technology to deliver DNA payload.”
Scholz explains that the key to delivering DNA is to have a non-toxic delivery system that can easily get into cells without causing damage. “These technologies are in humans now, and we expect it won’t be long before more pharmaceutical companies adapt this technology to deliver DNA payloads,” he states. DNA payloads permit an additional layer of selective expression, thus paving the way for encoded therapeutics that not only target and kill tumor cells while sparing healthy cells, but also deliver key immune modulators to healthy cells, including immune cells, which can enable multi-pronged therapeutic approaches, he adds.
Becraft also notes that there are a number of DNA-based approaches currently in development, including T-cell therapies, genome editing and writing, and clustered regularly interspaced short palindromic repeats (CRISPR). Becraft adds that viral-based gene therapies that use adeno-associated virus (AAV) for diseases such as muscular dystrophy use a DNA-based payload. “While DNA and RNA-based therapeutics have similarities, the main difference is that DNA needs to be delivered further into the nucleus. This presents a lot of challenges to overcome due to the strength and defense of the nuclear membrane,” says Becraft.
DNA offers a variety of benefits compared with RNA, remarks Lewis. Benefits from using DNA include more durable expression, the ability to design a single DNA therapy that expresses multiple therapeutic molecules, and less complex supply chain management. “To date, a challenge in realizing the full potential of DNA-based therapies has been developing safe and highly effective delivery systems,” says Lewis.
Lipid-based systems, for example, are associated with liver toxicity while viral delivery systems trigger immune responses (which can result in serious adverse events) that limit repeat dosing, Lewis explains. He further adds that, while the DNA-based gene therapies approved to date have shown promising long-term data, it remains to be seen if a single-dose provides life-long therapy or if repeat dosing will be needed following longer periods of time.
“Given the enormous potential of DNA-based therapies, there are many academic and industry efforts to develop new delivery strategies, either through innovation of novel delivery systems or improved approaches to mitigating the safety and efficacy limitations of current approaches,” says Lewis.
Larry Pitcher, senior director and general manager, microbial manufacturing services, Thermo Fisher Scientific also emphasizes the fact that plasmid DNA (pDNA)-based vaccines have been around since the dawn of recombinant DNA technology. He also notes that pDNA is increasingly being used as a raw material or therapeutic agent in gene therapies and certain vaccines, and that its use is driven by the growth and transformational benefits of cell and gene therapies as well as the expanded application of mRNA.
“Its [pDNA’s] advantages, including weak immunogenicity, increased safety, and ease of manufacture, have dramatically increased demand for materials and manufacturing capacity globally. Investments in state-of-the-art manufacturing ensure [that clients] have reliable access to the high-quality materials and capabilities that have become vital for the production of these new lifesaving medicines and the patients in need,” says Pitcher.
Among the key challenges for developers of nucleic acid-based therapeutics will be the need to scale up to meet current demand and to evolve as market needs change, highlights Serena Fries Smith, director of Market Development & Strategy, mRNA Vaccines and Therapeutics, Thermo Fisher Scientific. Smith explains that, to meet current demand and evolve with the market, developers need a reliable supply of high-quality, good manufacturing practice (GMP)-grade raw materials.
Without reliable materials supply, developers can encounter manufacturing disruptions, including deviations, that can result in costly delays.
“Another challenge is the need for in-region manufacturing capacity and distribution to improve global access, including for people in lower income regions. To help address barriers such as lack of a cold supply chain, we are seeing smaller facilities popping up globally,” Smith says. “Stability and shelf-life are also challenges, but researchers have learned to alter chemical features of the mRNA transcript to improve the stability. In addition, improvements in delivery can protect mRNA against degradation.”
Historical and current challenges to nucleic acid-based therapy development include the production process itself, in terms of scalability, standardized quality metrics, and delivery, emphasizes Jordana Henderson, associate director of Research and Development at TriLink Biotechnologies (part of Maravai LifeSciences), a contract development and manufacturing organization specializing in the synthesis of nucleic acids, nucleoside triphosphates, and mRNA capping analogs. Henderson notes, however, that mRNA production scale-up, specifically, is quickly being overcome through optimized in-vitro transcription reactions followed by simplified purification processes. Optimal on-target delivery and standardization of drug substance analytical procedures, however, are still a work in progress, she adds.
“To make a functional message, it is critical that the synthetic mRNA contains all the structural elements of a mature post-transcriptionally modified mRNA found in eukaryotes. This includes a 5’ cap structure (Cap 1 or 2), 5’ untranslated region (UTR), coding sequence, 3’ UTR, and poly-adenosine tail,” Henderson explains. She also specifies that generating the appropriate 5’ Cap 1 structure in vitro once required additional enzymes and purification steps in the manufacturing process. Now, however, using co-transcriptional cap analogues (e.g., CleanCap, TriLink) can initiate the formation of a Cap 1 structure in a one-pot reaction via the RNA polymerase that is used to transcribe the message of interest, she further elucidates.
“Such improvement to the IVT [in-vitro transcription] reaction has enabled streamlined and scaleable purification processes for quick manufacturing of high-quality mRNA products. Ongoing and future work on the mRNA platform is focused on developing safe modifications to the nucleic acid that increase the molecules stability and translatability,” says Henderson.
Henderson also points out that several groups, including academic and industry alike, are actively working on lipid nanoparticle delivery system improvements, while regulatory leaders, including the US Pharmacopeial Convention and FDA, are looking to standardize mRNA drug substance specifications. “Altogether, these efforts will contribute to much needed advances in medical treatment options,” Henderson states.
Becraft points to the specificity of therapeutic protein expression as another major challenge in developing nucleic-acid agents. By using synthetic biology, however, the industry can address this roadblock and improve safety and efficacy. “Synthetic biology coupled with mRNA guarantees that the molecule will be encoded with the correct genetic information. We can also engineer the mRNA to be context-dependent so the protein will only be expressed inside of certain cell types. This logic-based genetic programming allows us to have precise control of the location, timing, and level of protein expression,” Becraft states.
Lewis also hones in on the limitations in tolerability and immunogenicity, as well as scalability, of current delivery methods, as being among the major challenges to nucleic acid-based therapy development. “Lipid-based systems are associated with liver toxicities and viral-based systems lead to immune responses that can both limit initial transduction efficiency and prevent redosing. Entos was established to address these limitations and broadly enable the next generation of nucleic medicines,” he states.
“We are in the process of publishing preclinical data demonstrating the significant potential of our Fusogenix proteolipid vehicle (PLV) platform in diverse indications. These data show that this novel delivery system leads to broad distribution across tissue types and organ systems with minimal toxicity. Gene expression can be targeted using tissue-specific promoters, and delivery of DNA with the Fusogenix PLV platform [e.g., Entos’ DNA vaccine candidate] leads to sustained gene expression out to one-year post-dosing,” Lewis says.
The data from Entos’ as-yet-unpublished preclinical studies also demonstrate that the Fusogenix PLV platform can effectively deliver RNA as well. “Importantly, these studies also show that the low levels of immunogenicity and high levels of activity are maintained after extended periods of repeat dosing,” says Lewis. “Additional studies demonstrate the feasibility of using the Fusogenix platform to enable DNA vaccines against SARS-CoV-2.”
Entos’ DNA vaccine candidate includes the full-length viral spike protein, which may provide enhanced and broad immune responses compared with protein vaccines that use spike protein fragments, Lewis adds. DNA vaccines can also encode genetic adjuvants that can enhance neutralizing anti-SARS-CoV-2 antibody responses in mice and non-human primates. “Data demonstrates that our Fusogenix PLV DNA vaccine candidate protected against SARS-CoV-2 infection in an animal model. We are currently conducting a Phase II trial of our SARS-CoV-2 DNA vaccine candidate,” Lewis states.
Historically, the delivery of oligonucleotides into the cell has been an issue for RNA therapeutics, says Dispersyn. Companies have taken different approaches to address this issue, such as the use of lipid nanoparticles as the delivery vehicle leading to liver accumulation because of its role in first-pass metabolism. “As a consequence the current RNAi approaches most companies utilize are liver specific, even though RNAi can work equally well in non-liver targets if you can deliver the RNA molecules there. Phio’s solution to this non-liver delivery challenge was to develop a RNAi platform with structural and chemical modifications that allowes for spontaneous uptake to cells without the need for a delivery vehicle,” Dispersyn explains.
Oisin’s PLV delivery technology, meanwhile, has enabled the company to develop systemic DNA therapeutics. “Once the first generation of nucleic acid therapeutics have been developed for obvious disease targets, second-generation therapeutics will need to focus on delivering optimized DNA/RNA ‘software’ sequences for more complex diseases,” says Scholz. “Next-generation sequencing and analysis for new unique targets, along with software-based synthetic promoter design, will help us explore different programming techniques for specific disease burdens in the future.”
Of the nucleic acid-based approaches to biotherapeutic development, RNAi has been especially pegged for its potential role in IO therapies. For instance, RNAi can be introduced into T cells. Fundamentally, T cells are like any other cell in that they can be programmed by nucleic acid therapies, points out Scholz. Often in cancer, T cells are overwhelmed by tumor cells, and because of a built-in fail-safe mechanism, T cells can shut down when overwhelmed, that is, they can go into T cell exhaustion. T cell exhaustion can be counteracted with the help of RNAi, however. “In essence, with careful selection of the nucleic acid payload, delivery of RNAi to T cells could be used to combat T cell exhaustion by breaking the inhibitory circuit from the inside, releasing T cells to seek out and fight both the primary tumor and metastases alike,” Scholz emphasizes.
Scholz cautions, however, that RNAi technology has been limited in its ability to target and kill tumor cells directly simply because RNAi technology cannot distinguish a healthy cell from a tumor cell. “Our preclinical studies clearly demonstrate that a plasmid DNA-based suicide gene approach can distinguish tumor cells and effectively eliminate them. Using RNA and DNA in tumors has enormous potential. If you can deliver a precise payload to these tumors that target only that tumor’s microenvironment you could, in theory, come up with many different ways to attack cancer at the cellular level. This, in essence, could get to the root cause of cancer. Being able to target specific cancer traits systemically may also help to tackle metastatic cancers,” Scholz explains.
Conventional RNAi technology has limitations that prevent effective application in immuno-oncology, including challenges with delivery and stability, however, notes Dispersyn. Phio’s platform allows RNAi to be used for increasing the tumor cell killing activity of immune cells, as well as for increasing the vulnerability of tumor cells toward such cell killing. For example, Phio’s focus in adoptive cell therapy includes using its technology to reduce the therapeutic immune cell’s expression of immunosuppressive proteins, which can allow these immune cells to overcome tumor resistance mechanisms and improve their ability to destroy tumor cells.
“Similarly, we can reprogram cells used in adoptive cell therapy, such as CAR T-cells, to increase their actvity towards solid tumors. After expanding these immune cells ex vivo, and enhancing them with our RNAi compounds, the cells are returned to the patient for treatment,” Dispersyn says.
“RNAi could improve the efficacy and safety of current T-cell directed IO therapies by inhibiting the expression of cancer-related proteins rather than inhibiting active proteins contributing to disease pathology,” adds Lewis. Moreover, tissue-specific promoters may provide more precise control of this inhibitory activity to target cells and reduce off-target side effects. “As an example, some checkpoint inhibitors block CTLA-4 [checkpoints T-lymphocyte-associated protein 4] activity on T cells to overcome T-cell suppression and stimulate a T-cell response against the tumor. RNAi designed to inhibit CTLA-4 expression can achieve the same objective but be more effective because it prevents the CTLA-4 signaling pathway from turning on rather than inhibiting signaling once the pathway is active,” Lewis explains.
The value of mRNA therapeutics in immuno-oncology, meanwhile, lays in mRNA’s ability to express therapeutic proteins directly from inside the tumor, initiating the immune response that destroys cancer cells, adds Becraft. This approach enables the encoding of any specific protein sequence that can then be delivered to the tumor itself. In this case, the tumor begins to directly secrete its own anti-tumor IO therapy, and it is this localized expression that ensures the immune system can recognize and attack the tumor cells. What’s more, this immune response can commence without the off-target effects that plague current immunotherapies that must circulate in the blood stream before reaching the tumor microenvironment, notes Becraft.
“This same philosophy can also be adapted to CAR T-cell therapy, where synthetic protein receptors are expressed and embedded on the surface of a patient’s T cells that then can recognize and kill off certain types of cancer cells. However, to accomplish this, current CAR-T therapies use a production method where the cells are extracted from the patient’s body, genetically engineered, then transplanted back into the patient. This approach is difficult to scale and control due to infrastructure and reproducibility challenges,” Becraft states. “At Strand, we believe we can bypass this issue using our self-amplifying mRNA therapeutics to deliver the instructions directly into the patient so that the T cells can make these protein receptors and become armed with the tools to recognize and kill the cancer cells.”
Though the use of nucleic acids in biotherapeutic development has long been studied in research, the practical use of such therapeutics is still in its infancy at the industry level. However, progress toward commercialization is expected to move forward in light of growing interest, investment, and success thus far of early entrants, such as mRNA vaccines.
Feliza Mirasol is the science editor for BioPharm International.
Vol. 35, No. 4
When referring to this article, please cite it as F. Mirasol, “Nucleic Acid-based Therapeutics Are Stepping into the Spotlight,” BioPharm International 35 (4) 16–20 (2022).