Researchers based at the University of Toronto (U of T) Leslie Dan Faculty of Pharmacy have developed a novel ionizable lipid nanoparticle (LNP), called iso-A11B5C1, that can deliver mRNA specifically to muscle, while minimizing off-target delivery to other tissues, such as the liver and spleen. Through their newly reported study the researchers showed that a proof-of-concept mRNA melanoma cancer developed using the new LNP triggered potent cellular-level immune responses after intramuscular administration, even with limited expression observed in lymph nodes.

“Our study showcases for the first time that mRNA lipid nanoparticles can still effectively stimulate a cellular immune response and produce robust anti-tumor effects, even without direct targeting or transfecting lymph nodes,” said research lead Bowen Li, PhD, assistant professor, Leslie Dan Faculty of Pharmacy. “This finding challenges conventional understandings and suggests that high transfection efficiency in immune cells may not be the only path to developing effective mRNA vaccines for cancer.”

Li and colleagues reported their results in PNAS, in a paper titled “Combinatorial design of ionizable lipid nanoparticles for muscle-selective mRNA delivery with minimized off-target effects,” in which they suggest that their work advances methods for muscle-specific mRNA delivery and prompts rethinking of mRNA vaccine designs.

Ionizable lipid nanoparticles are crucial for delivering mRNA-based therapies, including COVID-19 mRNA vaccines that have been used worldwide during the recent global pandemic, the authors noted. However, many LNP designs can inadvertently result in substantial mRNA expression in off-target tissues and organs like the liver or heart, resulting in often treatable but unwanted side-effects The drive to improve the safety of mRNA therapies that have the potential to treat a broad range of diseases means there is an urgent need for LNPs designed to minimize these off-target effects, explained Li.  And as the authors further wrote “… the risk of mRNA delivery to off-target tissues highlights the necessity for LNPs with enhanced tissue selectivity … there is an urgent need for LNPs designed for highly muscle-specific mRNA delivery, minimizing off-target effects and addressing safety issues associated with current LNPs.”

The classical LNP formulation consists of four components: ionizable lipids, helper phospholipids, cholesterol, and lipid-conjugated polyethylene glycol (PEGylated lipids). “Of these, the ionizable lipid is pivotal in this complex,” the researchers explained,  but synthesizing these components remains a complex, time-consuming and costly process, so so fast, streamlined approaches are needed, they pointed out. “Toward this goal, combinatorial chemistry, employing multicomponent reactions, has emerged as an effective tool for high-throughput synthesis (HTS) of expansive and chemically diverse lipid libraries.”

Li and colleagues developed what they described as an advanced platform that enables, one-pot, high-yield fabrication of biodegradable, asymmetric ionizable lipids at room temperature. Using this platform, they identified iso-A11B5C1, an ionizable lipid that was “uniquely apt for muscle-specific mRNA delivery,” they noted.

The team’s research showed that, compared with the current benchmark LNP developed by Moderna, iso-A11B5C1 demonstrated a high level of muscle-specific mRNA delivery efficiency. “The Iso-A11B5C1 LNP demonstrated exceptional muscle specificity in mRNA delivery, exhibiting transfection efficiency comparable to the commercially available lipid SM-102 in the muscle,” they wrote. .It also triggered a different kind of immune response than those generated by vaccines used to treat infectious diseases, but still triggered a high cellular immune response, which prompted the team to test the LNP as part of an anticancer vaccine.

“Interestingly, iso-A11B5C1 triggered a lower humoral immune response, typically central to current antibody-focused vaccines, but still elicited a comparable cellular immune response,” said Li. “This finding led our team to further explore this as a potential cancer vaccine candidate in a melanoma model, where cellular immunity plays a pivotal role.”

Tests in the mouse melanoma model showed that iso-AB11B5C1 LNPs significantly slowed tumor growth and outperformed the MC3 LNP formulation. “In the B16-F10 melanoma model, iso-A11B5C1 LNP demonstrated a potent antitumor effect that outperformed MC3 LNP, as indicated by markedly reduced tumor progression, elevated levels of antigen-specific CD8+ T cells, and a substantial infiltration of CD8+ T cells into the tumor,” they wrote. The results, they said, “… substantiated the exceptional therapeutic efficacy of iso-A11B5C1 LNP, as demonstrated by its potent antitumor effects in a melanoma model, thereby solidifying its promise for the development of mRNA-based therapeutic vaccines.”

“Although iso-A11B5C1 showed limited capacity to trigger humoral immunity, it effectively initiated cellular immune responses through intramuscular injection,” said co-author Jingan Chen, a PhD trainee from the Institute of Biomedical Engineering at U of T, “The substantial anti-tumor effects observed with iso-A11B5C1 underscore its promise as a viable candidate for cancer vaccine development.”

The authors further concluded, “This study encourages rethinking of mRNA vaccine design principles, suggesting that achieving high immune cell transfection might not be the sole criterion for developing effective mRNA vaccines … Importantly, our investigations underscored the capacity of iso-A11B5C1 for efficient muscle-specific gene editing with undetectable off-target effects, and thus introducing a promising approach for safe and effective gene therapy targeting muscle diseases.”

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