Malvern Panalytical Pharmaceuticals

As we are physiologically, psychologically, and societally recovering from the COVID-19 pandemic, it can be easy to forget the tremendous impact the worldwide COVID-19 vaccination campaign has had on fighting the disease. Akin to a war effort, the whole pharma sector changed gear on vaccine research, prompting paradigm-shifting scientific discoveries like mRNA-based vaccines in a matter of months – a timeframe unheard in the pharmaceutical sector. One of the main reasons for this tour de force achieved by Moderna and Pfizer-BioNTech is lipid nanoparticle (LNP) technology.

LNPs are manufactured by formulating lipids (ionizable, PEGylated, and phospholipids) with cholesterol and the mRNA active pharmaceutical ingredient. The resulting supramolecular protective wrapper not only encapsulates mRNA, keeping it safe from ribonucleases, but it also facilitates its cellular internalisation and releases into the cytoplasm to enable its pharmacological action. First, brought to market in 2018 with the siRNA-based therapeutic ONPATTRO^TM for the treatment of transthyretin-induced amyloidosis, are LNPs about to bring to life the long-awaited golden age for oligonucleotide therapeutics?

COVID-19: a Stepping Stone for mRNA-LNP Therapies

LNPs can efficiently encapsulate a wide range of oligonucleotides, from tens of base pairs-long siRNA to whole genes. As such the technology can be applied to many therapeutic interventions other than COVID-19. No wonder clinical trials targeting other infectious diseases like influenza, HIV, RSV, Rabies, Zika, and Ebola are already underway. But what about beyond infectious diseases?

The Most Exciting Avenue for LNPs: Gene Therapy

Compared to the current viral-vector gold standard, LNPs can deliver much larger payloads, are less immunogenic, and are simpler to manufacture on a scale. As recently demonstrated for HPV-driven cervical cancers, the concomitant delivery of therapeutic mRNA with CRISPR/Cas9 using the LNP technology should provide attractive therapeutic opportunities for permanent and transient gene therapy.

One Step Closer to Personalised Cancer Vaccines

Liposomes have historically been used in oncology to improve small molecule efficacy by virtue of the enhanced permeability and retention effect of tumour microenvironments. Nowadays, many biotechs are trailblazing new immuno-oncology therapies, whereby immune checkpoint blockers are potentiated by cancer antigens delivered using mRNA-LNP vaccines. Many of these vaccines like the mRNA-4157/V940 – KEYTRUDA^® combination could help potentiate treatments for therapeutically challenging immunogenic cancers.

“LNPs can efficiently encapsulate a wide range of oligonucleotides, from tens of base pairs-long siRNA to whole genes. As such the technology can be applied to many therapeutic interventions other than COVID-19.”

LNP-mRNA Technology Come Thick and Fast.

Production methods commonly employed were reliant on ethanol injection or T-mixing, cannot generate truly monodispersed mRNA-LNPs and are often inconsistent from one batch to the other. These inconsistencies lead to uncertain levels of efficacy; a big handicap in LNP therapeutics. Microfluidics recently triggered a paradigm shift in LNP manufacturing by enhancing not only reproducibility but also scalability and affordability. Microfluidics is also well suited for automation and high-throughput manufacture--a useful feature when a new pandemic emerges. In addition, relentless progress in the sensitivity of instruments helping characterize LNPs in terms of size, morphology, dispersion, zeta-potential, or even mRNA payload LNP will play a major role in years to come.

The stability of the Comirnaty^® and SpikeVax^® COVID-19 vaccines is only achieved at sub-zero temperatures. This major drawback compared to traditional viral vectors has been extensively covered in the press. mRNA-LNP vaccine stability is a complex issue. Although LNPs are designed to keep their payload safe from physiological degradation, interactions with and between LNP excipients introduce additional risks for oligonucleotide degradation. If not stored correctly, mRNA will hydrolyse, oxidise, or react with impurities from within the LNP formulation. Many strategies are being developed to address these issues such as the introduction of stabilised nucleotides, reducing lipid-related impurities, and curbing water content by lyophilization. “mRNA printing” for point of use production could not only solve the stability issue but also offer opportunities to deliver personalised therapy.

Transfection efficiency is driven by the chemical structure, shape, and properties of the ionisable lipid component of the LNP formulation. Arguably, the most critical parameter is pKa. It needs to be finely tuned so the ionisable lipid is neutral in the plasma, keeping the formulation stable and is protonated in the acidified endosome to prompt the payload release required to elicit the desired pharmacological response. Despite much research aiming to optimise ionisable lipids and other LNP components, transfection efficiency remains stubbornly low. Solutions to this bottleneck include fusogenic proteins and polymers. These non-lipidic components prompt the fusion of the nanoparticle with the cell membrane, thus bypassing the need for endocytosis.

Immunological safety is another key issue associated with ionisable lipids. Whilst they are believed to elicit an inflammatory response contributing to potency, they also trigger side effects many of us have observed shortly after our COVID-19 jabs, such as pain, swelling, and fever. Similarly, although PEGylated lipids were initially introduced to the LNP formulation to make it invisible to phagocytes, PEG is also known to be immunogenic. The polymer is suspected to trigger some of the immediate allergic responses observed with the first shot or mRNA-LNP vaccines in patients with pre-existing PEG antibodies. One of the many alternatives under investigation includes biodegradable and non-toxic polymers such as poly sarcosine. These have similar physicochemical properties to PEG, but with much-reduced immunogenicity.

Off-target adverse events can occur as cells get indiscriminately transfected throughout the body and express the antigen encoded by the mRNA delivered via the LNP formulation. Their recognition by T cells can lead to severe systemic adverse events. Hence, specific targeting of cells of the immune system responsible for eliciting the desired pharmacology such as dendritic cells is being investigated. Examples include the conjugation of LNPs to antibodies like anti-Langerin and anti-CLEC9A. Targeting specific cells, tissues, or microenvironments like tumour-microenvironments may also stem from these studies and widen therapeutic indices.

As learnings made from the COVID-19 mRNA-LNP vaccines are being applied to related nano-delivery technologies such as nanostructured lipid carriers and the brain penetrant solid lipid nanoparticles, the whole family of LNP formulations is predicted to play pivotal roles in medicine.

With new scientific avenues being paved across imaging, theranostics, cosmetics, nutrition, agrochemicals, and even nanoreactors, the impact LNPs could have on our lives is promising to be tremendous.