Canadian Lipid Nanoparticle Research: The Key to COVID-19 mRNA Vaccines

Background

Messenger ribonucleic acid (mRNA) is a single-stranded molecule that corresponds to the genetic sequence of a gene that is read by a ribosome (molecular machines that link amino acids together in cells) in the process of synthesizing a specific protein. As early as the 1980s, it was thought that mRNA could be used to treat disease and make vaccines.

There are some 34 trillion cells in the human body, each surrounded by a thin membrane made up of a bilayer of lipids just 5 nanometers thick, which serves as the permeability barrier that divides the inside from the outside of cells. Enzymes in the environment and body can readily chop fragile molecules like mRNA into pieces, which makes lab experiments challenging and their delivery into cells difficult. Moreover, mRNA strands are large and negatively charged and can’t easily diffuse (or cross) the protective lipid membranes of cells. Scientists had to identify a way to deliver mRNA into cells.

Based on a detailed understanding of the biophysical properties and functions of lipids, the team of Pieter Cullis, Michael Hope and Thomas Madden first managed to solve the problem of efficiently producing lipid nanoparticles (LNPs). At the same time, they resolved how to package extremely delicate molecules in the form of cancer drugs, vaccine components and genetic material, such as mRNA, within LNPs and then consistently and safely deliver them into cells, and specifically into the cell cytoplasm, without any degradation.

Research Team

The beginning of the lipid nanoparticle (LNP) story can be traced to the biochemistry department at Utrecht University in the Netherlands, where Pieter Cullis met Michael Hope in 1977.

Pieter Cullis

Pieter Cullis was born in England in 1946 and moved to West Vancouver at the age of eight. His father was a physics teacher, and Cullis soon developed a similar passion for the subject, leading him to earn a PhD in physics from the University of British Columbia in 1972. Rather abruptly, he transitioned his research focus to biochemistry during his post-doctoral studies at the University of Oxford in the United Kingdom. While at Oxford, he built a nuclear magnetic resonance machine and adapted it to studying biological membranes, which are mainly comprised of lipids and proteins. However, as Cullis recalls in a 2021 article for The University of British Columbia Magazine, at the time he “didn’t know what a protein was, or DNA.” Nevertheless, looking back, Cullis said that he would not change a thing. He saw it as “a big advantage to switch fields,” he said. “It gives you a lot of confidence that you can try the next thing.”

Cullis then moved to the Netherlands to continue his research in the biochemistry department at Utrecht University, led by Professor Laurens L. M. van Deenen. Van Deenen’s lab was a kind of mecca for biomembrane research that drew many foreign post-docs. Professor Jack Lucy, at the University of London (UK) had proposed a model about how membrane lipids behaved during membrane fusion, but Cullis thought it was wrong and, to prove it, sought to collaborate a recent graduate of Lucy’s laboratory who was also doing postdoctoral research in van Deenen’s department.

Michael Hope

Michael Hope was born and raised in the United Kingdom. He had also been drawn to van Deenen’s lab after completing his PhD in biochemistry at the University of London with Jack Lucy. Hope, a “green post-doc” educated in the United Kingdom’s formal education system, was working on lipid fusion. Cullis invited Hope to help develop some ideas based on the application of a new technology and technique that he had developed called phosphorus nuclear magnetic resonance (NMR). According to Hope, Cullis was not cowed by the history and reputation of others who had worked in the field of membrane fusion for many years. He was incredibly focused, self-assured, and did not mind ruffling feathers. Coming from training in physics, Cullis had no preconceived ideas about how things should work in a biological setting. He could think more broadly than people who were formally trained in the field. “In those days, we were all trying to understand what lipids did in biological membranes,” Hope recalled in a 2022 interview with the author. “Why was this variety of lipids found in biological membranes? This technique of phosphorus NMR really enabled us to look at each individual lipid component and look at the structures it formed.”

Cullis and Hope worked well together at Utrecht University for about a year, at which point Cullis was appointed to a position at the University of British Columbia (UBC). The work with Hope had been exciting and productive and, once Cullis had settled in Vancouver, he invited Hope to continue their research at UBC. Hope moved to Vancouver in 1979, initially temporarily, to help set up the lab at UBC, but then never left.

Cullis never seemed to rest. While still in the Netherlands, late at night after running experiments, he and Hope would go to a local pub to discuss the work. “We have to build a research group that’s big enough to handle the big issues that science is facing right now and keep them together long enough so that we can really answer some of those questions,” Cullis would always say. He was quite disillusioned with the typical post-doc career track, with two years of work in one lab here and then three years at another and then trying to get an academic position somewhere. “He wanted to keep everybody — people he worked well with — together. That was his mindset right from the beginning.”

Thomas Madden

Thomas Madden joined Cullis and Hope at UBC in Vancouver in June 1980, shortly after completing his PhD in biochemistry at the University of London. Madden was also born in the United Kingdom and grew up in a working-class neighbourhood in London. After completing his PhD, Madden searched for fellowship opportunities to continue his biochemistry training. He had always wanted to go to North America and found himself drawn to the lipids work that Cullis was doing at UBC. Not unlike what happened with Hope, initial plans for a limited stay in Vancouver quickly gave way to a realization that he would not be going back to the U.K. Like Hope, Madden never left Canada.

Canadian Liposome Company

From the start, Pieter Cullis was successful at securing academic grants to support his laboratory research at UBC. However, the lab soon grew too large, and it became difficult to keep people based on the salaries provided by the grants. Cullis needed to offer better salaries and greater job security and thus proceeded to set up biotech initiatives that focused on developing clinical applications for the lipid delivery systems that his lab was researching. Indeed, Cullis started the first UBC spinoff company, known as the Canadian Liposome Company Inc. at the time. Cullis was determined to keep the work in Vancouver, despite overtures from biotech firms in the United States attempting to draw him and core members of his team south of the border. In doing so, Vancouver soon developed into a significant biotechnology hub as other companies and academic groups emerged in the city. The company worked with the Liposome Company Inc. of Princeton, New Jersey, based on an agreement that it would directly fund the research work in Cullis’s UBC lab, as well as its applied research.

Lipid Nanoparticle (LNP) Research

The focus of the research work in Pieter Cullis’s lab was to use lipid nanoparticles (LNPs) as a research tool and, in the process, better understand LNPs and find ways to make them, produce them faster and load molecules inside them. The original clinical application was using LNPs to deliver conventional drugs, especially cancer drugs. Research also included developing a LNP that could fuse and deliver larger molecules — not unlike what a virus can do — taking its nucleic acid payload into a cell. Indeed, the goal was, in effect, to develop an artificial virus but with no proteins associated with it, thus avoiding any immune response problems. “That proved to be one of the most difficult things to do,” as Michael Hope recalled during an interview with the author. It took some 35 to 40 or so years to make it work.

During the 1980s, while Hope and Thomas Madden were primarily focused on the biotech side of the LNP work, Cullis always maintained a foot in academia so that he could use his UBC lab as a training ground for graduate and post-doctoral students, as well as a backup when there was downtime in the biotech industry work. He wanted to make sure that the research was always moving forward. While many of the students and post-docs who worked in Cullis’s lab were soon hired at biotech facilities or involved in setting up new biotech ventures, several stayed with Cullis’s research team for many years, particularly the core group of Marcel B. Bally, Lawrence D. Mayer and Tom Redelmeier.

Rapid Extrusion Procedure

As Michael Hope and Thomas Madden recalled during their interview with the author, the early 1980s was an “interesting time in the lab,” with lots of papers being published on various aspects of their work. They were using LNPs daily, with Bally focused on making them regularly — although the process took two or three days with the available technology. Frustrated one day, Cullis proceeded to sketch a device that could produce LNPs faster. He sought a prototype and approached a self-styled part-time inventor, G. Webb, who assembled what would become known as the “extruder” out of plastic. Webb had a workshop at the Vancouver Aquarium, where he built devices to produce simulated rainforest storms in the jungle area. When the device was first used, Hope found that it produced what had previously taken several days in just five minutes. Importantly, the LNP were unilamellar, or spherical, and had a well-defined size that could be modulated by using membranes with different pore sizes. This was a “huge breakthrough,” but the high pressure involved in the process (1,000 psi) was a concern, as the plastic could suddenly fail and the device could blow up. When cracks appeared in the prototype, a second “extruder” was made using aluminum with the help of Webb and Sciema Technical Services Ltd. of Richmond, British Columbia.

Cullis’s first paper about the new extrusion process was submitted in April 1984 but not published until early 1985. “[The process involved] extruding these lipid mixtures through very narrow pores, and we needed high pressure to do that,” explained Hope during his interview with the author. “Once [we] did that, they spontaneously formed these small 100-nanometre LNPs, which is what we wanted.”

It did not take long before others working in the field heard about the “extruder” and wanted their own. To meet this demand and to generate funds to support the team, Cullis started a new company, Lipex Biomembranes. Cullis also found a machine shop to make the stainless-steel components for the device that were shipped to Hope’s Vancouver home, where, in the garage, Lipex was based. In the garage, usually in the evenings, Hope oversaw the cleaning and assembly of the components and testing of the completed devices, as there was insufficient space in Cullis’s UBC lab.

Before long, Lipex had a worldwide reputation and sold thousands of extruders. “And it wasn’t cheap,” Hope said, “in those days, $2,000 to $3,000 a piece.” Things worked out well and “helped keep the spirit of the group together,” which was Cullis’s key goal. Lipex board meetings were held at Hope’s house, with the company operating on a mission statement of being “purveyors of extruders and parties.” It was a successful approach to developing a biotech culture that kept a core team together in Vancouver for as long as possible.

LNP Delivery of Conventional Drugs

With the ability to produce LNPs more efficiently using the extruder, it was now possible to further basic research into their structure and capacity, as well as investigate their therapeutic utility for delivering packages, such as cancer drugs to tumour sites in the body. To advance this type of clinical application, Pieter Cullis, Michael Hope, Thomas Madden, Marcel Bally and Lawrence Mayer co-founded the Canadian Liposome Company Inc. (CLC) in 1987 as a subsidiary of the Liposome Company Inc. of Princeton, New Jersey (“TLC”). The primary focus was on increasing the effectiveness of cancer drugs while also reducing their side effects. In addition, new therapies were developed to control fungal infections that can develop in patients who undergo cancer therapies. However, in 1992, after a new CEO at the Liposome Company Inc. demanded that Cullis and his team relocate to Princeton, they objected and closed the Canadian subsidiary.

LNP Delivery of Nucleic Acids (DNA/RNA)

Pieter Cullis’s team proceeded to set up a new Vancouver-based biotech venture, Inex Pharmaceuticals Corporation, which maintained a focus on the delivery of cancer therapies. Soon, their attention shifted to adapting LNPs to the delivery of gene therapies and a line of research that focused on packaging DNA and RNA compounds into LNPs.

By 2005, the gene therapy work of Cullis’ team had accelerated, leading them to work with Alnylam Pharmaceuticals, Inc., based in Massachusetts, to develop and deliver a new RNA-based drug that could suppress a specific faulty gene in liver cells. Also, as a side project, the ever-energetic Cullis helped to establish the Centre for Drug Research and Development, based at the UBC campus in Vancouver, which later became known as adMare BioInnovations and played a significant role in building Canada’s life sciences sector. Marco Ciufolini, a UBC Chemistry professor, worked with Cullis at CDRD and made important contributions leading to more potent LNP systems. Cullis, Hope and Madden left Inex Pharmaceuticals (then Tekmira Pharmaceuticals) after its lead product was not approved by the FDA, but they continued working with Alnylam. This led them to establish another new venture in Vancouver, Acuitas Therapeutics, which began operations in 2009.

Development of mRNA Technology

Shortly after setting up Acuitas, Michael Hope was contacted by Drew Weissman, a biochemist and vaccine researcher based at the University of Pennsylvania, who, with Katalin Karikó, had discovered how to engineer mRNA. Weissman and Karikó wanted to utilize Acuitas’s LNP system to deliver vaccines into cells, starting with a potential vaccine against the Zika virus. At the experimental level, this vaccine delivery system worked quite well, but it was not developed any further. Later, a partnership was established between Acuitas and BioNTech, where Karikó was senior vice-president. The focus of this partnership was on developing mRNA-LNP to treat cancer and a mRNA-based influenza vaccine.

The emergence of the COVID-19 pandemic in 2019-20 quickly changed the mRNA vaccine R&D focus of the Cullis, Hope and Madden team and the payload that their intrepid and stealthy LNPs was bioengineered to deliver. Indeed, Acuitas was ideally poised to help the world fight off a pandemic. They provided the key. (See also COVID-19 Vaccines in Canada.)