Can you describe first researching lipid nanoparticles as a delivery vehicle?
I’ve been in this game for a long time, since the early 80s, in terms of drug delivery. Before that, I was doing more basic research and still am, to some extent, on the properties of lipids in membranes. In the early 1980’s, we used “model membranes” called liposomal systems. We found ways of loading anti-cancer drugs into them with very high efficiency. We pushed those forward as therapeutics, using liposomes to deliver cancer drugs more specifically to where they’re needed, namely tumors, without making your hair fall out and all the rest of the things chemotherapy does.
That was our main focus; we got two drugs approved by the FDA and the EMA in that process. But in the middle of the 1990s, in one of the companies I cofounded, the CEO said, “I can’t raise money putting old drugs into liposomes as carriers. We should be doing gene therapy.”
Can you tell us about some of the challenges?
At that point, it was known that lipid-based systems could introduce large macromolecules – DNA was the major focus – into cells in vitro using “cationic” lipids, but any application in vivo was really inhibited by the toxicity of these lipids. Cationic lipids are required because they are positively charged and nucleic acids are negatively charged, so cationic lipids provide a way of encapsulating the nucleic acid polymer in a lipid nanoparticle.
We had been doing some very basic research and had developed a lipid called an ionizable cationic lipid. At the pH in your body this lipid is not charged. But if you lower the pH to about the acidity of a lemon, say about pH four, then it becomes positively charged. So we thought, maybe we can use that lipid to encapsulate nucleic acids at the lower pH where it is positively charged, and perhaps the whole thing would hold together when we brought it back up to the pH in your body. That turned out to be the case.
After you discovered this to work, how did you apply these lipids?
The nanoparticles that resulted were relatively nontoxic and were stable in the blood. While working with Alnylam, we found that different species of the ionizable lipid gave optimized potency, causing us to develop a drug that delivered a piece of RNA called small interfering RNA, or siRNA, to silence a gene in the liver causing a disease known as transthyretininduced amyloidosis.
That disease is caused by expression of a mutated protein in the liver, which is secreted into the circulation. It then forms fibrils, which will deposit as plaques, particularly in heart and nerve tissue where they cause neuropathies and cardiac toxicities. Alnylam made an siRNA to silence that gene, and then used the optimized lipid nanocarrier system to deliver it to the liver. Alnylam took this drug into clinical trials in 2012, and the Phase III results were announced in 2017. The results were brilliant, and the drug, called Onpattro, was approved in 2018. That validated, in many ways, the LNP approach.
What was the challenge in shifting to delivering mRNA?
Most small molecule drugs have a molecular weight of 500, or in that range. siRNA is much larger with a molecular weight of 13,000. We thought that perhaps the LNP we developed for Onpattro could deliver messenger RNA, or mRNA, which has a weight of 100,000 or more, in order to express a protein in the liver, as opposed to silencing it.
That work was done in Acuitas, one of the companies I co-founded, resulting in better and better lipid nanoparticle formulations. Luckily, we were then contacted by Drew Weissman of the University of Pennsylvania, who wanted to try these systems as a vaccine. Instead of administering the LNP mRNA intravenously and trying to express a protein in the liver, he wanted to employ an mRNA coded for a viral protein and see if you could make a vaccine out of it administered intramuscularly.
First it was Zika virus, then influenza – several target systems – and worked very well. Drew was getting results from his collaborators, experts in various diseases, that were incredibly positive. That led to a collaboration between Acuitas and BioNTech, in Germany, for an influenza vaccine application.
BioNTech was also working with Pfizer on the influenza vaccine. And so, in January of 2020, when the pandemic hit, all of the efforts went to COVID-19 to develop a vaccine against SARS-CoV-2. And so the vaccine that entered the clinic is really enabled by the lipid nanoparticle that we developed with Acuitas.
It’s been a wild ride since then. The efficacy results last November were 95% potency for preventing COVID-19. It turned into something quite spectacular, and when I saw the efficacy, it was unbelievable. I mean, you do whatever you do in life, and then suddenly, wow, you’re a part of something that is affecting the world.
How does the success of the LNP for delivery impact the work you’re leading?
Certainly, for Acuitas this changes something. I would say the vaccine applications there are front and center. Many companies concerned with vaccines are coming to our door, to say, “Let’s work something out.”
An HIV application is definitely a real one, and there are several ongoing efforts in that. Another is a universal flu vaccine so you don’t have to take a new formulation every year. The list goes on and on. Other applications in terms of chronic disease, whether it’s cancer, heart disease, etc, are coming into focus. And then there is the rare disease aspect, because you can make the liver make any protein you want that may be missing. From a product point of view, it’s a gold rush. There are a lot of potential applications.
From my point of view, what we’re good at right now is gene therapy for the liver, particularly hepatocytes. We’re also good at vaccines, which requires intramuscular administration. However we need to expand the range of tissues that we can hit. That is either going in intravenously, to get to bone marrow. That opens it up for sickle cell anemia; various forms of leukemia come into focus. If we can get to the lung, we can tackle cystic fibrosis and other disorders. If we can get to the brain, diseases such as Alzheimer’s.
For all these diseases, with gene therapy you can tackle them at the root. It’s not like you’re dealing with some small molecule that is affecting a particular protein; you can actually make something very specific that will inhibit production of a protein or produce a protein. You can make a protein that isn’t being made normally, or is deficient. For Alzheimer’s, for example, you could make a protein that breaks down the amyloid plaques.
These things are now possible. It’s revolutionizing medicine. I refer to it as the third generation of pharmaceuticals: the first generation was small molecules, second generation was biologics and the third generation are these gene therapies.
What are the implications for delivering biologics?
In the case of other biologics, and particularly monoclonal antibodies, it gives you other avenues. As I said, you can make the liver make any protein you want. Using mRNA, that’s coding for whatever protein it is that you want to make. It seems like the liver is quite happy to do that. We may find out somewhere down the line that there’s deleterious effects there, but we haven’t yet.
So if you can make the body make any monoclonal antibodies you want a lot of opportunities arise. Instead of building a plant to make monoclonal antibodies that will go after cancer or other disorders, you can make the mRNA in a matter of a couple of months and then you have a really personalized therapy. Say you’re trying to treat cancer, you can figure out what the epitope is on that cancer surface you want to hit, there are ways of selecting very active antibodies for that. And then using mRNA, you’re getting your body to make them, instead of making them in a big plant.
In addition, CAR-T cell therapy is another good example. There is the possibility of doing that in vivo, as opposed to the ex vivo process now, where they expand and modify the T cells.
What is next in your research?
The field is at an interesting stage. We have something that clearly works, to some extent. We can transfect the liver, and we can get expression of proteins, and we have vaccines and so on. We know that if we have very high doses, we can get gene expression in bone marrow, for instance – this is in animal models. But we’re way too close to the point where we’re inducing toxicity by the carrier itself. It’s like playing with an anticancer drug, right? They work to some extent; doctors push it to the limit, trying not to kill the patient but kill the cancer.
But the goal here is to get the therapeutic index as high as you can. And so, we’re looking for ways to improve the potency of the system without increasing the toxicity of the system.
That is what we did with the liver in the project with Alnylam. We started off with formulations where we can see some gene silencing in the liver for the siRNA, but our therapeutic window wasn’t big enough. This was in a mouse model. And when we knew when we translated that to people, the window wasn’t big enough. We managed to improve the delivery technology to the point where we can see a therapeutic effect at a certain dose, but that we could give a 1000x higher dose before we saw any toxicity.
That meant the therapeutic index was about 1000 or so. At that point, you say, “I think we’ve got something here,” and you move it forward into nonhuman primates to see if it correlates and then into people. That’s the process we’re in now with other tissues. We know we can do something; we’ve seen the preliminary evidence but we have to make it more potent.