Transcript of interview with Professor Philip Felgner* by Dr Lara Marks, 24 June 2021

*Director of the University of California Irvine Vaccine Research and Development Center and the Protein Microarray Laboratory and Training Facility

(this transcript has been edited for clarity and brevity)

Philip Felgner

Photograph of Philip Felgner, credit Felgner.

Lara

Many congratulations on the news of your receiving the Princess of Asturias Award for Technical and Scientific Research in recognition of your contribution to designing COVID-19 vaccines (UCI).

Philip

Yeah. What a day. Oh my gosh. It was just such an experience. And I think it's continuing here. I'm just so looking forward to going there. I wasn't very familiar with that award before. But looking through the past recipients is amazing to see.

Lara

It's very exciting. And you get to see all these other really interesting people that are going to be receiving the award at the same time.

Philip

Yeah. That's one of the sort of lessons from this whole experience, because actually, the development of the technology, the point we're at today, began at least 35 years ago. It begins really with genetic engineering. And I happened to be up in the Bay Area, and my wife actually was recruited to go to Genentech. So that was in the early 1980s. So we were able to experience a lot of that. And all of that environment is what sparked this activity.

Lara

I feel it's important to document and chart the rise of biotechnology in different spheres and the stories of those who haven't been told before, because I always feel that genetic engineering dominates the space. But there's so many other stories out there that I think need to be documented.

Philip

That's really perfect for this. The other six recipients [of the Princess of Asturias Award] are the next generation that came along after we made some of those interesting findings.

Lara

Maybe you can tell me how you got involved with mRNA.

Philip

So, my educational background is I went to undergraduate school and got my masters and PhD at Michigan State University in East Lansing, Michigan.

Lara

Why did you go there?

Philip

My home was about an hour and a half away from there. A little German community.

Lara

Okay. And where were you born?

Philip

Frankenmuth. Michigan.

Lara

And what generation are you if you've come from a German family?

Philip

My grandpa came over from Germany, from Bavaria. That community was founded in 1850 when missionaries from Germany came over. And like in so many other places, they thought they were doing good things for the Indians in Michigan and then the Indians got sick. It's just one example of where the immigrants would come over into an area that had indigenous people and where those people weren't prepared for the infections coming along. But they built a beautiful community there. And it's the third most popular Michigan destination for people coming on the weekend to visit. It is a beautiful town.

Lara

And what was your parents' background?

Philip

Well, they were born in Frankenmuth. My dad was a president of an automobile insurance company, because in Michigan at that time, when he was in business, that was the centre of the economy of the world because of the automobile. The automobile industry started in Michigan, and all roads started to be built from Michigan out. Frankenmuth was a farming community so they developed an insurance business based on farm fire insurance that farmers had. First they had fire insurance because the barns would burn down. They teamed up on a fund so that they could absorb the shock of having to rebuild when things burned down. So they followed a similar model with automobile insurance. And so he was doing that. My mother was a housewife.

Lara

Do you have any siblings?

Philip

Two brothers, both older. One is seven years and the other is ten years older than me.

Lara

When did you get interested in science?

Philip

There was a lot of technology on display in Michigan and Frankenmuth. Henry Ford also made museums available. One of the things, for example, that he showed was Thomas Edison's laboratory. He brought it over from Indiana and set it up in a place called Greenfield Village. And you could see the actual laboratory where Thomas Edison developed so much of his technology. As a young kid that was just amazing to see. And a lot of things converged at that time because televisions were just becoming available. One of the things they showed on television was Thomas Edison and his findings. That was the most interesting thing to me. And Walt Disney was always showing off scientists who I found so interesting. I really admired and was always reading about Albert Einstein.

Lara

Is that what made you choose to go to Michigan State University to study science?

Philip

Yeah.

Lara

And when you went to Michigan, what did you decide to study?

Philip

It was biochemistry as an undergraduate.

Lara

Okay.

Philip

And I was really interested in Spanish classical guitar, too. I was playing that all the time and I've kept that up to this day. And there's one thing that I regret is that I don't have enough time to play it as much as I would like. Actually, I have two guitars from Spain. One I bought in Madrid in 1972 before I went to graduate school and then another one I bought recently in Granada. We loved our trips to Spain. So in the early years I was tossing around the alternatives.

Lara

At what point did you start getting interested in cell membrane research?

Philip

My project [at Michigan] involved mitochondrial membrane proteins.

Lara

What was the project?

Philip

At that time people were purifying enzymes, learning how the enzymes work. The enzyme I was working on was hexokinase. It actually binds to a membrane allosteric protein that affects the enzymatic activity of hexokinase. So we were studying that whole system. And because the receptor for the hexokinase was in the membrane that was an important topic. We wanted to purify that receptor and learn how to use surfactants to solubilize the membrane protein and then restore the structure of the membrane again, by removing the detergents.

Lara

What did you do after that?

Philip

Well, after I graduated from graduate school I began looking for a place to do my postdoc. I gravitated to Tom Thompson's lab in Charlottesville, Virginia, and he had a biophysics department that was interesting to me. When I went there at the time there were major gaps in our understanding of membranes. One of the things that was done, around 1980, was to learn how the lipid molecules pack together to form a bilayer. We got that down to the square angstrom. You can make what they call small unilamellar vesicles. Phosphatidylcholine is in the shape of a brick in 3 dimensional space . Then you can add up all the brick-like molecules that are in one vesicle. You can understand all the dimensions of that vesicle and how much capture volume there is inside it. So we got a very thorough understanding of membranes there using the most modern ways to do the characterisation.

One of the key things is that the study started out using phosphatidylcholine and then people were changing the lipids around and seeing how that affected the bilayer formation. And one of the things that was noticed was that phosphatidylethanolamine had much more diverse behaviour than phosphatidylcholine. Phosphatidylcholine was really very predictable about what it would produce. It would produce these bilayer membranes with all the right dimensions and everything. But phosphatidylethanolamine could adapt to something called a hexagonal two phase. And then as that was getting more and more understood, people understood how important phosphatidylethanolamine is in fusion and membrane fusion. And then following that, how important fusion is in biology. The whole process in a biophysics lab is going from the bottom up. You start by understanding the most basic things, getting clarity on that and then finding out how that applies to more complex systems like we have in biology. Those things are rooted in everybody's understanding now.

Lara

So when you went to Tom’s lab, that's what you were working on. And was it basic scientific research at this point? Were you looking at questions about the membrane structure and how it works? When did you learn about Alex Bangham’s work?

Philip

Yeah. Right. And then, because you brought up Alex Bangham, as we were doing this in Tom Thompson's lab there was a community of liposomes scientists. And Tom was always the pure biophysicist. He wasn't inclined to do the translational things. But Alex Bangham and Dimitri Papahadjopoulos and Francis Szoka were aiming to take advantage of these vesicles to deliver drugs to two particular sites.

Lara

How much contact did you have with Demetrius and what he was doing?

Philip

Like I was saying, this was a community of scientists. Peter Cullis was also in that group. He's really important now, because he is the founder of Acuitas, the company that is providing all these liposomes for the mRNA vaccine. Pieter Cullis is the founder of that company. It's in San Diego. It was really a boiling pot of technology and people motivated to make this field successful.

Lara

What period are we talking about now?

Philip

Beginning in 1982.

Lara

How did you come to land up at Syntex?

Philip

I was being recruited for some of the liposome companies and also Syntex. Syntex wasn’t a liposome company but they were really interested in liposomes there. And when I went to Syntex I liked the environment there. They had the Syntex Research Institute. It was very well established. You can do a history on Syntex.

Lara

I've written a book on the history of the contraceptive pill. So I know a lot about the original foundation.

Philip

So you can maybe understand why it was interesting to me when I went there. Because those original people were still there. It was interesting, for sure. And I was most experienced then in biophysics, and not studying physiology or pharmaceutical sciences. So when I went there I was flooded with information. I would go to our working group meetings or to seminars and had no idea what the speaker was talking about, so I'd have to go into the library and study.

Lara

What were you recruited to do at Syntex?

Philip

Well, they did have a pretty focused interest. The chemists there made a library of muramyl dipeptide analogues. A muramyl dipeptide is the building block of the bacterial cell wall and it was known that the cell wall component of bacteria has the ability to stimulate immune responses. And that originally came out of Julius Freund's adjuvant, you know that's used for vaccines. Turns out Freund's adjuvant was too toxic. But the chemists thought if they could make different analogues of the muramyl dipeptide, they could get some with acceptable toxicity.

Lara

Were they looking to use it as an antibiotic?

Philip

What they really had in mind was that if they boosted the inflammatory response in people, that people would be more resistant to infections.

Lara

Oh. Okay.

Philip

Yeah, that was the idea. What happened after years of experience with this, is that especially today, and as we get more and more sophisticated and sterile in our living circumstances, inflammation doesn't do a good thing for you. It's not something you want to have going on chronically. But anyway, it got started with this idea. They thought maybe there would be a vitamin that you could take, that would always be boosting your immune response. But Tony Allison then came on board. He was a very senior in the administration there and in the laboratory. An amazing scientist, I consider him a mentor to me. Tony is the person who reported in 1955, that sickle cell trait was associated with resistance to malaria. And he was brought up in Kenya, and noticed that and he reported it.

Lara

He was part of Syntex and he was your mentor.

Philip

Yeah. He was just amazing. He had made this discovery by observing the populations in Kenya. And then, in around 2000, the Wellcome Foundation funded a group to use a genome wide sequencing study to find something that would provide a genetic basis for resistance to malaria. They spent $100 million on that study and the only thing they found that was associated with it was sickle cell trait that he had discovered 45 years earlier.

Lara

Interesting.

Philip

So it was a great place to have experience. We developed an adjuvant called Syntex adjuvant formulation one (SAF-1) and it has one of those muramyl dipeptide analogues in it. And it's a squalene emulsion. The squalene emulsion was what Freund had offered. And a lot of people wonder why it is a squalene emulsion. But today there are hundreds of millions of people being vaccinated with a squalene emulsion. That was first brought to everybody's attention, I guess, in the early days in around 1950 by Freund. The reason why squalene emulsion was used is because before Freund they were trying to make a vaccine against tuberculosis. At the turn of the century, they were trying to grow the tuberculosis organism, and then make a vaccine for it. It turned out that the organism grows in butter and they found that the immune response they could get was better if they left it in the same media that it was growing in, rather than taking it out of that media. That's 120 years of continuity based on that very first observation.

Lara

So when you joined Syntex, at what point did you start working with liposomes? What led to your development of the lipofection system?

Philip

With the liposome the idea was to make a liposome that would be like a synthetic bacteria. And it would have these muramyl dipeptide units on the surface of it, which is just like the bacteria. So they made lipophilic muramyl dipeptide analogues that could be incorporated into the liposomes. That was a project that we set out to explore and it was successful in the sense that we had this Syntex adjuvant formulation 1 that went into a phase 1 clinical trial to show that it was safe. Then later the technology was sold to Novartis and to Glaxo and then they made their own versions of SAF-1. Today those are in widespread use. So that's that project. Then why the cationic lipids? Well, as I was learning from being at the pharmaceutical company, and also from my postdoc, cells are negatively charged. In fact all natural lipids are only either neutral or negatively charged. Sometime in evolution there was a decision made not to make cationic lipids. Our thinking was that if we made drug formulations with the cationic lipids, they would fuse with negatively charged cell membranes.

Lara

When you say cationic lipids do you mean by positively charged lipids?

Philip

Yeah. So there were no positively charged lipids around that could form liposomes. We knew something about what the molecular shape of the lipid needed to be like in order to form a liposome. I talked with the chemists and they said they could make that. There was another programme, that is quite peripheral [to the story]. But anyway, they had all the chemistry worked out so they knew how to make these lipids and we talked about it and they started making them right away.

Lara

What was the advantage of having positively charged lipids?

Philip

The idea was that you could have a drug encapsulated or incorporated into the membrane of a positively charged liposome. And then that liposome would be able to fuse to membranes and deliver the drug directly into the cell.

Lara

Okay, so with a negative one, it wouldn't fuse, is that the point?

Philip

Right. So we thought that since all of our cells are negatively charged, positively charged liposomes containing a drug would be like magnets and they would come together, and they wouldn't be distinct. The cells have to stay distinct. Our cells need to have a tendency to repel each other and then be brought together by more selective interactions. That's why I say that there had to have been a choice at some point in evolution to have either a negatively charged world or a positively charged world, and we have a negatively charged world. There's very few surfaces in biology that have a net positive charge. The shell of the crab has got a net positive charge, but that probably helps it build up the shell.

Lara

So was the idea that if you could get these positively charged lipids, you'd have a way of fusing the cationic liposome to deliver a drug?

Philip

Yeah, right. We envisioned a lot of things. Many drugs that have interesting activity, or are lipophilic, they're hydrophobic. So you really need two things, you need to be able to solubilize that and deliver the drug into the cell.

Lara

What do you mean by solubilise?

Philip

Hydrophobic drugs are not soluble in water. They will just form an insoluble precipitate and are not bioavailable. But if you formulate them in a liposome, the liposome performs the solubilization effect. These lipophilic molecules are inserted into the bilayer and then the liposome itself is able to interact well with water so then it's easy to make it more bioavailable. You can even make it more bioavailable by making it positively charged, because then it will be attracted to these negatively charged surfaces. That was the idea. You know, things you could do, was you could make the drugs fluorescent, you could put them on the ear of the mouse or rat, and then you could take sections of the ear and you could see that the drug was travelling across the stratum corneum and beneath the skin. That's an easy thing to imagine. And that works, you know, with these positively charged liposomes making even something applied to the surface of the skin more bioavailable.

Lara

When you say bioavailable, do you mean going across the membrane and into the bloodstream?

Philip

Yeah. So that was going on with their regular inventory of drugs that they were interested in developing. It was a nice formulation excipient to be used for testing, improving the performance of drugs. In pharmaceutical companies, it's a very common pathway. Nowadays, you do something in cell culture to see an effect that you're looking for. And then you want to do it in an animal. Then immediately when you do it in an animal, one of your first priorities is called pharmacokinetics and pharmacodynamics. So you need to know, does it get it in, and how much of it gets in and what percentage? That is bioavailability. After you've identified key drug molecules, that's the next step.

Lara

Right.

Philip

So we were involved in providing some of these novel liposomes systems to help with all of that. And then you're familiar with Papahadjopoulos [and his team] - they were taking it to another level. They were wanting to administer it intravenously, and have it go to the tumour tissue, or whatever like that. And that was another level of specificity trying to produce.

Okay, but here we have these positively charged liposomes for all of these reasons. And we also had a Genentech now entering into the genetic engineering space. There was a good molecular biologist across the hall from me - Hardy Chan. He could make these plasmids with reporter genes, beta galactosidase, and chloramphenicol acetyltransferase. And, about the same time, also, the liposomal, people were thinking, we've got the perfect vehicle, looks just like a virus, to do gene therapy with liposomes. There were some problems because the long dimension of the DNA is way longer than the internal diameter of the liposome. You can't put a square peg into a round hole. There just isn't the space there. So they were struggling with that, always trying to do something to improve the capturing efficiency. You know, if we're doing drug encapsulation of water soluble molecules, and if we got up to 50% encapsulation efficiency for a small molecule, we felt we were doing good. But it was way less than that. We were throwing away a lot of stuff that was valuable and everything.

So we thought that maybe we could take advantage of these positively charged liposomes because the nucleic acid was so oppositely negatively charged. So I made those positively charged liposomes and mixed them up with some of the plasmid that Chan had already made. And this is a really important issue here. Because I thought when we did that it would be really obvious that they were interacting with each other and the whole thing would turn into gelatin, or just everything would fall out of solution. But the shocking thing was that I mixed these two oppositely charged things together and I couldn't see any change at all. The question was how could that be because the interpretation was that they were not interacting because I couldn't see any change. And if you're going to have an interaction, then surely everything would fall out of solution.

To show they interacted we had to run a sucrose density gradient. You know DNA goes to the bottom of the gradient because it's dense and the lipids float. So the one thing you could have found is that DNA is still going all the way to the bottom and lipid is going to the top. But that's not what happened. All of the DNA was recovered on the top with the lipid. So it was interacting with the lipid. The interaction was occurring in a way that didn't produce particles of a larger size. If they were of a larger size we would have seen a change in the turbidity. So it self-assembled into a virus-like particle is what the bottom line here is. Pretty much you're encapsulating all these different plasmids, one plasmid at a time in each little vesicle. So you've got something that we then later called a ‘lipoplex’, because it wasn't a liposome. A liposome is a bilayer structure. There's a nice publication where we described this and named it a lipoplex (Felgner, Gadek, Holm ). Today, they're called lipid nanoparticles because at a certain point you could get more funding when you are working with something that is nano. But we had scientific reasons to call it a lipoplex.

Lara

Okay, so they were the very first lipoplexes to be developed in the world.

Philip

Yeah. The first one was that one that I said that went together and there was no change. And we had to prove that they were interacting with the sucrose density gradient.

Lara

When were you doing that research?

Philip

In 1984. That observation was, without anything else, already a revelation. The whole world of possibilities opened up to you right away. And the reason is, is because it solved a tremendous problem. Because everybody was trying to encapsulate the nucleic acid into the traditional liposomes and it just was not practical to do that. But here, suddenly everything became practical. We call it an extemporaneous mixture. You can take any gene you want, and you can mix it up, and you get what you're looking for. You don't even have to do anything more sophisticated than that. So it's extemporaneous.

Lara

So now you basically had the delivery mechanism for encapsulating the nucleic acid which was very easy and quick with what you call a lipoplex.

Philip

Yeah. The reason it opened things up was you could now think about making a virus-like particle and you could put anything on the surface of this liposome that would improve its delivery and be more and more like a virus. So that opened up. Then we said to Hardy let's see if we can get some expression from these reporter genes in cultured cells and we can get started and then we'll make it better by changing a lot of things. So, we made up these lipoplexes. The most simple ones and put them on cells and immediately got expression.

Lara

When you say you got expression, was it that the cell started to produce a particular product?

Philip

Yeah. It produced the protein that was encoded on the gene.

Lara

Okay.

Philip

That's called transfection. We were immediately producing these gene products. It was kind of a disappointment. Because we were getting all geared up to have a whole programme to make a sophisticated virus-like particle but it was working right off the bat. And the chemists had made all these different analogues. So we just had a screening programme, making lots of different formulations. We had ways of making hundreds of different formulations of this, and then we could optimise it.

But underlying all of this, is the knowledge now that we had a way to introduce any gene into any cell and affect the physiology of that cell. For basic science that was fantastic, you know, because whatever you were studying, you could have a cell that lacked that gene then you could understand the differences about having a different gene in there. So that's a basic science advantage. And many things have come out of this now. It's so practical and there are thousands of citations every year recording the use of these kinds of systems in basic research.

Lara

Is lipoplexes the word that would be used in those citations?

Philip

Well, lipofectin is the first one.

Lara

Okay.

Philip

And then lipofectamine is the one that Thermo Fisher has put so much energy behind to inform customers about its performance. They give a tremendous package of information about that one. So rather than somebody who's not really interested in transfection, per se, they can go to Thermo Fisher, because Thermo Fisher explains a lot of stuff. They've committed to that one.

Lara

You were at Syntex and you developed this system. What happened then? Did they patent it?

Philip

Yeah. We have a pretty amazing patent there. Because the patent anticipates all these uses. We did patent that. And as soon as we got this and we worked it out we got the paper published. I guess there were two things. One thing is I wanted to see it be used in a lot of other labs because I wanted to see what the breadth of the use is. That was one thing. The other thing we wanted to do was gene therapy with it. Because here we were, we were doing something in vitro, this is the pathway in drug development, and then after you do it in vitro you try to do something with it in vivo.

Lara

What you are doing is basically going from the test tube into an animal.

Philip

Yeah. And we wanted to do this for gene therapy. But the company was not going to support that. Hardy Chan said, 'That's for the year 2020'. That is the amazing thing to me, because I can remember standing next to him in the hallway and I said, 'you know we've got to convince these guys to support a project so that we can find out if this works in vivo. And he said, 'gene therapy is for the year 2020'. Then to have it actually appear this way in the year 2020 is just really ironic.

Lara

At that point did you feel you weren't getting anywhere within Syntex and that you wanted to broaden where you could go?

Philip

Yeah, I wanted to do something. I mean, there was a, there was a pathway at the company that I could have taken. You know, they were promoting me, but it wasn't going to be successful in my job there. By doing all the research that was necessary to develop this kind of gene therapy. That gets back to another generalisation. Because I don't think biotech companies assign enough value to research. When I went to Vical it turned out to be even worse. I probably would have more latitude in doing research at Syntex because they had a big company and products supporting their business. But at Vical as soon as we made these founding discoveries, the venture capitalists came in, and the venture capitalists are always suspicious that these guys that are in the lab all they want to do is their own research. And they're just going to take advantage of that thing that they bought into now. And they're going to get product developers to develop the product. The problem with it is you may not know enough yet to develop that product yet so you need a research engine there to help in the decision making. It's just how pervasive this matter is, it is very frustrating.

Lara

Can I take you one step back? What made you go to Vical?

Philip

Well, I was getting embroiled in this gene therapy community, which was really hot in the mid mid 1980s. I guess I had gotten some reputation started and a couple of faculty members at UC San Diego were aware of some of my work. And San Diego was a hotbed because of Hybritech which Ivor Royston founded. Hybritech was active there and everybody knew about it. What happened is they sold the company and it was big news. That here a biotech company made big, big bucks, you know $400 million was huge then. So that informed everybody how successful you could be if you invested in a biotechnology company. So this whole thing was brewing in the liposome field and then the Hybritech. So there was a community in San Diego and all the infrastructure. So when San Diego, like other places around the country, supports the biotechnology community you have everything you need. You have eating places, you have places where you can go to get things fixed, you have architects and contractors and all those things around there. One of the things we had was the founders of Hyberitech had money. Tom Wollaeger and Ted Green were beneficiaries of the Hybritech sale. They set up a venture capital group and founded Vical and they had me go there. My wife and I were among the first employees there.

Lara

What was the vision for Vical?

Philip

That was really really interesting. AIDS was an issue at that time. That was even before AZT was approved, or maybe it had just been approved, as a drug. By the way, AZT, Syntex also had AZT and chemistry, which Burroughs Wellcome then had to have an agreement for. So I guess I had that background too. And Doug Richman and Carl Hostetler at UCSD were making lipophilic derivatives of AZT. So you could make liposomes with AZT on them. That was important, because there was discussion about the reservoir of HIV. What cell population was the HIV living in that made it so difficult to get rid of it? One population was considered to be the macrophage and liposomes are known to be taken up by macrophages. We could do all of this. We did this and we made beautiful formulations. We got a contract with Burroughs Wellcome. We got about a million dollars a year. Then we got the formulation. They were able to manufacture it in their manufacturing facility to their satisfaction. We had quantities sufficient for the clinical trial, and it was going to be an infusion. At that time, they were prioritising infusions. But they had so many patients they needed something that worked orally so they wound up not prioritising it.

Lara

Okay, so you were preparing infusions, but they went for the oral one, which is why Wellcome went in a different direction.

Philip

Yeah. They stopped funding that project. But we were doing that. But then, at the very same time, as soon as I went to Vical, one of the things I asked the investors to do was to support this backburner project, which was the one I wanted to do at Syntex. And they begrudgingly agreed to pay for it. And I met a guy in Ted Friedman's lab, named John Wolff.

Lara

You hadn't worked with him before?

Philip

No, I, I just went over. I was so interested in doing this gene therapy thing and Ted Friedman was considered to be kind of the founder of the whole idea of gene therapy. His lab was right there in San Diego. The lab was right next to the library, and I was over at the library all the time so after going to the library I went over to Ted Friedman's lab. I thought I'd introduce myself to Ted. Then I ran into John Wolff and discussed this experiment that we wanted to do. I said we have to do something because we don't have any animal facilities at Vical right now. And he said, ‘Well, I’m finishing my post-doc in Ted’s lab taking a faculty position at Wisconsin, and they have the animal facilities there and I would like to work with you on this project. You know, it would be a great project for somebody just taking his first faculty appointment to try to get started. And he was interested in other kinds of gene therapy, applications in neuroscience. Anyway the first experiment was to get these reporter genes, and then make the lipoplexes that work so well in cell culture, and inject them all over the animal wherever you could get a needle and see whether you could get expression.

Lara

When you say reporter genes, what do you mean by that?

Philip

That's a gene that we can easily detect the gene product for.

Lara

Okay.

Philip

That's why some of our papers, one that is cited so much, it's an injection of genes into the skeletal muscle of a mouse. And you see these blue cells. That's because beta galactosidase was expressed in those cells and there's a substrate for the beta galactosidase that turns blue. So you can take a tissue section and put that substrate on there. If there's any beta galactosidase expression there, you'll see a blue cell.

Lara

Okay, so you've deliberately targeted that because you knew you could stain it and see it.

Philip

Yeah, right. So we injected this into mouse skeletal muscle. And John was good at doing tissue sections, so he could make the thin sections you need, and then go to the microscope and see those blue cells.

Lara

Were these live mice you were putting it into?

Philip

Yes.

Lara

And then what did you do?

Philip

So that was positive. We were overjoyed when we saw that. And we started writing a patent then for Vical because it didn't have any technology on this and we needed to get some technology, if we're going to get somebody to invest in it. So we were writing a patent. Then we continued to do experiments. The next experiment was to change the amount of cationic lipid that we had in the formulation. Because you need to have a lot of the cationic lipid for this to work, or a little. One of the groups was using no cationic lipid at all. What we found was that the one that had no cationic lipid also worked. So this cationic lipid that was necessary to achieve expression in cultured cells was not absolutely necessary in vivo. That was actually really exciting because it was completely unexpected. There was no prior art, essentially. If we had made a cationic lipid formulation, the prior art would have been Syntex’s patent. Because we already claimed in vivo gene therapy with cationic lipids. That gave a very powerful Naked DNA patent position for Vical.

Lara

What were you using? I've read what is called ‘naked DNA’. But what were you using in that technique that allowed you to introduce the gene.

Philip

It required no special delivery system. The only thing it required was a certain volume of the fluid injected into the muscle. What the fluid volume does is it breaks up the muscle tissue a little bit, and it allows space for the DNA to get into the cells. But then the muscle heals right away. So it turned out that it was a pressure mediated effect, not any kind of delivery system effect that everybody was striving for. That is what the venture capitalists and the board members are looking for is something that excludes everybody else from working in the field. And that was a patent that was the founding basis for Vical.

Lara

How does that lead to today's use for mRNA.

Philip

Well, everything that we did with DNA, we also did with RNA, messenger RNA. And it all worked the same way in the assays that we were running. You could get the same levels of expression with either DNA or RNA. The difference was that DNA expression was long lived, because the DNA would stick around. But the mRNA was being degraded so you would get a transient expression from the messenger RNA expression.

Lara

My question is this, you can't just isolate mRNA and then just put it into a muscle cell. It would degrade would it not?

Philip

Yes it does get degraded. But what we observed is that it degrades slowly enough that there's time for the protein to be made.

Lara

Okay, so all this time you were doing a lot of work around liposomes when in fact if you just isolated the DNA or the mRNA, and put it directly in, it would have worked anyway.

Philip

Yeah. But you would have never done the experiment because it would have been ridiculous to think that that would ever work.

Lara

Extraordinary. All these years playing around with lipids.

Philip

Yeah, but I think it's gonna come around again. But anyway, that was where we were in and the investment from the investors was to support that founding patent for which there was nothing like it at all. There was nothing to compete with it. So we set out to do immunisations that way, with DNA. And the DNA and the RNA were both in play.

But we got our funding from Merck. And Merck looked at the alternatives. There was a way to make the DNA and right away it seemed to be practical. RNA had too many obstacles, technical hurdles, to get over. And again, it's the same situation as before, they weren't going to invest in research. They weren't gonna invest in a technology that would take years of research. We know how many years it's taken to develop the mRNA, 20 years and tremendous commitment from at least three companies and billions of dollars. So Merck made the decision not to do that, but to do DNA. They took what we know about how to make DNA, and they took it to their manufacturing facility to do pilot batches. And in a matter of a few months they were convinced that they could do it. That was what they wanted. Something that they could make and test right away. So that was what was really driving that history.

But the thing that really interested Maurice Hilleman so much is that this could solve his manufacturing headaches that he faced every time he had another vaccine and it drove him nuts. What he had in mind was some vaccine that he wanted to develop, and everything was too slow for him because all those darn people in manufacturing couldn't get it right. He just was furious about all that stuff. But this was the answer. With DNA all you have to do is change this sequence of the DNA and you have a new vaccine. The vision was easy to manufacture and solve the manufacturing problem.

So we proceeded, we published a paper in Science showing that it worked for influenza in mice (Ulmer, Donnelly, Parker). But then not enough things converged. There's some very basic things that made it not work. The animal model system we used and published showing that it did work using the nucleocapsid protein from flu. It was working by a cell mediated mechanism. When we had to go to human use, we had to use the hemagglutinin from the flu, and that one is mediated by an antibody mechanism. When you go by antibody mechanism, it's very attractive from a clinical perspective, because they already had their benchmark of what your antibody titre needed to be in order to go to the next phase. We couldn't quite reach that antibody titre level. But actually, our demonstration didn't depend on antibody at all. It was a cell mediated response. But in their clinical development of flu vaccines, they didn't have a pathway for that. It didn't have a pathway to say, ‘okay, we like this now because it has a kind of cell mediated response we're looking for’. Because they didn't have that. One of the frustrating things is, I think if we were to use nucleocapsid protein in that study we'd probably be doing a flu vaccine with DNA right now. We're going to be exploring that kind of thing with coronavirus coming along.

Then the other thing that happens in any new biotechnology company is there's really only one thing you're going to be asked to do and that's to cure cancer. They're not interested in anything other than cancer. And it was especially bad at that time because even the NIH their interest in microbiology was declining. Even if you went to the NIH, and had a proposal to study an infectious disease vaccine, then the NIH said, ‘We're the National Institute of Health, not the International Institute of Health’. Because infection was something that was considered to be a problem outside the United States, but not a problem inside the United States, with the exception of this annoying HIV thing, but that was a side issue. Anyway, they had figured out how to cure that one with drugs. So vaccination was not a high priority whatsoever. We didn't have a lot of chances to look at different targets.

Lara

Can I just take you back to the mRNA? What foundation do you think your work at Vical meant to the development of the mRNA vaccine?

Philip

I think one thing that gets lost is the role of Bob Malone in sparking the mRNA experiments that we did at Vical. He had picked up through his training and experience how to make functional mRNA at a time when there were very few people around who could make that. He didn't discover all, but he was a graduate student, and he reached out to the right contacts, so that he could find out what would need to be done to make a functional messenger RNA. There was a lot of literature on it too. So he was reading that literature. So he understood from what was in the literature, what it would take to make a functional mRNA. And, he did it very effectively. It's in, in the PNAS paper that we published, and there's just classic experiments in there (Malone, Felgner, Verma). What they're doing today is essentially exactly the same thing.

Lara

So he was making a functional mRNA. But how were you then putting it into the animals?

Philip

To make the publication, it had to have the RNA that he made and the cationic liposomes, the lipofectin, that I provided. You know, we were in San Diego with this boiling community of gene therapy people were rubbing shoulders. And people became aware of the cationic lipids that I had that could introduce the nucleic acid into cells. Bob wanted to do that. He wanted to do it in Inder Verna’s lab, also a leader in the viral gene therapy field. They wanted to make a packaging system to package nucleic acid that would be for gene therapy purposes, but package it into a virus. Their idea was that they wanted to have those genes that are the proteins that make the viral package, have that encoded in RNA. That would be a really good thing to do, because you wouldn't have any risk of having the replicated virus emerging. All those proteins that made the virus are encoded on RNA and the RNA is gonna get degraded. So another way to make a viral gene therapy vector was to actually make artificial recombinant viruses in this way. That was what was initially motivating him to put so much effort into getting functional RNA that could express the gene product. But then he did two things. We collaborated on that paper to transfect cultured cells. That was the first time that was done. And then when you start doing that immediately your eyes are opening up to the possibility of doing RNA gene therapy. But then he took it one step further. He was a TA in a laboratory at UCSD, he was doing that to get some of his money to survive in graduate school. He had a bunch of xenopus and frog embryos that they were doing some experiments for the graduate students, I guess. So he had those embryos there and he said, ‘I wonder if I can use the lipofectin and transfer RNA into those embryos. And he did. He showed that. Christine Holt was the faculty member who he was teaching assistant for and Christine took that observation and extended it beyond just doing the transfections. She could transfect neurons and all kinds of cells that way. I guess, not so much to me, but maybe to Bob who sparked that work, it would have been annoying that he wasn't a co-author on Holt’s paper. He was mentioned as was I in the acknowledgments section. But yeah, those are things that happened.

References

Felgner, PL, Gadek TR, Holm M, et al. (Nov 1987), 'Lipofection: A highly efficient lipid mediated DNA-transfection procedure', Proceedings of the National Academy of Sciences USA.Back

Malone, RW, Felgner, PL, Verma, IM (1 Aug 1989), 'Cationic liposome-mediated RNA transfection', Proceedings of the National Academy of Sciences USA, 86/16, 6077-81.Back

UCI News (24 June 2021),Back

Ulmer, JB, Donnelly, JJ, Parker, SE et al (19 March 1993) 'Heterologous protection against influenza by injection of DNA encoding a viral protein', Science, 259/5102, 1745-49. Back

Respond to or comment on this page on our feeds on Facebook, Instagram or Twitter.