Robert Malone’s role in the invention of mRNA vaccines — an analysis

“Zero to one” or 0.5 to 0.51?

Dr. Robert Malone is back from obscurity on the news that he is suing the NYT over their recent article:

The Latest Covid Misinformation Star Says He Invented the Vaccines

I have been pointing out Malone’s dubious claims about the safety and efficacy of Covid vaccines for many months now [1, 2], but until recently I was never really curious enough to investigate his original claim to fame, namely that he has invented mRNA vaccines to begin with. So let’s do it. Let’s do a deep dive on Malone’s inventorship claims.

In particular, at various times he has repeatedly claimed to have invented mRNA vaccines, the field of “messenger mRNA [sic] therapeutics”, or even “RNA as a drug”. Intriguingly, until Dec 2020, Malone’s own website only modestly called him “one of the original inventors of ‘DNA vaccination’” and did not even mention RNA, but then in May 2021 it was changed to start calling Malone “the inventor of mRNA vaccines”, and by June 2021 the website claimed Malone invented the entire field:

In support of his inventorship claims, Malone cites Vical Inc. patents where he is one of the co-authors. However, the first author on all those patents is Dr. Phillip Felgner who was the one who actually invented the lipid encapsulation methodology, which is the methodology that makes it possible to deliver DNA and RNA to cells (the process is often called ‘lipofection’ which stands for lipid-mediated transfection).

When elaborating on the topic, Malone usually concedes that he didn’t invent any actual vaccines (i.e. approved therapies) but rather invented the underlying “mRNA vaccine technology”. However, as we just saw above, the lipid-mediated delivery technology that Malone used in late 1980s to deliver RNA into cells was not invented by him. Moreover, none of that particular technology is used in today’s mRNA vaccines.

How It All Began

So how close was Malone to the original invention of that delivery technology? Well, Dr. Phillip Felgner started working on his lipofection methodology in the early 1980s long before he knew Malone and by 1987 has demonstrated that it can be used to successfully deliver DNA and RNA into cells — as disclosed in one of Felgner’s patents filed in 1987, which provided examples of doing so for both DNA and RNA. Note that the above patent predates any patents that Felgner would later file with Malone as a co-author.

Felgner’s most famous lipid molecule is DOTMA, and it has since become a key ingredient of one widely used commercial lipofection reagent, Lipofectin™. DOTMA is what Felgner would later send to Malone to experiment with RNA in late 1987.

Note that DOTMA-mediated lipofection is just one of many possible lipofection methods — others have used liposomes to accomplish the goal of RNA delivery into cells almost a decade prior. For example, one group has demonstrated successful liposomal delivery of mRNA into murine cells in 1978, while another did the same for human cells. However, canonical liposomes were suboptimal for gene delivery purposes, particularly due to their negative charge, and it took Felgner’s breakthrough in creating positively charged lipids to catalyze the lipofection gene delivery approach.

Malone first started experimenting with using Felgner’s DOTMA lipid formulation to deliver RNA into cells in late 1987 — as part of his graduate work at Salk. In early 1988, Malone wrote a letter to Felgner, in which he thanked Felgner for the chance to contribute data to “[Felgner’s] DOTMA RNA transfer project” and for sending over the actual lipids for Malone to do so. In the same letter Malone fully acknowledged that Felgner has already tried his own method on RNA but was having problems:

So based on his own words, Malone’s contribution was figuring out how to use the DOTMA methodology for mRNA lipofection and diagnosing why Felgner’s earlier attempts to do so were unsuccessful: judging by the above letter, Felgner used mRNA that didn’t have a poly(A) tail — a feature that is important for mRNA stability in eukaryotes. Also, the reporter gene Felgner used to verify that his mRNA was actually being translated into protein was hard to detect at low levels.

Why did Felgner collaborate with Malone on the DOTMA RNA experiments in the first place? It seems that Malone had a skill that was quite rare at the time — he mastered a recently developed method of RNA synthesis. It was developed in 1984 by the groups of Krieg and Melton at Harvard, and turned custom RNA synthesis from a prohibitively complex process into something even a grad student could do. Of course, being one of the first to master a novel method someone else has invented is laudable but not an invention in itself.

So, in a nutshell, it seems that Malone’s contribution to Felgner’s DOTMA project was synthesizing the right mRNA that would be stable and detectable in eukaryotic cells, mixing that mRNA with the DOTMA lipids provided by Felgner, and confirming that the same cellular uptake and expression of lipid-encapsulated nucleotides which Felgner has previously observed with DNA and RNA was also happening with mRNA synthesized by Malone.

An Inventor is (Not) Born

Excited by his success, Malone then wrote an internal Salk memo in which he mused that DOTMA-mediated mRNA delivery could provide “a way to treat RNA as a drug”. It seems that Malone believes that this letter marks the point at which he has invented “RNA as a drug” in 1988:

Does this letter really grant Malone the title of having invented RNA as a drug? Does his contribution to Felgner’s DOTMA project really make Malone the inventor of mRNA vaccines?

Speaking of vaccines, they were an interest of Felgner long before he met Malone. Felgner was working on vaccine adjuvants at Syntex since the mid-1980s and one of the applications of the DOTMA methodology was to deliver vaccine adjuvants — in fact, the DOTMA RNA lipofection example provided in Felgner’s 1987 DOTMA patent demonstrates delivering RNA for a known vaccine adjuvant, poly I:C. So it is only natural to assume that it was just as obvious to Felgner, as it was to Malone, that the DOTMA technology (or any other technology) to deliver DNA or RNA into animals can be used to deliver genes on which the animals’ immune system can be trained to produce protective immunity, i.e. as a vaccine.

Of course, in biotech, an idea is nothing without execution, and for patenting purposes Felgner needed to provide animal data which would have demonstrated that the DNA or RNA they administer to animals do, in fact, produce the desired therapeutic response, be it the production of a needed protein, or an immune response to one.

To generate such animal data Felgner engaged several collaborators, one of which was Dr. Jon Wolff, who ultimately ran some of Vical’s animal studies at his fledgling lab at the University of Wisconsin-Madison. Wolff’s initial animal studies did not investigate any potential therapeutic effects but just tried to confirm that the reporter gene (CAT) encoded by DNA or RNA liposomes administered to animals does end up being successfully taken up by their cells and translated into protein.

That work was by then done under the umbrella of Vical — a startup which Felgner joined in late 1988 from Syntex and brought over his lipofection platform. In early 1989 Felgner also recruited Malone, who by then had a falling out with his supervisors at both Salk and UCSD, and decided to drop out of his PhD program. However, at Vical, Malone also lasted only a few months before resigning in August of 1989 due to perceived unjust claiming of credit by Felgner for Malone’s “intellectual contributions to the gene therapy project”:

In his brief time at Vical, one of Malone’s roles relevant to our analysis was to produce lipid-encapsulated DNA and RNA formulations used by Wolff in his animal experiments, as well as “naked” (i.e. not encapsulated) DNA and RNA to be used as experimental controls. The data from those experiments was then used for Vical’s only patent application 07/326,305 filed while Malone was still with the company. That application would go on to provide the priority date for several of Vical’s subsequent lipofection gene delivery patents.

Intriguingly, as Vical’s patent application was being written, Malone wrote a competing patent application for Salk, which was filed on the same day (March 21, 1989) as the Vical patent application, and which tried to shoot down Felgner’s previous claims of successful RNA transfection via his DOTMA method:

It is interesting to see Malone’s above dig at Felgner — “no experimental results or even theoretical considerations are disclosed in support of this speculation”. It looks like Malone was unaware that DOTMA-mediated RNA transfection was actually disclosed in Felgner’s patent US4897355 in 1987.

The Salk application written by Malone looks even more ethically and legally dubious when one considers that Malone (who was already employed by Vical at the time) was carbon-copied by Felgner on a Vical memo to its patent lawyers regarding Vical’s own application:

The very same memo by Felgner spelled out vaccine applications of the lipofection platform in question:

Moreover, whereas Malone’s application did not even mention vaccination or provide any in vivo data on gene delivery (in vitro RNA transfection of embryos which were then lysed hours after RNA delivery doesn’t really qualify as in vivo data), Vical’s application provided actual examples of vaccinating animals by mRNA:

I believe Vical’s focus on HIV vaccines was inspired by its cofounder Dennis Carson (who is probably most known for having co-invented an anti-cancer drug). Prior to co-founding Vical, Carson was at Scripps where he worked on anti-HIV treatments for several years. He also looked into the Epstein-Barr virus’ gp110 envelope protein with Gary Rhodes who would later also join Vical and lead its DNA vaccine efforts targeted at HIV’s gp120 envelope protein. In any case, only mRNA (as per above) and not DNA vaccination was part of the original 1989 Vical application, and Rhodes would only be added as a co-inventor to DNA vaccination patents in 1990. Curiously, as Vical’s internal meeting notes show, gp120 mRNA for those studies was produced by Jon Wolf’s own lab and not by Malone (see bullet VI):

So it was Wolff’s lab that both prepared the HIV mRNA formulations and ran the HIV vaccination animal studies. As Malone’s lab notes show, the only animal experiments he was involved in (via preparing DNA/RNA formulations for them) were reporter gene experiments (CAT and luciferase) and not any clinically relevant animal studies. For the latter studies — like experiments involving HIV (gp120 or NEF genes), polio or EMC viruses — it was Wolff’s lab that was making its own DNA/RNA formulations.

Thus, as Malone was not involved in any vaccination studies during his time at Vical, it is unclear how he can claim any credit as even a co-inventor of mRNA vaccination, let alone call himself “the inventor of mRNA vaccines”.

So What Did Malone Invent?

Putting aside the bad look (and legal aspects) of Malone trying to scoop his own company’s patent application for Felgner’s lipofection platform, let us come back to the potential novelty of what Malone has done in using lipofection to deliver RNA into cells.

Even back then it was clear that Malone was far from the first to do so: just a few months after the Salk application, in his joint paper with Felgner and his former Salk PhD supervisor, Dr. Inder Verma, Malone acknowledged that other groups have previously succeeded at delivering RNA into cells: “progress in introducing RNA molecules into cells has been very slow and restricted to a few cases (1–4).” Curiously, in those 4 references of prior works Malone did not include the two 1978 papers (i.e. a decade prior) that used liposomes to deliver mRNA into human and murine cells, which I cited earlier.

The animal experiments conducted by Wolff which showed that DOTMA-mediated lipofection can be used to deliver mRNA not just into cells but into adult animals — and, more importantly, to get that mRNA translated into detectable quantities of protein — were subsequently published in 1990 in a joint paper with Malone, Felgner and four other authors. By then Malone had already left Vical and went back to Northwestern to complete his medical degree.

That paper was to be his last collaboration with Felgner or Wolff. However, lipofection work at Vical was only beginning. In fact, all of Vical’s patents [1, 2, 3, 4, 5, 6, 7, 8, 9] on which Malone is named as a co-inventor were filed after he left, in some cases many years after. The original patent application 07/326,305 filed during Malone’s tenure at Vical was ultimately abandoned and only used as a priority date anchor for subsequent divisional or continuation-in-part applications.

Scientific publications on DNA and RNA lipofection by Felgner and his team also continued. Judging by the signatures on the relevant Vical lab notebook pages posted by Malone, his responsibilities on the DNA and RNA lipofection project were taken over by Felgner’s wife, Dr. Jiin Felgner:

She was one of Vical’s first employees, and has stayed with the company for a decade. In the 1990s, she has also co-authored several of Vical liposomal patents [1, 2, 3] that do not have Malone as a co-inventor, and has also co-authored several academic papers on the topic [1, 2, 3, 4].

Coming back to Phil Felgner, I think one of the best measuring sticks of the relative impact of Malone vs. Felgner on the field of RNA delivery is provided in the 1998 paper by Katalin Kariko, who many believe might get a Nobel Prize for her role in the development of mRNA therapeutics. In that paper Kariko et al. cite Felgner 10 times in connection with mRNA lipofection, while only citing Malone once and even then in co-authorship with Felgner. I should mention that Kariko has herself managed to successfully deliver RNA into cells long before Malone — as early as 1985. Notably, both Felgner and Kariko have been recognized in 2021 for their contributions to COVID-19 mRNA vaccines by Spain’s Princess of Asturias Award for Scientific Research — a very prestigious scientific award sometimes dubbed as “Spain’s Nobel Prize”.

Moreover, most of Malone’s academic citations come from papers and patents in co-authorship with Felgner, namely 2 papers and 9 patents, which, as I mentioned above, were all filed after Malone left the company in 1989. Google Scholar shows 12,847 citations for Malone, with 5172 of those coming from the Wolff et al. paper on in vivo RNA and DNA delivery using Felgner’s lipofection. Another 3607 of Malone’s citations come from Vical patents filed between 1996 and 2007. For comparison, Felgner has 41,355 citations in Google Scholar, with his pre-Vical 1987 lipofection paper having the most citations at 6258.

Naked DNA and RNA Delivery

The story of Malone and Felgner would be incomplete without the naked DNA/RNA delivery saga. During animal experiments that Wolff was running for Vical on in vivo delivery of liposomal DNA and RNA formulations, it was accidentally discovered that you could actually deliver naked DNA and RNA i.e. one not encapsulated in lipids.

Initially such naked DNA and RNA was meant to be used as control by Wolff, i.e. no activity was expected to be observed from such formulations. However, Wolff’s experimental readouts have shown that even naked DNA/RNA get into animal cells and result in protein expression. This was so unexpected that it was initially dismissed as an experimental error. However, after repeating the experiment, Vical was confident they had indeed stumbled upon a monumental discovery. They have filed appropriate patents and have made an announcement in the press in March of 1990:

The experiment was so elementary, and the results so surprising, that researchers working with San Diego’s Vical Inc. couldn’t really believe what they were seeing. It all seemed too simple.

They had been injecting submicroscopic fatty globules containing DNA or RNA into mice to see what would happen. The idea was that the fat globules, called liposomes, would be taken up by cells. The cells would use the genetic material inside to make proteins they couldn’t otherwise make.

The researchers found moderate success with that, but the rigors of science demanded that the experiment have a “control” portion — injecting the raw DNA or RNA into the mice to show that the liposomes themselves were making it possible for the new genes to be incorporated into the cell’s processes.

It turned out the cells like the raw material even better and began making the new proteins for as long as six months.

“This was a big surprise, and that’s really what you’re looking for in this area,” said Philip L. Felgner, director of product development at Vical. Felgner worked on the experiment with Dr. Jon Wolff and others at the University of Wisconsin at Madison.

…

Vical hopes that the results of this checking and double-checking, reported in today’s issue of the journal Science, will convert the company from a bare-bones start-up to a major player in the ranks of San Diego’s biotechnology community.

The above article barely mentioned Malone as an also-ran, and this must have caused him great irritation. In fact, years later, in 2001, he even wrote to Vical’s CEO making a case that it was Malone and not Jon Wolff who meets the inventorship criteria for discovering the naked DNA/RNA delivery methodology:

In the process, Malone seems to shoot down the very validity of the discovery:

As the 1990 LA Times article mentioned, Vical considered the naked DNA delivery discovery as very important. In particular, it was thought to pave the way for “DNA vaccination” — a term that became quite popular in the 1990s, and which Malone originally claimed to have co-invented, before he converted himself into “the inventor of mRNA vaccines” or “RNA as a drug”. The 1996 Lancet article explains why Vical felt the naked DNA delivery held so much potential:

In fact, what Felgner and Wolf originally thought was a laboratory error turned out to be a seminal observation — one that may lead to a revolutionary new type of vaccine — the naked DNA vaccine.

Only a small amount of protein was produced after DNA injection, but Felgner and Wolf immediately realised that it might be enough to generate an immune response to an antigenic protein. They tested this idea by injecting a plasmid containing the gene for the HIV envelope protein gp120 and found that they induced both antibody and cytotoxic immune responses in mice.

Vical’s co-founder and co-author of its gene delivery patents, Dennis Carson, in a 2006 interview claims to have recognized the potential of naked DNA delivery for “DNA vaccination immediately”:

In our initial experiments, it was discovered serendipitously that naked DNA, without the liposome delivery system, gave better expression in the muscle than the DNA encapsulated in a liposome. I wrote a letter — I still have it — in 1988, to the CEO of the company, saying this was a major discovery and it could have very interesting applications in immunology. … I realized that injecting naked DNA with a vector simply mimicked what a virus did.

As I mentioned above, Carson was an immunologist previously working on HIV therapies, and Felgner has worked on vaccines long before he met Malone. The original March 1989 Vical patent application not only mentioned vaccination as one of the applications of DNA/RNA delivery but provided a concrete example of vaccinating mice by mRNA coding for HIV’s gp120 protein. Those experiments did not involve Malone at all. Moreover, after Malone left, Vical has entered into a collaboration with Chiron to test their gp120 DNA (rather than mRNA) formulation. Gary Rhodes led those animal studies at Vical in-house and has earned a spot as a co-inventor on relevant DNA vaccination patents. In fact, it seems that at around the same time DNA vaccines became the focus of the industry and have remained so for many years, as evidenced by Merck licensing the Vical patents with the goal of producing their own DNA vaccines.

One possible reason for the popularity of DNA vaccines over RNA ones is RNA cytotoxicity due to excessive inflammation — an issue that seemed prohibitive to the use of “RNA as a drug” until in 2008 Katalin Kariko and Drew Weissman have figured out how to circumvent it with pseudo-uridine modRNA.

Maybe that relative obscurity of RNA therapies over DNA ones until essentially 2020 is why until May 2021 Malone has referred to himself only as a co-inventor of the DNA vaccine technology, as evidenced by his website metamorphosis which I highlighted in the beginning of this article, as well as his 2017 CV:

As I’ve already mentioned, the original Vical patent application only provides examples of mRNA vaccination, and DNA vaccination work was done by Vical after Malone had left the company. Of course, his name was still retained on DNA vaccination patents which were filed by Vical after Malone left because those patents were continuations of the original Vical application, but that doesn’t mean Malone did any actual work on DNA vaccination. Even the mRNA vaccination experiments — i.e. experiments trying to elicit expression of a therapeutically relevant protein rather than just a reporter gene like CAT — that are provided in the original Vical application were done without Malone’s involvement.

So why in May 2021 did Malone precipitously abandon his self-appointed title of co-inventor of “DNA vaccination” in favour of “the inventor of mRNA vaccines”? Does that mean he no longer believes he has invented DNA vaccination? He could have easily claimed to have invented both DNA and mRNA vaccination. Was his sudden rebranding brought on by the success of mRNA Covid vaccines? After all, Malone’s wife claimed that they were “now saving the world from COVID-19”. That is quite ironic in itself, given that Malone is one of the loudest voices producing all sorts of anti-vaccine rhetoric about these very vaccines: from claims that the spike protein encoded by them is cytotoxic and will often “cause permanent damage in children’s critical organs” to claims that these vaccines are unsafe for women or that they cause ADE.

As a bit of an aside but a relevant point on Malone’s character, I found it quite interesting that one recurring theme in recollections by Malone or his wife is how often he felt that he was treated unfairly by nearly everyone he worked with. One quote that I found particularly illustrative on this point comes from Malone’s letter to UCSD that he sent a few weeks before resigning from Vical. In that letter he was “begging for reinstatement back into the PhD Program”, as per Malone’s website, and had this to say about his former colleagues:

That seems like quite a long list of people Malone felt victimized by in his short time at UCSD.

In any case, Malone’s own motivations for making his claims aside, I hope my analysis has provided enough evidence for people to draw their own conclusions about the role he has played in the invention of mRNA vaccines or RNA as a drug.