mRNA-LNP platforms: the inflammatory features of the LNPs and the role of EVs as carriers of synthetic genetic material were already known in 2019

More evidence from a Nature publication

Thank you for reading my Substack! I promised a follow-up on the environmental impact of mRNA vaccines. Dealing with computer issues and others, and trying to turn this also into an article, things have been busy. However, I just came across a publication in Nature Communications from 2019 which blew my mind.

It is sobering to see what was, and what was not, known about mRNA-LNP platforms before the COVID-19 mRNA vaccine rollout!

Open Questions and Issues in 2019

Titled 'Linkage between endosomal escape of LNP-mRNA and loading into EVs for transport to other cells," Maugeri and coauthors aim to address a few major challenges for "RNA therapy" - which surprisingly seemed to be fully resolved for the CIVID-19 mRNA vaccines. (They define RNA-based therapeutics as functioning by either silencing pathological genes through the delivery of siRNA or expressing therapeutic proteins through the delivery of exogenous mRNA to cells. In essence, this step is the same for the mRNA "vaccines" that intend to express antigenic proteins instead). So, back in 2019, some of the known challenges were recognized as follows:

• "RNA is highly unstable in extracellular fluids because of the presence of nucleases and the fact that mRNA/siRNA needs to be taken up by the right cells and must be able to escape the endosomes to be translocated into the cytosol, for protein expression or gene silencing to occur."

• "[T]he transport of mRNA/siRNA to the cytoplasm of recipient cells requires safe and efficient delivery vehicles."

• As for lipid nanoparticles (LNPs) for mRNA delivery, "their limited capacity to undergo endosomal escape limits the use of LNPs as RNA delivery vehicles, as only a small fraction of RNA efficiently escapes endosomes to reach the cytoplasm of cells."

• Specifically, only a tiny amount of the siRNA administered via LNPs escapes the endosomes - and is therefore useful for the planned activity: "Despite the fact that, a major proportion of LNPs (95%) is endocytosed (taken up) by cells within half hour6, it is estimated that <2% of the siRNA administered via LNPs escapes the endosomes to reach the cytosol."

• What's happening next is also not clear: "Thereafter, the fate of endocytosed LNPs ... is not completely understood."

• Neither is clear what happens to the LNP-delivered mRNA.

What Happened Next?

I not only find it shocking that these foundational issues were still unresolved in 2019 but also what the purported consensus is a few years later. We are either told it's all totally figured out and settled, or else, previous knowledge related to these difficulties has disappeared from public view.

For example, in my book, I described how researchers emphasized the many open questions even after the mRNA injections were rolled out/mandated. Specifically, Sonia Ndeupe et al. write that by that point, including the clinical trials, the potential inflammatory nature of these LNPs had not been assessed.

However, Maugeri et al. clearly state the opposite in their 2019 paper, emphasizing that:

• Ionizable lipids "despite being important compounds for LNP manufacturing, can be toxic."

• LNPs are known to cause the expression of pro-inflammatory cytokines.

• "[I]onizable lipids ... are partially immunogenic to recipient cells and elicit immune responses in the host."

The important findings by Maugeri and colleagues

Trying to address these open questions, Maugeri et al. develop some important hypotheses, which they also validate. Their main goal was to try to use EVs rather than LNPs for the delivery of synthetic mRNA.

Their utilization of EVs is even more memorable when we consider how Bansal and colleagues, after they had essentially confirmed some of what Maugeri et al. had done, backpedaled and tried to divert their previous findings relative to the importance of EVs in the COVID-19 mRNA vaccines (and hence, apparently, to stop discussions on the shedding of these products via EVs).

By contrast, Maugeri and colleagues deliberately utilize EVs.

To recall, Extracellular vesicles (EVs) are a heterogeneous population of nano- and micro-sized vesicles, including microvesicles, exosomes,  and others.

Based on their previous work, Maugeri and collaborators knew that EVs can transport and transfer RNA between cells. Thus, they specifically aimed to exploit these vesicles as delivery vehicles for exogenous RNAs.

Specifically, their experiment involved LNP-delivered modified mRNA encoding human erythropoietin (hEPO protein).

Their first and key conjecture concerns the LNP-mRNA that does not escape the endosomes and which therefore cannot produce the intended (therapeutic) protein. They hypothesized that the hEPO-mRNA and ionizable lipids when they do not escape the endosome,

"can be incorporated into intraluminal vesicles of the late endosomes, and could subsequently be secreted into the extracellular environment as EVs."

This hypothesized mechanism is critical. The EVs are given a special name. Specifically, they refer to the EVs secreted after the endocytosis of LNP-mRNA as endo-EVs (see their figure, copied below).

(From Fig 1.e in Maugeri et al.)

Here is what they found about the LNP-hEPO mRNA and ionizable lipid components of LNPs, which had not been dissociated/escaped into the cytoplasm and which had not been degraded in early endosomes. It is astonishing indeed.

• Their study demonstrated that part of these mRNA and ionizable lipids were packaged into endo-EVs and secreted outside the cell.

• The endo-EVs can act as RNA delivery vehicles because:

  • The hEPO mRNA was confirmed inside these EVs.

  • They protect exogenous mRNA during in vivo transport.

  • They can transport and take synthetic RNAs into distant cells. Specifically, they were shown to deliver the intact hEPO-mRNA to the cytoplasm of recipient cells.

  • Via endo-EVs, exogenous mRNA can be delivered to different cell types, including epithelial and immune cells (even though immune cells are generally more difficult to transfect with RNA using other delivery vehicles).

  • In both in vitro and mice, the delivered mRNA was shown to be functional and produced human EPO protein.

• When mice were injected intravenously, with EVs containing exogenous hEPO mRNA, the hEPO protein was detected in plasma and organs, revealing systemic delivery of EVs.

• Comparing the EV-based delivery with LNPs, they found that in some organs there was no substantial difference in the amount of hEPO protein produced, whereas, in some others, the LNP-based delivery was somewhat better.

Ironically, in 2019, it was known that both vehicles that were tested (the LNPs and the EVs), were systemically delivered. While this was consequent to intravenous injection, they demonstrated several issues that are highly relevant for the COVID-19 mRNA vaccines (or, rather, should have been relevant before their roll-out).

• Both vehicles caused systemic delivery of the exogenous mRNA and caused the expression of pro-inflammatory cytokines.

• Nonetheless, they did suggest that EVs might be better tolerated by the recipient mice since EVs contain fewer ionizable lipid molecules per mRNA nucleotides. Indeed, endo-EVs induced fewer inflammatory cytokine responses than LNPs upon transferring an equal dose of hEPO mRNA to mice.

Ironically, the systemic distribution was clearly demonstrated, as was the highly inflammatory nature of these carriers - a fact that many are still trying to conceal these days for the mRNA COVID-19 vaccines. And no, the Nobel-prized mRNA modifications cannot resolve issues inherent to the delivery system, whether they are known or unknown.

The inherently important role of EVs

Maugeri et al. thus suggested an alternative carrier to the LNPs. They further argued that "in contrast to LNPs, EVs are natural biological products, and might be better tolerated by the host" and that EVs for mRNA delivery "might elicit a milder immune response in the host."

A few years later, it seems hard to believe that EVs were shown to play such an important role, even as a deliberate carrier vesicle! (What a contrast! Nowadays the mere mention of EVs in the context of the jabs is being fact-checked and debunked).

The final observation by Maugeri et al. is even more amazing. They suggest they have found a new way to "load exogenous mRNA into EVs," as follows:

"Our method exploits the direct link for transport of molecules between endocytosis of LNPs containing mRNA and exocytosis, where endo-EVs acquire different LNP molecules, such as mRNA and ionizable lipids, directly from the endosomal pathway."

What they are really saying is that naturally, exogenous material will be further disseminated in the body, carrying the biological activity onward!

Their final conclusion (expectation) says it all:

"LNPs with the same ionizable lipid used in the present study are currently being tested in human clinical trials. As shown in the present study, a similar scenario may occur in humans: when mRNA is delivered via LNPs, the LNPs alone may not deliver mRNA to all cells that express the protein; part of the RNA delivery may be achieved via endo-EVs secreted by cells that internalize the LNPs (Fig. 7c). Additional studies are needed to determine how much of the LNP-delivery is actually achieved by the LNPs’ own contribution and not from the endo-EVs originating from LNP-treated individuals."

(From Fig. 7c in Maugeri et al.)

Conclusion

Even in 2019, a few things were astoundingly clear:

1) It was obvious that the search for carriers was not at all resolved. They knew that LNP carriers were highly inflammatory.

2) EVs were suggested as an alternative biological vehicle. They were found to elicit inflammatory responses too, but it was hoped that, being a natural substance, this would be better tolerated than the synthetic LNPs.

3) Maugeri et al. demonstrated that EVs can indeed act as carriers of synthetic mRNA, that the mRNA was thereby protected, able to take their cargo to distant organs and cells, including those that are known to be difficult to transfect. The mRNA was systemically disseminated and taken up by distant cells. Furthermore, it was shown to be functional and produced the desired protein in mice.

4) They provide some explanations for their hypothesis that the original mRNA-LNP material that does not escape endosomes to reach the cytoplasm of cells will get naturally recycled. According to their model, the packaged EVs that originate from endosomes are secreted again from cells. Notably, these EVs (endosomal and plasma membrane origin) were shown to contain the synthetic mRNA; naturally then, the EVs would be able to take these into different cells where they would be expressed.

Overall, one can conclude that regardless of how the original exogenic RNA was delivered (in their case intravenously via LNPs), EVs play an important role in propagating biologically active material. In an ongoing process, it is expected that endo-EVs deliver the synthetic genetic material, and thereby, their biological activity, into additional cells.

In fact, they believe that a large part of the delivery of synthetic RNAs is achieved by such EVs.

(I think this delivery/recycling of the LNP-mRNA as suggested by Maugeri et al. answers a question Maria Gutschi recently had and also raises tons of new ones, e.g. related to the larger impact on the host and the environment more generally).

Nonetheless, the hope that the biological nature of EVs would eliminate inflammatory immune responses and host toxicity (right-hand side of the figure above), was proven to be false. What matters is not only the vehicle alone. If the protein product produced is toxic, as is the spike from the COVID-19 vaccines, then the end result is still negative.