A RESPONSE FROM CHAD MIRKIN’S GROUP [follow up #1/n]

Some readers might wonder why I am going on about this, so let me tell you: this is a considerably more important story than Stripy Nanoparticles Revisited. If, as I am arguing, some of this science is shaky, then it is not only the way we evaluate scientists and spend public money which are put into question, but the foundation of ongoing clinical trials.

Back to basics: in the section of Mirkin’s group PhD dissertation (previous post) that respond to our critique of their work on Spherical Nucleic Acid / SmartFlare / StickyFlare, they wrote the following:

Additionally, since the commercialization and sale of the nanoflare platform under the trade name Smartflare (Millipore), dozens of researchers around the world have participated in successful sequence-specific gene detection.[80]

Reference [80] correspond to six (half a dozen) articles, 80a to 80f (see below for details and links). Out of these six, only two are actual research papers, and, for both, the SmartFlares are a very minor addition to the work. Out of these two, only one is completely independent of Mirkin/EMD Millipore (the other one comes from Northwestern).

80a) is not primary research; it is an advertorial produced by EMD Millipore.

80b) is not primary research: it is a 300 words congress abstract (no figure). A follow up paper by the same group is discussed here.

80c) is a review and it is a collaboration between Northwestern (Mirkin’s University) and EMD Millipore. CoI statement from the paper: “D. Weldon is the R&D Manager at EMD Millipore responsible for the production of SmartFlares. Patents related to therapeutically targeting Nodal in tumor cells have been awarded to E.A. Seftor, R.E.B. Seftor, and M.J.C. Hendrix.

80d) is a research paper. It does not show in any way that SmartFlares work. It assumes it does. The SmartFlare is a minor part of the article.

80e) is not primary research: it is an advertorial in a magazine funded by company advertising (including EMD Millipore in that very issue). The author is a journalist working for the magazine, not a practicing scientist.

80f) is a research paper. It does not show in any way that SmartFlares work. It assumes it does. SmartFlares are a very minor part of the article. The authors are from Northwestern, i.e. Mirkin’s University.

 

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19 comments

  1. Calling the clinical trials for spherical nucleic acids into question is disingenuous. They are being tested for antisense gene regulation, which has been demonstrated both in vivo and in vitro. I’d be careful about your indiscriminate bashing of Prof. Mirkin’s work.

    Liked by 1 person

    1. Antisense gene regulation cannot happen without the particles going out of the endosomes.

      Yet, in spite of the massive amount of funding, the dozens of articles, the upcoming clinical trials, and the fact that the technology required to establish endosomal escape has been available for more than half of century and is, literally, routine, the data are lacking.

      Mine is not an indiscriminate bashing but a careful candid critique, based on published papers (including their massive holes), basic knowledge of cell biology, as well as our own results: https://www.scienceopen.com/document?vid=a6754b9a-273e-4ccb-b965-2c98d96ac087

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      1. Considering the vast number of published articles demonstrating efficient gene knockdown with oligo-AuNP conjugates, I’d wager that some small fraction of either particle or free oligo is able to escape the endosome and interact with cytosolic mRNA. I’m not talking about the flare technology here, strictly gene regulation. It seems you are unfamiliar with the literature.

        Liked by 1 person

      2. I’ll fill you in briefly:
        pubs.acs.org/doi/abs/10.1021/ja306854d (72 citations)
        pubs.acs.org/doi/abs/10.1021/ja808719p (490 citations)
        science.sciencemag.org/content/312/5776/1027 (1491 citations)
        stm.sciencemag.org/content/5/209/209ra152 (173 citations)
        And of course many groups have reproduced these results with spherical nucleic acid structures of varying core compositions. I think you’ll just have to admit you’re wrong here.

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    2. Polyplex,
      Can you summarize the mechanism by which the oligonucleotides escape the endosome and interact with intracellular RNA? Do the oligonucleotides enter with or without the AuNPs?

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  2. Polyplex,
    Can you summarize the mechanism by which the oligonucleotides escape the endosome and interact with intracellular RNA? Do the oligonucleotides enter with or without the AuNPs?

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    1. In the case of AuNP-oligo conjugates think the most logical explanation is desorption of oligos from the surface since the Au-S bond is not quite as strong as a covalent bond, only 40-50 kcal/mol. But keep in mind that the same gene regulation effect can be seen with multivalent oligonucleotide nanoparticles of varying cores, such as self-assembled micellar structures, e.g. https://www.ncbi.nlm.nih.gov/pubmed/24827740 – one would not expect the self-assembled structure to remain fully intact upon endocytosis. In fact, taking advantage of lower endosomal pH to dissociate nucleic acid carriers is a well-known strategy for facilitating endosomal escape of oligonucleotides. https://www.ncbi.nlm.nih.gov/pubmed/11356938

      Liked by 1 person

      1. Interesting.

        If this “most logical explanation” is correct then all of the nanoflare/smartflare/stickyflare articles are wrong: they all rely on separation of oligos from particle due to specific base-pairing competition due to presence of target. If desorption occurs due to breaking of the Au-S bond, there cannot be specificity.

        Please note that the chemistry is the same in all of those articles whether they are “gene regulation” articles or “smartflare” articles.

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  3. Good questions Suomynonan: looking forward to polyplex’s answers (or anyone else).

    Polyplex, you write “I’d wager that some small fraction of either particle or free oligo is able to escape the endosome and interact with cytosolic mRNA”.

    This is the most basic, surprising, unexpected and critical feature of these materials. Why is there no direct evidence when it would be so easy to do and even (with some efforts) quantify?

    Leaving this question of absence of direct evidence for a moment, and, accepting your ‘wagering’: let say “some small fraction” is 1-2%, i.e. similar to what was found for optimized lipid nanoparticles (https://dx.doi.org/10.1038/nbt.2612). If this is the case, it is strictly impossible that the smartflare can work because the background signal from the 98-99% would be large in comparison to the signal generated by these escapees. Therefore we would be in a position where dozens of papers on the basic technology underpinning the clinical trials are, at the very least, questionable. Given that we are talking about experiments on humans, I would have thought that this is a pretty serious concern.

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    1. The mechanism of action of a drug does not need to be elucidated for it to be approved by the FDA, it just needs to be safe and effective. And no, the papers on nano-flares are not underpinning the clinical trials, the papers on gene regulation are.

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    2. In response to your comment above: “If desorption occurs due to breaking of the Au-S bond, there cannot be specificity.”
      I’m not commenting here on the efficacy of the smartflare/stickyflare technology. I happen to agree with you that the flares lighting up in the endosome is a serious issue to be addressed and most certainly interferes with accurate detection of mRNA. It’s fair to be skeptical of what they are really “detecting.” However, since the gene regulation effect of spherical nucleic acid-type constructs is well-documented, it’s kind of like comparing apples to oranges. There may only be a couple mRNA copies in the cytosol that need to interact with a complementary oligo in order to observe knockdown. Here’s a good review to read on the topic: http://www.sciencedirect.com/science/article/pii/S1525001616306967#bib70

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  4. Last thought: One way the nano-flare “light-up” in the endosome vs cytosol could be deconvoluted is by employing a fluorophore that remains dark under endosomal conditions but “turns-on” in the cytosol. This could be accomplished either through a pH-dependent mechanism or in response to something like glutathione which has a much higher concentration in the cytosol: http://pubs.acs.org/doi/abs/10.1021/acsami.5b01934

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    1. Indeed but pH sensitive liposomes are a rather different thing than a DNA coated AuNP. I am not sure if I am repeating a citation that is already been provided in this post but low pH seems to stabilize the S-AuNP bond: http://pubs.acs.org.sri.idm.oclc.org/doi/full/10.1021/la200241d

      “At pH 4 and 5, the amount of released DNA was the lowest,”

      I’m not sure how it makes sense to suggest that since pH sensitive micells/liposomes can deliver nucleic acids so can AuNPs. The former have a plausible (though certainly not well characterized) mechanism: https://www.intechopen.com/books/molecular-regulation-of-endocytosis/endosomal-escape-pathways-for-non-viral-nucleic-acid-delivery-systems and there doesn’t seem to be a plausible mechanism for the latter. If I am not mistaken, all of your references (like the paper on LNAs) could be explained by the “proton sponge” theory.

      Agreed that the FDA doesn’t need a mechanism for a molecule to win approval, but perhaps Russell Katz’s statement is more comprehensive (mechanism isn’t meaningless):

      “Theories about mechanism of action of a drug or disease mechanisms play important parts in drug development and approval, but they are entirely subsidiary to the fundamental questions that must be answered in the course of drug approval; namely, is a drug effective, and is it safe in use.”
      https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534930/

      I’m glad there is a consensus on the fact that we should be skeptical of what SmartFlares are detecting. So it seem like at least either Smart/Sticky Flares aren’t doing what a few papers say they are doing or there is effective anti-sense nucleic acid delivery (which doesn’t need to deliver much payload). Do you think I got this right?

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      1. Let me refine those last sentences:

        So it seem like at least either Smart/Sticky Flares aren’t doing what a few papers say they are doing *but* there is effective anti-sense nucleic acid delivery (which doesn’t need to deliver much payload). Do you think I got this right?

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  5. I would say that reason is what is telling us that we should be concerned.

    We have a batch of papers that claim that AuNPs coated with nucleic acids representatively escape the endosome and get inside cells (Smart/Sticky flares) when this doesn’t make any sense. Running a clinical trial based on that technology that is based on such a flawed premise *could work* but it sets dangerous a rather dangerous example (not that this hasn’t’ been done before…ugh…that is an understatement).

    Why don’t we all just publish positive results that are based on impaired reasoning and then just push for a clinical trial with our technology? You never know, it *could* work. If we don’t believe that reasoning gives us a better shot at finding something that works, why not randomly translate bird songs into molecular structures and see if they work as anti cancer drugs in clinical trials?

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    1. Well, you said it yourself: “there is effective anti-sense nucleic acid delivery (which doesn’t need to deliver much payload)” – if that’s the understanding the clinical trial is based on, I don’t see the problem. The FDA does not require a drug’s mechanism of action to be fully elucidated for the drug to be approved.

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