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Monday, November 19, 2012

Proteins as gene carriers


By now you probably know everything about pluripotent stem cells, right? They are the hot topic in genetics right now, to the point that the fear of being scooped has pushed some people to lie about their results. Pluripotent stem cells are cells that have the ability to divide into a specialized cell and another undifferentiated cell. This of course is greatly useful in repairing damaged organs and/or regenerating tissue, and has great potential in medicine.

Lately there has been a lot of buzz on the notion that pluripotency could be re-induced in already differentiated cells. Studies have shown that four reprogramming factors can indeed reprogram fibroblast cells into pluripotent stem cells when over-expressed.

But how to over-express these factors?

The typical route is to transfect the genes into the cells by means of a viral vector. Basically, the genes are delivered into the cell using a retrovirus. Though effective, this poses the question of side effects: whenever you introduce foreign DNA into a cell you have the potential to silence secondary genes or disrupt the usual gene regulation. Unanticipated epigenetic changes in the cell can occur. A recent study [1] shows a safer alternative: cell-permeant proteins, or CPPs. These are small proteins that can cross the cell membrane and carry peptides inside the cell in a process called "protein transduction," thus offering a valid alternative to viral vectors.

By comparing the two methods (CPPs and viral delivery) on human fibroblast cells, Lee et al. noticed that gene expression was achieved much faster when using the viral vector. Puzzled by this difference, they wondered what was so special about the viral route that made the gene delivery so much more efficient. There had to be something in the viral vector that aided the delivery of the genes. Lee et al. hypothesized that this could be linked to the fact that the viral vector somehow activated an inflammatory pathway in the cells which in turn aided the delivery of the genes. So the next question was: can we enrich the CPPs so they too activate the inflammatory pathway?

Indeed they could! They used TLR3 agonists, molecules that activate the TLR3, or Toll-like receptor 3, a receptor that recognizes double-stranded RNA generated by retroviruses and thus activates inflammatory pathways. Once combined with the TLR3 antagonists, over-expression of the reprogramming factors was achieved faster through CPPs than it was with the viral vectors, validating the hypothesis that the gene delivery has to be achieved via the activation of the immune pathway. In fact, the contrary was also true: when TLR3 was knocked down (biology jargon to say that the gene was silenced), the viral vector was also inefficient in delivering the genes.
"TLR3 activation enables epigenetic alterations, including changes in methylation status of the Oct4 and Sox2 promoters as well as changes in the expression of epigenetic effectors, that promote an open chromatin configuration. The knowledge that the activation of innate immune response affects nuclear reprogramming permitted us to enhance the efficiency and yield of human induce pluripotent stem cells by using reprogramming factors in the form of CPPs."
Lee et al. conclude:
"Our observations highlight a previously unrecognized role for innate immunity activation in nuclear reprogramming. The viral vectors constructs used to induce pluripotency are more than mere vehicles for the reprogramming factors. Innate immune activation causes striking changes in epigenetic modifiers that favor an open chromatin configuration. These changes enable a fluidity of cell phenotype that contributes to successful nuclear reprogramming."

[1] Lee, J., Sayed, N., Hunter, A., Au, K., Wong, W., Mocarski, E., Pera, R., Yakubov, E., & Cooke, J. (2012). Activation of Innate Immunity Is Required for Efficient Nuclear Reprogramming Cell, 151 (3), 547-558 DOI: 10.1016/j.cell.2012.09.034

ResearchBlogging.org

6 comments:

  1. That's very cool. It seems like methods for introducing gene alterations (and the understanding of the mechanisms involved) are progressing by leaps at the moment.

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    1. I had never heard of this method before, but when I looked up the literature I found a paper from 2001, so It appears the method has been known for at least a decade but, apparently, it wasn't giving better results than viral vectors. I think this new discovery of making the proteins activate the inflammatory pathway just like viruses do will make this new methodology much more appealing. It's very exciting! :-)

      Thanks for your comment!

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  2. antisocialbutterflieNovember 19, 2012 at 7:16 PM

    This brings up an interesting issue that I hadn't thought about before now. Messing with immune responses can have some pretty significant downstream signalling effects. Chronic inflammation has been pretty extensively shown to induce tumorigenic transformation. I wonder how this has potentially skewed results in the literature over the years. I guess it would depend on what you're trying to measure.

    As an aside I know one of the labs that does in vivo studies on the retro-differentiation of hemopoietic stem cells into other cell lineages. They single-handedly have the most horrifying IRB protocol I have ever seen.

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    1. wow, that's intriguing... not to mention worrisome! what does IRB stand for?

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    2. antisocialbutterflieNovember 19, 2012 at 8:07 PM

      It's the Institutional Review Board. Whenever you do animal studies you have to get your protocol cleared in conjunction with federal, state, and institutional regulations.

      Their protocol involved taking mice, destroying their immune system, giving them GFP spiked bone marrow transplants, waiting for them to heal, laser ablating their retinas, and then seeing if the eyes heal with GFP present. From what I saw it works pretty well but it seems like a horrible thing to do to the poor mice. I suppose this is why I don't do animal work anymore.

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    3. Argh! :-(
      And that's why I'm a theoretician...

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