Debunking myths on genetics and DNA

Thursday, October 11, 2012

Gene therapy goes... topical

The paper I'm discussing today is so cool, I don't know how I missed it when it came out last July. As the name implies, gene therapy is a technique used to "fix" defective genes either by replacing them with fully functional ones or by silencing them with the use of antisense RNA.

Defective genes either fail to produce the proteins they code for, or produce defective proteins, thus causing genetic disorders. A defective gene can be silenced (so that it will no longer produce the defective protein) using antisense RNA. The antisense RNA binds to the mRNA from the defective gene, thus preventing it from being translated into the protein. Small interfering RNAs, or siRNAs, are short double-stranded RNAs that can successfully deliver antisense RNA to the target genes and effectively suppress gene expression.

There are several ways to deliver either DNA or RNA, each with advantages as well as disadvantages. Viral vectors (genetically modified viruses that instead of carrying viral DNA they carry the therapeutic DNA or RNA to be delivered inside the cell) are great ways to deliver genes, but have to overcome the barrier imposed by the host's immune system. Furthermore, some vectors may have toxic side effects. Other delivery means include conjugate agents, i.e. particles such as lipids, polymers, or nanoparticles that bind to the RNA and have high penetrability. Though such therapies have been quite promising, they pose a challenge: they can be toxic when delivered intravenously or orally, while the topical route is inefficient because the skin won't let through anything greater than a few daltons.

That's too bad, though, because the skin would be the less invasive and easiest way to deliver therapy. Just imagine it: applying genes in the morning just like a daily moisturizer! :-)

In [1] Zheng et al. show that by conjugating siRNAs with inorganic gold nanoparticles, they can defeat the epidermic barrier and successfully reduce the expression of the target genes.
"Recently, we introduced spherical nucleic acid nanoparticle conjugates (SNA-NCs, inorganic gold nanoparticles densely coated with highly oriented oligonucleotides) as agents capable of simultaneous transfection and gene regulation. [. . .] SNA-NCs enter almost 100% of cells in more than 50 cell lines and primary cells tested to date, as well as cultured tissues and whole organs."
The researchers measured uptake, safety, and gene suppression efficacy of SNA-NCs in human keratinocytes, a cell line that constitutes 95% of the epidermis. They found no morphological difference between treated skin cells and controls. Furthermore, they studied the ability of SNA-NCs to aid the silencing of EGFR, or epidermal growth factor receptor, a cell-surface receptor that has been shown to be mutated and up-regulated in several types of cancers, including lung, anus, and 30% of skin cancers. After 3 weeks of treatment, EGFR expression was suppressed by 65% in hairless mice. Human skin is known to be thicker and more difficult to penetrate, so the treatment was also tested on 3D raft cultures that simulate in vivo human epidermis. EGFR mRNA expression was lowered by 52% and EGFR protein expression by 72%.

Zheng et al. conclude:
"Our data from blood serum and mouse skin show that siRNAs, when densely conjugated to the nanoparticles in the form of SNA-NCs, are minimally stimulatory and have far fewer off-target effects than the free siRNAs of the same sequence introduced by traditional methods. Furthermore, our studies show rapid clearance from skin, minimal accumulation in viscera, and no evidence of histological changes in internal organs after topical delivery. The low immunogenicity and few off-target effects, coupled with low toxicity and high efficacy, point to a significant advantage for using SNA-NC technology to introduce siRNAs."

Dan Zheng, David A. Giljohann, David L. Chen, Matthew D. Massich, Xiao-Qi Wang, Hristo Iordanov, Chad A. Mirkina, & Amy S. Paller (2012). Topical delivery of siRNA-based spherical nucleic acid nanoparticle conjugates for gene regulation PNAS DOI: 10.1073/pnas.1118425109


  1. That sounds pretty promising. How large of an siRNA structure does this allow?

  2. Excellent question, in the supporting info they say "1.5 nM siRNA duplexes"... Of course, being myself a theoretician, I have no idea what that amounts to... Maybe Kat can help? Compared to the size of a viral vector?

  3. antisocialbutterflieOctober 11, 2012 at 7:14 PM

    You called? 1.5 nM is a concentration not a size. siRNAs are usually about 21-23 nucleotides and are used as a sort of homing beacon for the RISC complex to cleave up the offending mRNA. It would only be good for turning off bad genes (or eliminating the bad copies in heterozygous individuals). As you alluded to in your intro this is an off switch but it won't put good copies in. You'll need the more traditional route for that. But I can think of a bunch of things this would help with including lots of the fibrosis based diseases.

  4. Thanks so much for the info, Kat! Yes, I figured it'd be for heterozygous. So, they have to be pretty short, right? "Cause as I was reading this I couldn't help but think about DNA vaccines, whether this would help with that too, though I assume those would have to be pretty long stretches of DNA, as viral envelopes at least...

  5. antisocialbutterflieOctober 11, 2012 at 8:44 PM

    Definitely not vaccines. The size translates to seven amino acids or two turns of a helix which probably wouldn't be enough to make a good antibody epitope (but I could be wrong). Without a 5' cap or an IRES it wouldn't get translated anyways. It would be a good experiment to see how big the oligonucleotide could be to get trafficked in this manner.

    This would be more useful for heterozygous genetic defects or overproduction issues. The paper mentions EGFR which when overproduced leads to scarring. Off of the top of my head stopping EGFR production can prevent retinal scarring after lasik procedures.

  6. Thanks! I wonder if the technique of using nano-particles as carriers of genetic material slipping past antigen responses can be extended to longer length sequences or, as antisocialbutterfly alluded, as part of a one two set up mechanism.
    Cool stuff.

  7. Yeah, seven amino acids seems pretty short... I mean, it's undoubtedly cool as it is, but I too, like Steve, wonder if in the future they'll be able to extend it to larger sizes... of course, that'd be fantastic and scary at the same time, if you think of the consequences...

  8. antisocialbutterflieOctober 12, 2012 at 10:36 AM

    I got bored watching water drip from a tube so I did a bit of literature scavenger hunting. They are pretty strict about the oligonucleotide part (~25 bp). There are linker regions for particle attachment to allow for higher surface density of RNA on the particle but that seems to affect the transfection efficiency. I can't find any systematic studies on spontaneous Au-NP transfection rates dependent on nt size. It looks like for larger fragments (>200 bp) the nanoparticle is transfected ballistically (i.e. the gene gun also they called it biollistics which just seems silly) There is some stuff on the ideal particle diameter for uptake which is around 50 nm but I didn't see whether that included the nucleotides. I would expect that since this is receptor-mediated endocytosis driven that there would be a finite size that could be transported into the cell.

  9. That makes a lot of sense actually. And I take back what I was saying about DNA vaccines because my supervisor tells me that they are readily absorbed by the skin cells, so in that case penetration is not the issue, the problem is that they then get degraded and cleared...

    I find the word biollistic rather cool, I'll have to look it up... :-)

    And water dripping down a tube sounds like a nice subject to photograph... :-)

    Thanks so much for the info, very cool stuff.


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