I have another question regarding cystic fibrosis.

I understand that gene therapy is currently being talked about in the cure for cystic fibrosis. I know that Eric Alton at imperial college London is working on delivering genes using liposomes and viruses like lentiviruses but I am confused over the advantages and disadvantages of those two methods.

In addition, is there an alternative 3rd method for injecting genes that don't involved viruses or liposomes. (An alternative type of vector) ?


  • $\begingroup$ There are many types of non-viral vectors. They generally fall into cationic lipids and cationic polymers. The positive charge binds to the negative charges on DNA and forms stable particles. Getting these to work in the body is harder. I've been working at it for 5.5 years and far better scientists than me have spent far longer. Some researchers working on non-viral gene delivery include Dexi Liu, Leaf Huang, and Mark Kay. $\endgroup$ – user137 Oct 29 '14 at 17:10
  • $\begingroup$ Just thought of a couple guys who are doing research on cystic fibrosis specifically, John Engelhardt has a ferret model and Joseph Zabner is using pigs. Neither animal perfectly replicates the human form of the disease, but they're both better than current mouse models. $\endgroup$ – user137 Oct 29 '14 at 20:21
  • $\begingroup$ Why are the ferret and pig models better than mouse models? $\endgroup$ – user46725 Oct 30 '14 at 17:36
  • $\begingroup$ I don't exactly know, but Engelhardt and Zabner have papers describing the animal models. If I remember right the mice don't exhibit the same inflammation and immunity symptoms. $\endgroup$ – user137 Oct 30 '14 at 17:41

There are many other methods that are used for transfecting cells in culture. These include:

  • Cationic lipid mediated (as mentioned by user137)
  • Calcium Phosphate mediated transfection
  • Biolistic methods
  • Electroporation

Newer techniques include optical transfection (using lasers to bore a hole through membrane) and impalefection (using nanotubes). Wikipedia page on transfection nicely summarizes these techniques.

Induced pluripotent stem cells can be cultured from a patient, transformed using the plasmid and transplanted back to the tissue where it can replace the old cells with "corrected" cells.

There is another somewhat recent technique called in-vivo electroporation using which Nucleic Acid can be delivered directly in to the desired tissue.

However, to fix a mutation or introduce a transgene, just transfection is not enough; you have to make sure that the desired gene is inserted in the chromosome at the right location. Faulty gene can be replaced by the transfected functional gene via homologous recombination(HR), but rates of recombination are generally very low. Targeted gene insertion methods using genome editing tools such as Zinc-Finger Nuclease (ZFN), TALEN, and Crispr-Cas can increase this rate by introducing a break in the DNA at that region; this causes DNA repair systems to "repair" the break using HR.
There is another type of DNA repair system called NHEJ (Non-Homologous End Joining), and this is not what we are interested in. NHEJ can, in fact cause mutation through indels. Presence of the transfected DNA would shift the preference towards HR, however it has also been shown that different stages of cell cycle have different preference towards HR and NHEJ (cant find the reference as of now but I am sure about it because I was thinking at some point of time, to work on this).

Having said that, I would add that it is essential to screen the cells to see if the gene is incorporated or not. If some optimal protocol is devised then one can proceed to in-vivo electroporation.

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  • $\begingroup$ Consider adding viral transduction to the delivery methods. If that could be effective to silence the mutation. Then introduce the wt gene by viral delivery again. It's done in vitro, even with CFTR However the pathophysiology behind CF would not allow something this simple to work. Otherwise +1 for good answer $\endgroup$ – rhill45 Jan 15 '15 at 0:17
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    $\begingroup$ @rhill45 OP was interested in non-viral methods, that is why I did not include it $\endgroup$ – WYSIWYG Jan 15 '15 at 4:25
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    $\begingroup$ @ChrisStronks In vivo electroporation has been used for experimental purposes (I have provided a JOVE link for it) but I don't think they have been used for therapeutic purposes. As such gene therapy is still in its inception. Stereotactic injections are also used to deliver a gene to a desired tissue but afaik, they mostly use viral vectors $\endgroup$ – WYSIWYG Jan 15 '15 at 5:30
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    $\begingroup$ All of these methods are good for delivery in vitro, but in vivo is a much harder problem, especially for nonviral methods. I've reviewed a paper about endoscopic electroporation in pig intestine, but never heard of it in the lung, which would be where you want the DNA to go for CF. Hydrodynamic Injection is a fun one that works well in mouse liver, but in larger animals it turns into a major surgical hassle, and probably wouldn't translate well to the fragile tissues in the lungs, not to mention pumping them full of fluid. $\endgroup$ – user137 Jan 15 '15 at 6:38
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    $\begingroup$ I think this is that electropration paper, looks like it got published. $\endgroup$ – user137 Jan 15 '15 at 6:42

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