In-vivo gene silencing

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Andy Resnick
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Using the Cre/LoxP method can also conditionally knock out genes in vivo. Also, by coupling the Cre/loxP to an tissue-specific promotor, it's possible to conditionally knock out a gene in a specific organ:

http://jasn.asnjournals.org/content/15/8/2237.full
 
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Thanks.

Although more effective, it seems the Cre/LoxP method only works on "made-for-experiment" subjects. (Those with LoxP spliced into their genome).

siRNA, from my understanding, can interfere with gene expression post-transcriptionally and therefore can be used on an unprimed (if you will) genome. I feel this route has more promise for clinical applications. A few questions.

Is there any leakage from unbinded mRNA fragments that then do get translated into proteins?
Could one tailor a virus to splice the siRNA DNA into a target cell so the siRNA could be manufactured inter-cellularly? (I.E. Lytic cycle without the virus parts)
 
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Thanks.
Is there any leakage from unbinded mRNA fragments that then do get translated into proteins?
Could one tailor a virus to splice the siRNA DNA into a target cell so the siRNA could be manufactured inter-cellularly? (I.E. Lytic cycle without the virus parts)
This study was designed as a "proof of concept" and was published in 2003. I only cited it to answer your initial question. I'm not active in research anymore so I can't advise you of the current status of this approach. You might try to contact the authors.
 
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Andy Resnick
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siRNA, from my understanding, can interfere with gene expression post-transcriptionally and therefore can be used on an unprimed (if you will) genome. I feel this route has more promise for clinical applications. A few questions.
Gene therapy has been pursued for more than a decade, with not much success:

http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml

I'm (somewhat) familiar with the case of Jesse Gelsinger. He had a variant of cystic fibrosis, which was identified as a candidate disease for gene therapy since the lung is an easy organ to target (inhale the carrier).

People are still trying various approaches with some success, but progress is very slow.
 
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Gene therapy has been pursued for more than a decade, with not much success:

http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml

I'm (somewhat) familiar with the case of Jesse Gelsinger. He had a variant of cystic fibrosis, which was identified as a candidate disease for gene therapy since the lung is an easy organ to target (inhale the carrier).

People are still trying various approaches with some success, but progress is very slow.
****. The immune response didn't even cross my mind
 
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Ygggdrasil
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Is there any leakage from unbinded mRNA fragments that then do get translated into proteins?
Yes, you can still have low level protein expression even thought the mRNA from is being silenced by RNA interference. In fact, biologists make a distinction between "knocking out" a gene (removing or disrupting a gene such that the cell is incapable of producing the mRNA for the target protein) and "knocking down" a gene (using RNA interference to silence a gene). Incomplete knock-down of a gene is a potential problems faced by those using RNA interference. However, if this is the case, one can still design better siRNAs or combine multiple siRNAs to achieve a better knock down of the target protein.

Could one tailor a virus to splice the siRNA DNA into a target cell so the siRNA could be manufactured inter-cellularly? (I.E. Lytic cycle without the virus parts)
Yes, people engineer lentiviruses (the same family of viruses as HIV) to integrate sequences that express the appropriate siRNA into cells. This technique works well for cultured cells, but as Andy said, there are problems with using this method in living humans.

Another approach being developed to possibly knock-out genes (as opposed to knock-down) is by engineering enzymes that will bind to specific sequences in the cell's DNA and cut the DNA to either introduce mutations that might inactivate the gene or to allow foreign DNA to insert into the cut site. The two general classes of enzymes being developed for these purposes are called "zinc-finger nucleases" and "homing endonucleases."

While these methods have had some success in a laboratory setting using cultured cells, it would likely be difficult to use these in living human patients.
 

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