anyone here work in genetics or Molecular Bio?

[milo hobgoblin]

Its been years since Ive done any research but I have a question regarding CCR5 mutations and gene therapy. (for those who dont know CCR5 homogenous mutations confer complete resistance to HIV ~10-14% of Northern European stock have this.. only 1% of Americans appear to be homogenous) and yes being heterogenous does confer "some" resistance and significantly extend the lifespan.... (I have a feeling.. people who completely "clear" the viral load are heterougenous, although no one is sure they are actually clear)

When I did research (mosty on red seaweed of all things) we could be somewhat aware using a viral vector (e.coli if I remember correctly) as to where that gene landed. I can only imagine that in the last ten years specificity has gotten better through enzyme manipulation.

And as far as wiping out the old CCR5 mutation.. isnt it possible using another viral vector to disable or destroy the existing CCR5 mutation??

again its been a long time and I know work in computer science so if Im just way off base please explain..

thanks.

Im gonna post this on the HIV board too.. but I seem to remember there being a couple of other Bio-nerds here.

 

[Becoming]

And as far as wiping out the old CCR5 mutation.. isnt it possible using another viral vector to disable or destroy the existing CCR5 mutation??

again its been a long time and I know work in computer science so if Im just way off base please explain..

are you asking if it is possible to eliminate the benefits of carrying a CCR5 mutation by introducing a normal CCR5 gene (and then expressing it) into affected individuals? (thereby making them suseptible to HIV, where they had not been before?)

if so, yes, it would be possible, at least in theory. I don't know if anyone has pursued this to any degree. even if it was only to see if cells of individuals carrying the mutation could be infected if they were induced to express the CCR5 receptor.

 

[milo hobgoblin]

LOL becoming.. I was hoping for the opposite.. isolating the mutated CCR5 gene and by using a viral vector not only introducing it into non CCR5 mutos conferring resistance to the host and also shutting off their old CCR5 (which would be required to confer comlete immunity)

 

[Becoming]

LOL becoming.. I was hoping for the opposite.. isolating the mutated CCR5 gene and by using a viral vector not only introducing it into non CCR5 mutos conferring resistance to the host and also shutting off their old CCR5 (which would be required to confer comlete immunity)

oh. and here I thought you wanted to make sure everyone was unable to escape HIV... (you evil mastermind you)

well, I don't know a ton about the CCR5 problem specifically, but here is how I understand it...

As you say, the CCR5 gene product is basically a receptor on immune cells that the HIV virus uses to attach onto and introduce it's genetic material into a cell. in theory, by adding the mutated CCR5 gene to a normal person, you would be causing them to express a mutated CCR5 receptor that the HIV virus cannot use. the problem is that without shutting off expression of the normal CCR5 gene (and production of the normal receptor) the cells would still be infectable by HIV... (as you alluded to in your last post)

There might be a couple methods where by one could shut off normal CCR5 expression... but this would be more difficult than introducing a mutated gene for expression...

one method might be utilizing anti-sense rna (basically you introduce a gene that codes for the anti-sense version of the rna - which will bind the normal CCR5 rna, resulting in double stranded RNA, which woudl not be translated into receptor protein) of course the presence of double stranded RNA also signals to the immune system that the cell is infected by virus and signals that it should be eliminated by fellow immune cells.

I don't know how the whole anti-sense rna thing is working out, but last I heard it wasn't donig to well outside of the lab. but then again I haven't read up on it in a while.

I can't think of any other methods off the top of my head that might work to help eliminate the normal CCR5 expression or that could otherwise be used as a "vaccine"

 

[milo hobgoblin]

Would it be possible to tailor a splicing enzyme specific to unique base pair sequences in the standard CCR5 gene and simply insert dead genetic material .. thereby shutting it off.

again its been so long I dont know how advanced they have giotten with enzyme manipulation.

 

[Becoming]

Would it be possible to tailor a splicing enzyme specific to unique base pair sequences in the standard CCR5 gene and simply insert dead genetic material .. thereby shutting it off.

again its been so long I dont know how advanced they have giotten with enzyme manipulation.

the simple answer I think is no. the geneome in higher organisms is way too complicated to try something like this. any combination of base pairs that could be used woudl no doubt occur hundreds if not thousands of times and the effects woudl be unpredicable and probably not survivable (e coli and such simple organisms you can clip out genes etc because there are only 100s-1000s of base pairs)

to try another approach (viral mediated gene therapy) basically when you transduce a human cell with a virus, what you do is you hijack a virus, taking out all its genetic material that is undesirable and then insert your gene after its promotor (or add in your own promoter). you then infect the cells with the virus, which randomly inserts its genetic material to be transcribed and translated... even when you insert a gene into a cell of a higher organism (with most viruses) you don't really have any control over "where" it inserts for the most part (and it will only insert into a % of cells overall anyways, not 100%). in addition, it is possible to get "multiple" infections because of this. there will be occasions where it will randomly insert into other functional genes etc, making a nonfunctional product or other anomoly. there are times when it will kill the cell, and there are times when it will work as you wish.

I have only used viruses, so I know how those work (they are most efficent)... I believe that most plasmids also insert at multiple locations...

I believe the answer lies in whether or not you could get the insertion to occur at only one point in the genome, and for that one point to be the specific point you desire. due to the complexity of the human genome, I believe the answer is no (at least not that I know of)

from what I know as things now stand, you would have to work on something more downstream, either inhibiting the rna, protein or protein processing to interrupt the expression of the gene.

note-I just did a google search and it appears that Epstein-Barr virus might insert at only one point (if that is correct) so I suppose there is the possibility that someone might figure out how to do with a virus what you are asking about if they were so inclined. I have never myself heard of it with human cells however. (not that it does not exist)

>>>To get back to your idea of how to approch it...
I suppose you could use something like homologous recombination to replace the normal gene with the mutated one, or attempt to specifically mutate the normal gene but how you would go about this on this specific gene is way over my head.... that is something for the hardcore molecular biologists (if it is even possible/suitable for anything outside of a petri dish is another issue all together)