gravenewworld said:
This was going to be my next question. I guess it wouldn't really matter if it were a multi gene or single gene related disease, I mean what happens to an exogenously delivered gene once it is inside the nucleus? I mean does the cell have some sort of machinery that it can take a little snippet of DNA and incorporate it at the exact precise location on the chromosome? I'd imagine that if you deliver a piece of DNA in the wrong spot on a chromosome you'd cause all sorts of other problems like frame shift mutations. All the work being done now seems to be entire focused on just trying to deliver DNA with cell specificity. What happens at the subcellular level? Is there even any research being done on manipulating the cell machinery so that you can have guided incorporation of a gene into the right location of the genome?
Yes, there is certainly research being done on making gene insertion less dangerous.
As Andy points out, we provide all the necessary "stuff" with the gene when it is delivered. For researchers and some gene therapies, this often means using a viral promoter sequence--As they tend to get good "active" results for the genes you want to be expressed.
For long term viability of gene therapy though ("real" gene therapy) then we'd need to gene into a cell, where it is subject to all those "normal" gene regulatory regions. This gets complex and is slow research because we have found that gene regulation is much, much more complicated than anyone thought. Its complicated in prokaryotes, but gets to a whole new level of complexity when you start looking at things like mammals.
Plasmids seem a good alternative to not "wrecking" the genome at the moment, but the problem with them, is your cells have little "policing" agents which like to, over time, kick the plasmids out of the party. This means, that gene therapy with plasmids would need to be an ongoing treatment. Also, as I mentioned above, the way we deliver plasmids for gene therapy (liposomes) can cause an immune reaction against them. Which renders the therapy rather mute.
Andy Resnick said:
That's an interesting question, and I can only give an approximate answer:
The genes that I insert into my cells are not isolated genes (for say, GFP-tagged proteins), but also contain "promoter sequences", "antibiotic resistance genes", and a few other things. What I mean is that a functional gene is not just an isolated snippet of DNA- there is also a promoter region (and possibly an inhibitor region) that controls how much that gene is read and expressed.
http://en.wikipedia.org/wiki/TATA_box
So, the specific location in a chromosome (or selection of a specific chromosome) does not appear to be that important- but altering the promoter sequence can change a gene from being silenced to being over-expressed.
Just to clarify for the OP Andy, gene insertion
in vitro (in the lab) and
in vivo (like we'd need for clinical usage) are really different things.
I've transfected lots of immortal cell lines and bacteria. When you use something like a reverse transcriptase to do the transfection, you
do inevitably wreck some chromosomes and cause some cells to die. The difference, in vitro, is you are dealing with log scales of bacterial or transformed cells and loosing a few is small fish.
Clinically then; it becomes a problem because while we can certainly think of people as "giant petri dishes of cells", the function of the organism
does depend on the function of it's cells. Wrecking some, may not be well tolerated by the organism or lead to undesirable clinical outcomes (why does everything always seem easier in lab
)
For example, the some of the first clinical applications of gene therapy where in individuals lacking Adenosine deaminase, also known as severe combined immunodeficiency (SCID) aka: "bubble boy".
Functional Adenosine deaminase was RT'd into cell lines in the patients and it seemed at first, to work out great. The problem, which pops up a couple of years down the road--is they start getting cancers.
Now, to me, this is one of those "risk/reward" scenarios where the reward, certainly outweighs the risk. We can treat those cancers and prolong life--However, you cannot live without an immune system. Case in point, people with SCID rarely life past their second birthday.
However, looking at someone with CF--Who is likely to live into their 40's with current treatments, for aggressive gene therapy the risk of doing it at an early age certainly outweighs the reward (hence, we've moved toward less aggressive gene therapies such as plasmid introduction through liposomes).
For gene therapy to really be successful in the long term, we are going to need a way which provides us with some sort of control regarding the introduction of genes into cell lines--Otherwise well always have that pink elephant (
what did we wreck?) in the room.
