If people with this polymorphism are highly resistant to HIV-1, why can’t scientist purposely “knockout” this gene in those infected with HIV-1? I work in a lab where we use (well, I don’t, I’m more of the guy who autoclaves stuff) “knockout” mice that lack certain receptors.
Well, the short answer is that given current technology, there is no ethically feasible way to do it.
To create a knockout mouse you need to establish a culture of embryonic stem cells. One then engineers a knockout construct (DNA encoding the mutation you wish to introduce with some selective markers), which is fairly easy to do, and then introduces it into the ES cells. One selects for correct insertion based (usually) on the two selectable markers in the construct – one for correct insertion, and one for targeting.
One then overovulates mice to harvest oocytes, which are allowed to develop into blastocysts. The transgenic ES cells are then injected into the inner cell mass. This is implanted into the uterus of a pseudopregnant mouse and allowed to develop. The resulting mouse is a chimera of regular and transgenic cells. Hopefully their germ-line is a chimera as well, and it is bred out, producing whole individuals who are heterozygous for the altered locus. These can then be interbred to get homozygotes.
There are obviously a lot of ethical objections to doing the same in humans. ES cells are a big ethical deal right now, and research on human ES cells cannot be federally funded. Clear that hurdle and deal with harvesting and modifying human eggs and letting them develop in a dish. Keep in mind that every step is imperfect and you are talking about killing a great proportion of the eggs. Remember that there is a debate now about whether we should consider these things human lives. Next, you are putting into a host uterus which may not be a huge deal, but then purposefully creating chimeras. Then, you have to ask the chimeras to breed out and somehow give special preference to only the ones that you have modified.
There are ways, perhaps, to do it in a more reasonable fashion, but not very. Cloning or oocyte nuclear transfer after modification comes to mind, but of course that has a whole thicket of ethical problems as well. Given that CCR5delta32 is found in something like 15% of the population (IIRC), it seems far easier to let nature take its course: if HIV becomes a long-term negative selective factor, the allele frequency for CCR5delta32 will increase. You know, evolution and all that.
CCR5 delta 32 is a 32 base deletion in the normal CCR5 co-receptor(to CD4). HIV infection is greatly aided when the Wildtype receptor is present. Without it infection is still possible, but occurs at a much lower rate. Once infected however fuck all matters. that virus is staying in your system. It’ll just progress slower with the ccr5 delta 32 receptor.
last I heard there is a drug out there that specifically blocks the binding of ccr5 to cd4 and thus has the effect of a knockout ccr5 organism. I’m not sure, but since CCR5 is essential to a healthy effective immune system CCR5 knockout effectively immunosupresses people anyway.
Theres also RNAi (interfering RNA) which can effectively knock out an existing gene in a living organism, but I have no idea how effective it is in humans(I’m guessing it isn’t).
I should think it would be possible to harvest pluripotent bone marrow stem cells from an individual, and used targeted deletion and knock-in methods to modify these cells to express only the delta-32 mutant (perhaps clever insertion of LoxP sites flanking the intron containing the relevant coding sequence for the delta32 mutant, with subsequent replacement of the sequence with a Cre-expressing vector containing the proper donor sequence to be swapped in). Kill off the person’s bone marrow as you would any leukemia patient, and then reintroduce the modified stem cells. Voila: You now have only resistant T-cells that still function pretty well for immunity.
Not that I’d volunteer for this program, or anything…safe sex seems a better way to go.
The easiest way to do your protocol would be standard homologous recombination by selectable marker. I don’t think you need to engineer a gene-switch type mechanism since you are not looking to create a conditional knockout. Actually, since it seems like the delta-32 polymorphism is dominant, you may be able to do this using retroviral knock-in, although there are always cancer and other fears associated with this (Jesse Gelsinger, anyone?)
My knowledge of HIV initial infection fails me, but isn’t there a role for tissue macrophages and quiescent CD4 cells in lymph nodes? If this is the case, you may have a very hard time getting complete ablation of the entire immune system.
The method I propose wouldn’t be conditional, just a somewhat Rube Goldbergian method of replacing one stretch of DNA more-or-less completely with another, leaving the overall structure of the gene essentially intact. All the modifications are in vitro, and once the switch is made, that’s all that matters. You just need to introduce a vector that transiently expresses the cre for the modification step. There are methods now to use modified LoxP sites to allow for the transfer of minimal vector sequence to the genome, once the Cre-recombinase-mediated excission and replacement of LoxP-flanked seqeunce is completed. As the delta32 mutant is dominant, selection, post removal of the initial selection-gene-bearing sequences I used to introduce the LoxP sites in the first place, could be carried out by FACS sorting. Hell, I don’t even know if this could work, it’s just an approach I’ve thought of for other applications, where I generate a line with a modification that allows for the introduction of whatever I want relatively easily (once the LoxP sites have been put in their proper place, which will be no mean feat) and “cleanly” (meaning not much is left of the modification in the non-coding portions of the gene once the swap is completed). Are there other, more-resistant mutants of CCR5 in this particular region that preserve some level of T-cell function, yet confer even greater resistance to HIV infection? That I don’t know, but perhaps a program for screening such mutants would progress relatively quickly once a readily-modifiable stem cell line is established.
As for the imprefection of this approach at at fundamental level: Yep. HIV also infects dendritic cells, which, last I heard, probably provide the primary route of efficient infection, and subsequent transmission, through the mucosa, what with these being the most potent antigen-presenting cell type there is, sitting right in the path of initial contact. But, unless I’m mistaken, dendritic cells can be bone-marrow derived, so there could be some rapid replacement if you could deplete the mucosa of these so-called Langerhans cells with some kind of treatment. My understanding is these, and dermal dentritic cells, are a likely culprit in the development of GVHD in bone-marrow transplant patients. Simple UV light exposure can get rid of Langerhans cells in the skin, for instance. At any rate, the method only slows down the process, and perhaps helps prevent initial infection. Those born with the mutation are not completely safe themselves.