X-linked adrenoleukodystrophy (X-linked ALD) is a demyelination disease in little boys. The kids are usually dead before they hit adolescence.
The demyelination mechanism is not the same as the demyelination disease you all have heard of, MS. With X-linked ALD, the kids dont have a functional ABCD1 gene. This means they cant break down very-long-chain fatty acids (VLCFAs) and they end up accumulating in the brain and screwing with the cells in charge of keeping up myelination, oligodendrocytes and microglia. Screw those guys up, screw up your myelin, screw up signal transduction in your brain and CNS.
X-linked ALD can be treated with allogeneic hematopoietic cell transplantation. You all can tell from the Wikipedia page, this isnt something you want to do unless you literally have no other options–
Hematopoietic stem cell transplantation remains a risky procedure with many possible complications; it has traditionally been reserved for patients with life-threatening diseases. While occasionally used experimentally in nonmalignant and nonhematologic indications such as severe disabling auto-immune disease and cardiovascular disease, the risk of fatal complications appears too high to gain wider acceptance.
Know what might be a better option, especially for kids who cant find transplant matches?
Retroviral gene therapy.
Okay, so retroviral gene therapy has been tried before, and though it cured the kids of X-SCID, some of them ended up getting leukemia instead (funny how MLV causes leukemia in these kids and you know, mice, but ‘causes‘ weird vague symptoms in CFS patients. isnt that weird? *blink*).
How is this trial any different?
First, they isolated hematopoietic stem cells cells from a couple of X-ALD kids who couldnt find transplant matches (in other words, the kids were as good as dead if they didnt try this).
They then infected the cells with a gene therapy vector composed of three different viruses:
1. VSV-G– If VSV were a retrovirus, we would call VSV-G its ‘env‘. Know how any virus has to have the right key (env) to your cells locks (receptors) to initiate infection? VSV-G is like a super master skeleton key for any mammal. It can get into any and every cell. This is a way to ensure that as many target cells are infected with your gene therapy vector as possible.
2. HIV-1– This HIV-1 genome did not have env (env was going to be provided in trans with VSV-G, this is only good for one round of infection), or most of HIV-1s accessory genes (vif, vpr, vpu, nef, tat). Basically, they just wanted HIV-1 for its muscles– gag and pol. Gag provides all the structural genes this viral vector needs. Pol provides the reverse transcriptase and importantly, the integrase. Lentiviral integrases will give this vector some tricks that a simple retrovirus, MLV, wont have. One of those tricks is better integration into hematopoietic stem cells.
One source of cancer retroviral gene therapists need to worry about is LTRs. Remember? One LTR drives transcription of the virus, and the second LTR… can do anything. If an oncogene is downstream of the retroviral insertion site, youre screwed. So, the HIV-1 genome they use here has been crippled so that the LTRs do not act as promoters. Like I said, this is one round of infection– we dont want to make progeny viruses anyway. We just want the virus to get in. After its in, we want the LTR dead.
… But then how do you get transcription of the healthy ABCD1 gene?
3. MPSV, myeloproliferative sarcoma virus– All you need to know is that it provides the promoter for the ABCD1 gene.
So to summarize the gene therapy vector–
1. VSV-G delivers the ABCD1 RNA into the cell.
2. HIV-1 reverse transcribes the ABCD1 gene into DNA, and inserts it into the genome.
3. MPSV promoter gets the normal ABCD1 gene transcribed.
Once the healthy gene is integrated into the stem cells, you put the stem cells back in the kids, and any of the new microglia the retrovirally transduced cell makes will be able to break down VLCFAs, thus they will be able to take care of myelin!
And thats exactly what happened when they put the altered stem cells back in the kids!
They were able to break down excess VLCFAs, and their disease progression slowed, and then stopped! Showed the kids brain-scans vs another kid who didnt get treatment… it was sad/scary what that disease does when left unchecked… but still, good news, the kids who got this treatment are doing great!
But wait! Theres more!
Wayward LTRs arent the only way retroviruses can cause cancer. They might also cause cancer if they accedentally plop themselves into a tumor suppressor gene. So, the scientists capitalized on the fact retroviral insertion sites are random and mapped where the retroviral vector inserted itself in lots and lots and lots of cells, and followed the frequency of those insertion sites over time.
Lets say, there were 10 insertion events in 10 stem cells. Every cell those stem cells produces will have the same retrovirus in the same spot. If everything is fine, you should find relatively equal distribution of retroviral insertion sites in all the cells you investigate– 1/10 cells you investigate should have the same insertion site.
IF something has gone wrong with cell cycle regulation, the ‘cancer’ stem cell will replicate too much. The cancer cell will be over represented in your investigation– maybe 15/20 cells you sample has the same retroviral insertion site, or 99/100. That would have been a clear sign that something had gone terribly wrong in these kids… but… NOPE! Things are actually looking pretty damn happy hunky dory for the two kids that got this therapy +2 years post-treatment!