Safety concerns in using replication incompetent HIV based lentiviruses for DNA tranduction.

So hello everyone. I stumbled upon this group while looking for information about using lentiviruses to modify the human genome. I've been considering for some time now injecting myself with a replication incompetent lentiviruses to transduce the gene for telomerase, as well as a promoter for the hTERT gene, into my own genome. As far fetched as it seems there are a couple of companies that sell such lentiviruses pre made (and relatively cheaply). It would literally be a matter of buying this virus, thawing it, and intravenously injecting it. Here's the actual page of one of the companies where this virus can be bought:

However this lentiviruses is based on HIV so there is the risk that a replication competent form of HIV could develop if I injected it. There is also the risk that the viral vector could integrate itself into my genome in such a way as to disrupt a tumor suppressor or stop the suppression of an oncogene. This actually happened a while back when murine leukemia virus was used in a clinical trial ( However there was a clinical trial on a group of HIV positive individuals using HIV based vectors and there weren't any negative side effects noted ( Plus wild type HIV infections don't really correlate with outbreaks of leukemia, so I'm not so much worried about the possibility of a HIV based lentivirus inducing cancer. However there still is the problem of such a virus causing HIV.

Now the lentivirus that I posted a link to is a so called 3rd generation self inactivating virus. It supposedly (though the page doesn't go into specifics) is missing genes that are essential to HIV replication as well as a deletion in the 3' LTR of the virus which some how or another makes the virus replication incompetent, but again I can't find specifics about the exact lentivirus being sold. In addition the virus doesn't use the protein envelope used by HIV, it uses the VSV-G envelope. This increases the viruses tropism (allows it to affect a larger variety of cells) so even if a replication competent virus did emerge it shouldn't behave in the same way HIV does (though it might actually be worse given that it can infect a larger variety of cell types).

I could only find one clinical trial (or rather the abstract of the paper about it) where an HIV based lentivirus was used on HIV negative individuals ( Everything went well in that trial but the transduction was performed ex vivo whereas I'm considering in vivo usage.

So I'm wondering if anyone here might know anything about how safe HIV based lentiviruses are. I'd really love to find some more clinical trials where these viruses where used on people who are HIV negative.


  • edited May 2012
    I'm still an undergrad so don't take what I say too seriously, but I doubt a genetics company would market anything able to give people AIDS. I'd be far more concerned about the way it inserts telomeres at double-stranded breaks. You could end up as one massive, immortal tumor if things really go wrong.

    Is it possible to do using a different vector? I assume it doesn't matter where it is located on the genome. And can you test it on a mouse or something? (Using mouseTERT, I guess. Expensive project. :P ).
  • I'll try and make a list, from least to most pessimistic.

    1. Catching the HIV is the least of your concerns; the producer cells have never seen most of the HIV genome, as all the genes sourced from HIV would be synthetic. However, if you're injecting this into yourself, the company would not be liable for any side effects.

    2. The vector co-expresses GFP, which is non-human and will likely generate an immune response against transduced cells, resulting in their death. Certainly you could ask the supplier to make one without the GFP gene, but I doubt they'd do it. Antigenicity of the vector coat itself is also a concern.

    3. $450 may seem like a low, low price for your viral needs, but that aliquot is two million viral particles. Your body has several trillion cells, and a repeat and/or large-scale exposure is likely to be met with a massive neutralizing immune response.

    I should add that the last two points can be somewhat managed by producing your own vector, since the commercial markup on recombinant protein/DNA products is anywhere from 10- to 1000-fold. That's a whole other topic.

    4. Integration is always a concern; wild-type HIV is busy killing the cells it infects, so they're unlikely to become cancerous. In the case of the SCID kids who died during that infamous trial, it's possible that their non-functional immune system could have prevented the cancer, had it been working. With the number of viruses you'd need to do anything effective, the chance of an integration somewhere bad goes up.

    5. Telomerase is not technically an oncogene itself, but it does enable oncogenes to flourish. Telomeric degradation is a significant part of the regulatory network to eliminate cells which have likely accumulated a lot of mutations. I can sort of understand the obsession with telomerase among certain elements of the life extension community, but shooting massive amounts of it into yourself won't help.
  • Some very good points. As you said telomerase isn't an oncogene. It is more the case that telomerase expresion is necessary for most cancers to occur but is not in of itself sufficient to cause cancer. If somatic cells did constitutively express telomerase then it could be argued that the risk of cancer developing would increase.

    However I'm not quite sure what you mean by "shooting massive amounts of it" into myself as that is not what I'm doing. Simply injecting the telomerase enzyme would do nothing as it would not be able to bypass cell membranes and enter the cytoplasm. I'm talking about integrating the gene for telomerase and a promoter for it exogenously into my genome and having it expressed from within my cells. Why exactly wouldn't introducing telomerase into the cell help?

    Now concerning integration the link I posted ( describes the use of replication incompetent HIV which integrated a gene coding for antisense HIV RNA in HIV positive individuals. Obviously the sample size is extremely small but at least in this case there are no apprent consequences from integration. But yeah your right I can't really say that because wild type HIV doesn't cause cancer that replication incompetent HIV won't. I really need to find more clinical trials where these types of lentiviruses are used to rule out oncogenesis.

    I hadn't considered GFP's effect. The company I linked to will make a lentivirus with just the telomerase gene and promoter (at four times the cost...) but I was sort of planning on using GFP as a measure of successful transduction. Do you know if GFP expression as a fusion protein with telomerase would mitigate an immune response? If not I could get the hTERT only virus but I don't know of any cheap and practical way of determining whether or not transduction was successful.

    Now I wasn't expecting very many cells to be successfully transduced from just one injection. I was sort of planning on injecting once, checking for GFP fluorescence, and then injecting more if I found any. Immunosuppresants could be used to mitigate diminishing returns do to immunity. But if I can't use GFP I'm at a loss. I'd have to blindly inject and just hope it's working. If the immune response isn't especially fast I might be able to use GFP to confirm the first injection and then use pure hTERT from there out but as far as I know there's no easy way to confirm long term effectiveness.

    Really not being able to use GFP is a nail in the coffin, unless someone knows a cheap and easy way to test for telomerase activity? I'll read up on immune response to transgenes and see if I can't find some ad hoc solution in the meantime.

  • edited May 2012
    Have you considered an adeno-associated virus vector? 

    Edit: And if I am not mistaken GFP is cytotoxic 
  • The main reason I want to use a lentivirus is that they can transduce non dividing cells whereas adenoviruses can only transduce dividing cells. Do you know if a fusion protein of GFP and some other protein is necessarily cytotoxic?
  • By "massive amounts" I was referring to your approach, not injecting the protein intravenously, which would obviously do nothing.

    The potential hazards of integration aren't especially worrying by themselves, but combined with telomerase expression, they become more of an issue. If you have a cell near the injection site with a high multiplicity of infection, and the extremely strong CMV/EF1a promoter regions potentially landing near oncogenes, telomeric shortening is one of the ways by which such a cell would normally be prevented from runaway division.

    As for actually using GFP as a reporter, most of the transduction would occur in internal solid tissue, which you'd have a hard time assaying anyway. I suppose you could keep track of transduced immune cells in blood samples and use that as a proxy, but you'd have to count a lot of cells before finding a positive one, considering the sheer number of circulating immune cells. You could try doing quantitative PCR of the CMV-TERT sequence from blood lysate, but again the number will likely be too low to be reliable.

    A TERT-GFP fusion protein would do nothing to blunt GFP's antigenicity or toxicity.

    AAV vectors are the current darling of viral gene therapy, but most everyone will already have a strong neutralizing response due to prior exposure, necessitating the use of immunosuppressants from the beginning.
  • edited May 2012
    That was awesome. It is good to see more bio people here! I think I'll bump some of our old gene threads in tribute.

    I'm way out of my element here, but is replication deficient HSV-1 an alternative?

  • @1101 I might be able to talk my grandmother into being a lab rat for you if you want. She's in her late 80's so she really doesn't have much to lose.
  • My Grandma is down. At first she said no, but then she said yes when I asked her again 10 minutes later. <Thank you early stage Alzheimers>

    Btw, you know a forum kicks ass when nobody objects to injecting a grandmother with HIV in order to make her into an immortal mutant tumor. I wonder if I could collect her social security checks for eternity?
  • Ladies and gentlemen, biohacking's own mad scientist^
  • I have a... 'friend' (acquaintance/client? I may have hacked her ex-husband's accounts to help her win a custody dispute...) who's elderly father might also be open to such research.  
  • Herpesviruses are an option, but are far less investigated than lentiviruses for gene therapy, for various reasons. They do have the ability to repress the immune response against infected cells, but my concern is that injecting a large number of viral particles will trigger an extracellular response anyway. They do have very large genomes though, which is always a bonus, though not necessarily in this case.

    I should add that viruses really aren't my expertise; they're efficient and all, but the cost and hassle is far beyond the realm of biohacking, so I tend to prefer non-viral approaches like plasmid electrotransfer.

    I won't get into complaining about how administering telomerase is useless with our current tech - maybe doing something at the germline level would be effective, but for already-grown humans, we're still a few years away from a virus that can replicate autonomously in the body and deliver the gene to every important cell while also evading an immune response.

  • edited May 2012

    Herpes simplex is
    some sort of retrovirus so I'm not sure whether or not it can bypass the
    nuclear membrane which means it might only be able to integrate into dividing
    cells only.

    However my main concern at this point is cost effectiveness. As yashgorath
    pointed there is no practical way to deliver a gene vector to every single cell
    in the body. Now with many forms of gene therapy this is fine as even transducing
    a small portion of cells type is enough to elicit a therapeutic response. For
    instance you can imagine some sort of gene therapy that targeted bone marrow
    and corrected some defect in blood cell production (that might for instance
    cause anemia or something). Even if just a small fraction of cells are
    transduced it impacts the health of the entire body since the newly produced
    healthy blood cells are going to benefit every cell in the body. However
    telomerase produced by a single cell has no organism wide benefit so every
    single cell would need to be transduced.

    Now I could just keep injecting viral particles while taking immunosuppressants
    and eventually I will have probably transduced a sizeable amount of my own
    cells, however this would be impractically expensive and I'm assuming that no
    immune response would arise. What I wanted to do originally was just use one
    injection and measure the response from that based on GFP production. But lukas
    pointed out that GFP would be cytotoxic. Now I could integrate telomerase that
    is not GFP tagged but the only remotely practical way of detecting its presence
    would be (as yashgaroth said) qPCR. And just to get telomerase expression up to
    a level I could detect with qPCR would require many, many aliquots of the virus (think in terms of orders of magnitude).

    Now I could try producing the virus myself but it's difficult. It's three transfections (unless someone can point me to some company selling a ready made cell line) and even then I'm stuck shelling out for purification kits to isolate the viral supernatent. Now this is still something that is very much doable, it's just difficult and expensive. At this point in time It just seems easier to wait and see what comes on the market in the next couple of years. 

    Also I wouldn't recommend testing this on any elderly people. It would support the safety of using viruses for gene therapy but it's unlikely they will see any benefits from the treatment, without injecting an absolutely massive amount of viral particles. Plus at this point it seems doubtful that the symptoms associated with aging are caused by any one thing. You might be able to fix the problem of telomere shortening but then you are still left with things like mutations in chromosomal and mitochondrial DNA, buildup of intercellular aggregates, and what have you.

    But on a more positive note, a very similar type of gene therapy/augmentation was just successfully implemented in mice. Though they used a replication competent adenovirus rather than incompetent HIV:

  • DirectorX directed me here, so hello everyone.

    1101: that article on telomerase in mice is amazing. I didn't think telomerase would also delay osteoporosis and type-2 diabetes. This is exactly the sort of thing the anti-aging community has been trying to develop. Oh and the virus they used was replication INcompitent AAV9. I think an even more elegant gene therapy would be to reactivate the copy of telomerase you've already got. Human telomerase is located on chromosome 5p15.33. You would splice in an appropriate promoter in front of telomerase to reactivate the gene. On the other hand you may also have to do some epigenetic modifications to reactivate telomerase as well.

    From Lukas' article, I'm not convinced GFP is cytotoxic. The study didn't say they had a control. If they had two cell lines - one transformed with GFP on plasmids, and the other transformed with "placebo plasmids" and the GFP ones died, then I'd be convinced. As of this study though, it could have been just injecting a plasmid that killed them, or maybe all their cells just died anyway and they blamed it on GFP. To make a claim like they're making, you have to do a controlled comparison. Also, GFP is used all the time to make glow in the dark animals, who live apparently normal lives. I also just recently saw a time-lapse of neurons growing into a network while glowing with GFP ( starting at 4:26). But... do you really want to glow green permanently?

    There are several other genes that would probably lengthen mean and maximum life span in humans. One being inserting additional copies of the promoter for the nrf2 gene which controls the several genes which repair proteins. Over-expressing nrf2 in C. elegans raised mean lifespan by 30%, while silencing the gene reduced it by 40%. Another gene that would be good for us to have is gulo, which in other mammals is active in liver cells and is the enzyme for making our own vitamin c. This gene is still functional in most mammals, but monkeys and apes (including us) have inherited a dysfunctional version on chromosome 8p21. Additionally, there was a study that used gene therapy to put the gulo gene in human liver cells and the cells really did make vitamin c, so we know that one gene is the only missing link. The biggest benefit to vitamin c related to longevity is it acts as an antioxidant, so it keeps oxidative stress low. Oxidative stress is one of the causes of protein damage and also an outcome of chronic inflammation which itself is a culprit in many degenerative diseases. You could think of it as rust-proofing your body.

    Another way to possibly prolong life is just to take 80 mg of aspirin a day. In C. elegans, 1 mM of aspirin prolonged mean lifespan by 28%. They figured out it worked by altering gene expression of insulin receptor like genes (they named FOXO). This probably isn't translatable to humans, but 80 mg per day in humans has already been found to lower the rates of cancer, diabetes, cardiovascular disease, stroke, and delays Alzheimer's. This video is pretty technical but goes into the details on how aspirin enhances lifespan in c. elegans
  • bumping topic to see if anyone moved forward with this 
  • Lukas, I can definitely say I agree, and so far as I'm aware GFP is toxic, but I can verify that with a check into some of my textbooks. I have a few suggestions.
    Have you considered using the flu as a viral vector instead? It's what we used in my freshman year when we created glowing mice.
    As far as the generation of telomerase, don't you think you'd come out better by simply applying the promoter itself to your own genes instead. I'm aware it might actually cost a bit, but have you thought of actually having some gene assays done on a group of individuals who have a marginally longer life span and comparing it with those who come from stock of a relatively normal life span to compare, and then cross-reference that with some animals who have an equally long life span just for good measure? I mean, these are just thoughts, and biology was only my minor so I'm sure I'm still quite lacking in comparison to others.
    Personally, though, I'd rather work towards improving the biology in general before attempting to enhance life span, since the capacity of the body to deal effectively with given stressors will directly impact the life span to some extent anyway.
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