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Thread: Engineered M.O.

  1. #1 Engineered M.O. 
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    Is it possible to engineer or mutate a microorganism to only attack a certain type of cells?

    Or a microorganism to act as a container of a chemical and attach itself to certain type of cells?

    Can I get an insight on how this is done?


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  3. #2  
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    It's possible. HIV is programmed that way.
    I'm not a biology major yet, so I wouldn't know how to go about doing that. But think of the human body like a machine and the DNA like the programming.


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    That's a virus. It's a simple and kinda lifeless container for transporting genetic bad news between cells. The bad news being directions to the stupid target cell for manufacturing a spew of these containers, spreading more bad news, and so on.

    The cell in this scenario is a factory, hijacked. You want to have it build your virus transporting some drug, I think. But then how do you grow more? I think we want a virus with a zipper, so once the little vessels have saturated the blood, we introduce a catalyst to split them open.

    That's talking beyond my knowledge BTW.
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  5. #4  
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    Yes, it's a virus, but they target T cell's. They don't really attack any other cell to my knowledge.
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    Quote Originally Posted by verzen
    Yes, it's a virus, but they target T cell's. They don't really attack any other cell to my knowledge.
    Dendritc cells, macrophages and neurons also. HIV initially tends to infect the first two before moving on to T cells. Neuron infection appears to be a form of latent infection strategy. Look up tropism shifting in HIV for more on that. It's essentially a form of high-speed, high pressure evolution.

    The tropism (ie the cell(s) they like) of viruses is determined by the viral capsid (its protein shell) or by the lipid envelope around that if present. It's possible to change the tropism of certain viruses to be specific for certain cell types or even broaden the specificity so that it is less fussy. We can do this by changing the coat protein sequence or modifying protein receptors in the envelope. This is quite useful in gene therapy research where one may wish to target a therapeutic virus to a specific organ. The process is often called pseudotyping as it alters the "serotype" (binding characteristics) of the virus.
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    If we're using virus as a vehicle, then we want an innocuous one that, first, efficiently gets itself well established, and second - by some trigger - performs a kind of useful suicide. Can we program such transformations yet?

    I'm imagining a 2nd stage where the capsid's just a bag of drugs not genes and easily popped by catalyst.
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    The tropism (ie the cell(s) they like) of viruses is determined by the viral capsid (its protein shell) or by the lipid envelope around that if present. It's possible to change the tropism of certain viruses to be specific for certain cell types or even broaden the specificity so that it is less fussy. We can do this by changing the coat protein sequence or modifying protein receptors in the envelope. This is quite useful in gene therapy research where one may wish to target a therapeutic virus to a specific organ. The process is often called pseudotyping as it alters the "serotype" (binding characteristics) of the virus.
    Are we highly capable of doing these? I think not yet. These can be the answers to a lot of diseases.


    @Pong

    Maybe we can make the protein shell weaker or reactive to certain medicines or stimuli for it to self-destruct.
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  9. #8  
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    Quote Originally Posted by Pong
    If we're using virus as a vehicle, then we want an innocuous one that, first, efficiently gets itself well established, and second - by some trigger - performs a kind of useful suicide. Can we program such transformations yet?

    I'm imagining a 2nd stage where the capsid's just a bag of drugs not genes and easily popped by catalyst.
    The way we currently use viruses for gene therapy is as replication-disabled vectors. So, for a disease such as cystic fibrosis (which is the result of being homozygous for defective CFTR gene), the idea would be that we package a functional CFTR gene inside the virus (with the virus replicative genes removed) and then infect the patient. In the case of cystic fibrosis we would ideally infect via inhalation. Since the virus will not replicate, we use a fairly high dose in order to genetically transform as many cells as possible.

    A considerable number of viral vectors (such as AAV, adenovirus and lentiviruses) have been investigated for the treatment of cystic fibrosis as well as numerous other diseases such as haemophilia. We can re-sequence the capsids of these viruses to re-target them to different tissues. All of the above has reached clinical trial stage for a number of diseases.

    Quote Originally Posted by CoolEJ
    Are we highly capable of doing these? I think not yet. These can be the answers to a lot of diseases.
    We can easily give our viral genome a coat from a related virus that changes its tissue specificity. Going further, we can also make pretty much whatever recombinant protein sequence we like. Re-sequencing a viral capsid is no exception. This has been done (at least for AAV), though I'm not sure whether that technique has proceeded to clinical trials yet.
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    If we can do that, we should not be far in curing cancer.
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  11. #10  
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    Quote Originally Posted by TheBiologista
    We can easily give our viral genome a coat from a related virus that changes its tissue specificity. Going further, we can also make pretty much whatever recombinant protein sequence we like. Re-sequencing a viral capsid is no exception.
    So we could build a capsid that, with the right key, breaks and releases its contents. Just when we want it to.

    I know that seems pointless when injection accomplishes pretty much the same, directly. But I'm thinking the virus (the patient's own cells really) could be manufacturing medication this way, and forever replenishing a reservoir. And since the medication is delivered internally one could administer, hypothetically, a daily dose just by ingesting theobromine (chocolate!) ...or whatever we make the capids susceptible to. Maybe pop the capsids with a neurotransmitter so people get the dose each morning when they wake.

    Quote Originally Posted by TheBiologista
    The way we currently use viruses for gene therapy is as replication-disabled vectors...

    Since the virus will not replicate, we use a fairly high dose in order to genetically transform as many cells as possible.
    A virus that first does replicate might go down easier, if only 'cause we're not flooding people with a lot of junk (it's an IV therapy right?). I guess one serious challenge is making a virus with two distinct stages: replication, and medication. And a greater challenge is coaxing cells to actually build that funny second stage. But then one little prick and you're infected for life, with your own treatment.

    Is a capsid full of insulin even plausible?
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    Insulin for diabetes? We may not need that because insulin can be introduced directly to the blood stream.

    Chemotherapy though affects all cells. As you kill all the cancer cells, you also kill the good cells. If we use that gene-engineered virus, we can control it to react only with the cancer cells.

    And same goes through all the other infections.
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  13. #12  
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    Quote Originally Posted by CoolEJ
    Insulin for diabetes? We may not need that because insulin can be introduced directly to the blood stream.
    I'm sure most of diabetics would much rather a single injection that restores insulin production to their body than multiple injections every day for life.

    Quote Originally Posted by CoolEJ
    Chemotherapy though affects all cells. As you kill all the cancer cells, you also kill the good cells. If we use that gene-engineered virus, we can control it to react only with the cancer cells.
    Quote Originally Posted by CoolEJ
    If we can do that, we should not be far in curing cancer.
    The problem with cancers is that firstly they are a highly diverse set of diseases. So a single "cure for cancer" is a myth. There will be no magic bullet for this one, no more than we could have a single-shot "cure for viruses". The second problem, and one which comes up every time some tries to develop a selective therapy for cancer, is that cancer cells are difficult to differentiate from normal cells in terms of receptors. Typically they express all the normal human receptors and soluble factors but in different combinations and at different rates.
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  14. #13  
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    Quote Originally Posted by Pong
    So we could build a capsid that, with the right key, breaks and releases its contents. Just when we want it to.

    I know that seems pointless when injection accomplishes pretty much the same, directly. But I'm thinking the virus (the patient's own cells really) could be manufacturing medication this way, and forever replenishing a reservoir. And since the medication is delivered internally one could administer, hypothetically, a daily dose just by ingesting theobromine (chocolate!) ...or whatever we make the capids susceptible to. Maybe pop the capsids with a neurotransmitter so people get the dose each morning when they wake.
    Why bother with that? The idea is that infection genetically modifies the target cells. If you can make that modification stable (say by viral integration into a chromosome) then their cells will just continue to make the fixed protein. So it's a single dose, once.

    Quote Originally Posted by Pong
    A virus that first does replicate might go down easier, if only 'cause we're not flooding people with a lot of junk (it's an IV therapy right?).
    The problem with repeatedly administering a viral vector is that the patient's immune system will generate an adaptive response and start destroying both the virus and potentially any cells it has modified. You want to get it in there once and you really don't want it to replicate because that will also trigger a response.

    Quote Originally Posted by Pong
    I guess one serious challenge is making a virus with two distinct stages: replication, and medication. And a greater challenge is coaxing cells to actually build that funny second stage. But then one little prick and you're infected for life, with your own treatment.
    It's not a bad idea, but generally viruses either damage or kill the cells that they replicate in. They'll also trigger interferons which will get the antiviral immune response started. I'll stand by my comment that the best strategy is to get a replication-disabled virus to a target tissue and set up a stable genetic transformation.

    Quote Originally Posted by Pong
    Is a capsid full of insulin even plausible?
    Yes, but probably not very useful. If you're talking about packaging insulin inside a person then that would make little sense. If you've gotten the person to the point that they can produce insulin then you may as well get them to just secrete it into their blood.

    So what you actually want is a capsid with the insulin gene inside it. Preferably with regulatory sequences that make translation of that gene into protein inducible based on blood sugar levels and tissue location of the modified cell. I'm pretty sure that very idea is being researched as I type, possibly even at clinical level.
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    There is also work being done on genetic vaccines, where you infect macrophages with a virus carrying genes for antigens, which would then be presented on the MHC-II by the macrophage and hopefully produce an adaptive immune response to a pathogen.
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  16. #15  
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    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    Is a capsid full of insulin even plausible?
    Yes, but probably not very useful. If you're talking about packaging insulin inside a person then that would make little sense. If you've gotten the person to the point that they can produce insulin then you may as well get them to just secrete it into their blood.
    We don't want a constant saturation. Healthy insulin level even oscillates minute by minute (unless you're diabetic and just get crude dose with each meal). Insulin bearing capsids, though, we could regulate nicely.
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  17. #16  
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    Quote Originally Posted by Pong
    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    Is a capsid full of insulin even plausible?
    Yes, but probably not very useful. If you're talking about packaging insulin inside a person then that would make little sense. If you've gotten the person to the point that they can produce insulin then you may as well get them to just secrete it into their blood.
    We don't want a constant saturation. Healthy insulin level even oscillates minute by minute (unless you're diabetic and just get crude dose with each meal). Insulin bearing capsids, though, we could regulate nicely.
    It will only be a constant production if we put the gene under the control of a constitutive promoter such as CMV. We can easily make our transformed cells inducible in reaction to blood sugar levels. That's a trivial change to make and would be an automatic part of any diabetes gene therapy. So this way we do a single set of injections and the patient has self-regulating insulin production for life. No more injections.

    Your idea of constantly re-administering capsids full of insulin is little better for the patient's quality of life than injecting insulin. Probably worse, since administering virus would probably require a hospital visit each time whereas the patient can inject insulin any time and anywhere. Also a virus full of insulin will be vastly more expensive to produce and much more expensive to store.
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  18. #17  
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    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    Is a capsid full of insulin even plausible?
    Yes, but probably not very useful. If you're talking about packaging insulin inside a person then that would make little sense. If you've gotten the person to the point that they can produce insulin then you may as well get them to just secrete it into their blood.
    We don't want a constant saturation. Healthy insulin level even oscillates minute by minute (unless you're diabetic and just get crude dose with each meal). Insulin bearing capsids, though, we could regulate nicely.
    It will only be a constant production if we put the gene under the control of a constitutive promoter such as CMV. We can easily make our transformed cells inducible in reaction to blood sugar levels. That's a trivial change to make and would be an automatic part of any diabetes gene therapy. So this way we do a single set of injections and the patient has self-regulating insulin production for life. No more injections.

    Your idea of constantly re-administering capsids full of insulin is little better for the patient's quality of life than injecting insulin. Probably worse, since administering virus would probably require a hospital visit each time whereas the patient can inject insulin any time and anywhere. Also a virus full of insulin will be vastly more expensive to produce and much more expensive to store.
    I meant a chronic infection would manufacture the insulin capsids in the patient. The body's store of insulin is accumulated in the 2nd stage (non-replicating) virus capsids. It's a byproduct of the infection. Then whenever we wish, we pop some of those bags with quickly degrading catalyst (like chocolate ). I totally agree that just injecting lab-grown non-replicating capsids is pointless extra packaging.

    I'm suggesting viral insulin in case gene therapy to repair or regulate normal production (cure diabetes) is impossible. For example with the pancreas kaput no amount of gene repair is going to fix that. Diabetes is just one scenario. My idea essentially is to accomplish by symbiosis what the body can't.
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    Quote Originally Posted by i_feel_tiredsleepy
    There is also work being done on genetic vaccines, where you infect macrophages with a virus carrying genes for antigens, which would then be presented on the MHC-II by the macrophage and hopefully produce an adaptive immune response to a pathogen.
    Sounds dangerous. Playing around with genes should require extreme analysis of possible side effects.

    It can backfire and cause a plague or some sort of deadly changes.
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  20. #19  
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    Quote Originally Posted by Pong
    I meant a chronic infection would manufacture the insulin capsids in the patient.
    Which would result in an innate followed by adaptive immune response to the capsids. That could easily include IgG3-mediated complement destruction of the capids in serum. The result would be an uncontrolled and uncontrollable massive release of insulin into the patient's circulation. Best case scenario, you get a follow-up cytotoxic T cell response that eliminates all your transformed, capsid-producing cells. Worst case, the patient goes into hypoglycaemic shock and dies.

    Quote Originally Posted by Pong
    The body's store of insulin is accumulated in the 2nd stage (non-replicating) virus capsids. It's a byproduct of the infection. Then whenever we wish, we pop some of those bags with quickly degrading catalyst (like chocolate ).
    But what advantage does this have over simply putting an insulin gene under the control of a promoter that responds to blood glucose level? My way, you introduce foreign antigens only once- at the single replication disabled infection stage. In your case, new foreign antigen is produced constantly by a chronic primary infection. You'd have to include a tolerizing step of some kind and the end result would be the same as my method but with more steps and more hospital time.

    Quote Originally Posted by Pong
    I'm suggesting viral insulin in case gene therapy to repair or regulate normal production (cure diabetes) is impossible.
    It's already been done in phase 1 human trials. It works.

    Quote Originally Posted by Pong
    For example with the pancreas kaput no amount of gene repair is going to fix that.
    It can be fixed by viral transfection of the liver or introduction of pre-transformed, insulin producing cells to that organ. The pancreas is not a good target, but it's not the only suitable organ. The liver work has been done and seems to be very stable. Further trials will show us if it's really viable.
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  21. #20  
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    Quote Originally Posted by CoolEJ
    Quote Originally Posted by i_feel_tiredsleepy
    There is also work being done on genetic vaccines, where you infect macrophages with a virus carrying genes for antigens, which would then be presented on the MHC-II by the macrophage and hopefully produce an adaptive immune response to a pathogen.
    Sounds dangerous. Playing around with genes should require extreme analysis of possible side effects.
    That's what clinical trials are for. Viral infection of macrophages is actually one of the natural pathways by which viral immunity is induced, so this is not by any means a crazy technique

    Quote Originally Posted by CoolEJ
    It can backfire and cause a plague or some sort of deadly changes.
    How? I really can't imagine a mechanism by which this could happen.
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  22. #21  
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    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    I meant a chronic infection...
    Which would result in an innate followed by adaptive immune response to the capsids.
    I did say chronic. We have strategies to elude or suppress immune response. And note the 2nd stage (insulin bearing) virus need not survive long. But yeah that's another complication.

    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    The body's store of insulin is accumulated in the 2nd stage (non-replicating) virus capsids. It's a byproduct of the infection. Then whenever we wish, we pop some of those bags with quickly degrading catalyst (like chocolate ).
    But what advantage does this have over simply putting an insulin gene under the control of a promoter that responds to blood glucose level? My way, you introduce foreign antigens only once- at the single replication disabled infection stage. In your case, new foreign antigen is produced constantly by a chronic primary infection. You'd have to include a tolerizing step of some kind and the end result would be the same as my method but with more steps and more hospital time.
    I'm not understanding "your way" and want to.

    An insulin gene? You mean changing cells so they produce insulin?

    A gene promoter that responds to glucose? Just how timely would the response be? Remember our goal here is, like, dude sips pop & gets insulin within minutes.

    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    I'm suggesting viral insulin in case gene therapy to repair or regulate normal production (cure diabetes) is impossible.
    It's already been done in phase 1 human trials. It works.
    With viral vector, yes and I'm curious to know if that's really practical for dude and cola. But my way isn't a vector.

    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    For example with the pancreas kaput no amount of gene repair is going to fix that.
    It can be fixed by viral transfection of the liver or introduction of pre-transformed, insulin producing cells to that organ. The pancreas is not a good target, but it's not the only suitable organ. The liver work has been done and seems to be very stable. Further trials will show us if it's really viable.
    That does sound better. That's building a new gland by genes alone? Impossible, I thought.

    ***

    Diabetes is good example, but self-manufactured agent-bearing capsids could be used in weird situations, because the body accumulates a reserve of ...well, anything we'd like to have bagged and ready, for quick release on cue. You could get a rush of dopamine every time you eat cilantro. :-D
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  23. #22  
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    Quote Originally Posted by Pong
    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    I meant a chronic infection...
    Which would result in an innate followed by adaptive immune response to the capsids.
    I did say chronic. We have strategies to elude or suppress immune response. And note the 2nd stage (insulin bearing) virus need not survive long. But yeah that's another complication.
    Immune suppression is very dangerous and only done when absolutely required. For that reason alone the method described by Biologista would be hugely preferable.

    I imagine that she is also describing the insertion of a plasmid containing the insulin gene with appropriate promoter attached into a cell's nucleus, which it would then replicate every time it splits and replicates its own DNA. Either that or actual insertion of the above sequence into the genomic DNA itself, which I think is a little more risky as we cannot control where it goes.
    Man can will nothing unless he has first understood that he must count on no one but himself; that he is alone, abandoned on earth in the midst of his infinite responsibilities, without help, with no other aim than the one he sets himself, with no other destiny than the one he forges for himself on this earth.
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    Quote Originally Posted by TheBiologista
    Quote Originally Posted by CoolEJ
    Quote Originally Posted by i_feel_tiredsleepy
    There is also work being done on genetic vaccines, where you infect macrophages with a virus carrying genes for antigens, which would then be presented on the MHC-II by the macrophage and hopefully produce an adaptive immune response to a pathogen.
    Sounds dangerous. Playing around with genes should require extreme analysis of possible side effects.
    That's what clinical trials are for. Viral infection of macrophages is actually one of the natural pathways by which viral immunity is induced, so this is not by any means a crazy technique

    Quote Originally Posted by CoolEJ
    It can backfire and cause a plague or some sort of deadly changes.
    How? I really can't imagine a mechanism by which this could happen.

    Something like certain effects of the genetic changes that were not captured during trials, that can lead to harm us.
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  25. #24  
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    Quote Originally Posted by Pong
    Quote Originally Posted by TheBiologista
    Which would result in an innate followed by adaptive immune response to the capsids.
    I did say chronic. We have strategies to elude or suppress immune response. And note the 2nd stage (insulin bearing) virus need not survive long. But yeah that's another complication.
    You'd need to administer immuno suppressants constantly to prevent adaptive recognition. That will make your patient very ill indeed.

    Quote Originally Posted by Pong
    Quote Originally Posted by TheBiologista
    But what advantage does this have over simply putting an insulin gene under the control of a promoter that responds to blood glucose level? My way, you introduce foreign antigens only once- at the single replication disabled infection stage. In your case, new foreign antigen is produced constantly by a chronic primary infection. You'd have to include a tolerizing step of some kind and the end result would be the same as my method but with more steps and more hospital time.
    I'm not understanding "your way" and want to.

    An insulin gene? You mean changing cells so they produce insulin?
    Yes. That would be the preferred means, as it limits our patient's contact with foreign antigen to a single transient administration. If immuno suppressives were actually needed in this case, it would be for a matter of weeks and never again after successful treatment.

    Quote Originally Posted by Pong
    A gene promoter that responds to glucose? Just how timely would the response be? Remember our goal here is, like, dude sips pop & gets insulin within minutes.
    We'd ideally use the same promoters as the human insulin gene. I believe there are about 15 or so, though they should be very short. That would give us most of the responsiveness of the natural gene, though natural regulation is also control at translation and secretion stages. How well this works would depend a lot on the target cell type.

    Quote Originally Posted by Pong
    Quote Originally Posted by TheBiologista
    It can be fixed by viral transfection of the liver or introduction of pre-transformed, insulin producing cells to that organ. The pancreas is not a good target, but it's not the only suitable organ. The liver work has been done and seems to be very stable. Further trials will show us if it's really viable.
    That does sound better. That's building a new gland by genes alone? Impossible, I thought.
    Not really, after all a gland is not a highly differentiated organ. We're really just talking about changing some liver cells so they make insulin in response to glucose. Liver is the main target as it takes up vectors quite well and is heavily vascularised.

    Quote Originally Posted by Pong
    Diabetes is good example, but self-manufactured agent-bearing capsids could be used in weird situations, because the body accumulates a reserve of ...well, anything we'd like to have bagged and ready, for quick release on cue. You could get a rush of dopamine every time you eat cilantro. :-D
    It might be better to transform the patients cells so that they produce the desired protein in response to whatever stimulus. That can include storing the protein for rapid release. Capids won't be at all as stable and will be susceptible to immune destruction.
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    Quote Originally Posted by paralith
    Immune suppression is very dangerous and only done when absolutely required. For that reason alone the method described by Biologista would be hugely preferable.

    I imagine that she is also describing the insertion of a plasmid containing the insulin gene with appropriate promoter attached into a cell's nucleus, which it would then replicate every time it splits and replicates its own DNA. Either that or actual insertion of the above sequence into the genomic DNA itself, which I think is a little more risky as we cannot control where it goes.
    "He" :wink:

    I realise that "Biologista" suggests female if you follow Spanish grammar, but it's pretty much a made-up word (as far as I know).

    As to controlled integration, we can do that with the AAV virus. It integrates (albeit at a low frequency) into a well defined single site on chromosome 19. It's not too much of a stretch to imagine we can increase the efficiency and change the integration site if needed.
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    Quote Originally Posted by CoolEJ
    Quote Originally Posted by TheBiologista
    Quote Originally Posted by CoolEJ
    Quote Originally Posted by i_feel_tiredsleepy
    There is also work being done on genetic vaccines, where you infect macrophages with a virus carrying genes for antigens, which would then be presented on the MHC-II by the macrophage and hopefully produce an adaptive immune response to a pathogen.
    Sounds dangerous. Playing around with genes should require extreme analysis of possible side effects.
    That's what clinical trials are for. Viral infection of macrophages is actually one of the natural pathways by which viral immunity is induced, so this is not by any means a crazy technique

    Quote Originally Posted by CoolEJ
    It can backfire and cause a plague or some sort of deadly changes.
    How? I really can't imagine a mechanism by which this could happen.

    Something like certain effects of the genetic changes that were not captured during trials, that can lead to harm us.
    That's really... vague. The biggest threats in terms of gene therapy are tumor formation and runaway immune responses. Quite well understood things that we're very careful about. The formation of civilization-ending superviruses is way down the list of stuff likely to happen. I'd be much more worried about that happening due to the natural infections. Those happen much more frequently and are totally chaotic and uncontrolled.
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  28. #27  
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    Quote Originally Posted by TheBiologista
    Quote Originally Posted by paralith
    Immune suppression is very dangerous and only done when absolutely required. For that reason alone the method described by Biologista would be hugely preferable.

    I imagine that she is also describing the insertion of a plasmid containing the insulin gene with appropriate promoter attached into a cell's nucleus, which it would then replicate every time it splits and replicates its own DNA. Either that or actual insertion of the above sequence into the genomic DNA itself, which I think is a little more risky as we cannot control where it goes.
    "He" :wink:

    I realise that "Biologista" suggests female if you follow Spanish grammar, but it's pretty much a made-up word (as far as I know).

    As to controlled integration, we can do that with the AAV virus. It integrates (albeit at a low frequency) into a well defined single site on chromosome 19. It's not too much of a stretch to imagine we can increase the efficiency and change the integration site if needed.
    Haha - how ironic. People usually assume I'm a guy, and here I go assuming you're a girl.

    I probably should have realized that some retroviruses may have specific integration sites. I was always concerned about the danger of a plasmid segment getting inserted into some essential gene for cell regulation and setting off cancer. Though if the integration is, as you say, at a low frequency, that would probably require several treatments of virus in order to transform a sufficient number of cells. Is the frequency a function of the specificity of the integration site, or of the location itself?
    Man can will nothing unless he has first understood that he must count on no one but himself; that he is alone, abandoned on earth in the midst of his infinite responsibilities, without help, with no other aim than the one he sets himself, with no other destiny than the one he forges for himself on this earth.
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  29. #28  
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    Quote Originally Posted by paralith
    Quote Originally Posted by TheBiologista
    "He" :wink:

    I realise that "Biologista" suggests female if you follow Spanish grammar, but it's pretty much a made-up word (as far as I know).

    As to controlled integration, we can do that with the AAV virus. It integrates (albeit at a low frequency) into a well defined single site on chromosome 19. It's not too much of a stretch to imagine we can increase the efficiency and change the integration site if needed.
    Haha - how ironic. People usually assume I'm a guy, and here I go assuming you're a girl.
    Easily done. I've been asked the question before... "biologo/biologa?". I just like the name, and Biologisto doesn't have the same ring to it at all!

    Quote Originally Posted by paralith
    I probably should have realized that some retroviruses may have specific integration sites. I was always concerned about the danger of a plasmid segment getting inserted into some essential gene for cell regulation and setting off cancer. Though if the integration is, as you say, at a low frequency, that would probably require several treatments of virus in order to transform a sufficient number of cells. Is the frequency a function of the specificity of the integration site, or of the location itself?
    The specificity is high, though I'm not sure how accessible the site is. I think it's more to do with the efficiency of the AAV integration machinery in general, but really I'm not too clear on why the process is as rare as it is. I can tell you that AAV only opts for integration when it cannot replicate normally. It is a means of going dormant. To replicate, AAV needs to be in the same cell as one of its helper viruses- it is replication deficient. That's one of the reasons that it makes quite a nice gene therapy vector. Even if you don't scoop out all its genes, it's not going anywhere.
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  30. #29  
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    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    chronic
    You'd need to administer immuno suppressants constantly to prevent adaptive recognition. That will make your patient very ill indeed.
    Alright, I'm convinced.

    Quote Originally Posted by TheBiologista
    We're really just talking about changing some liver cells so they make insulin in response to glucose.
    How? And won't these funny cells get attacked?
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  31. #30  
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    Quote Originally Posted by Pong
    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    chronic
    You'd need to administer immuno suppressants constantly to prevent adaptive recognition. That will make your patient very ill indeed.
    Alright, I'm convinced.

    Quote Originally Posted by TheBiologista
    We're really just talking about changing some liver cells so they make insulin in response to glucose.
    How? And won't these funny cells get attacked?
    That depends on how the cells "present" viral protein bits (antigens) on their cell surfaces. There's a chance that if the cells present viral antigens that they may be targeted by cytotoxic t cells (cells which kill viral infected cells under normal circumstances). Usually though, tissue cells such as liver cells or pancreatic cells won't present antigens from viruses just due to viral entry into the cell (though white blood cells can do this)- instead they present newly-generated viral proteins (viruses hijack the cell's protein manufacturing machinery if they successfully infect). However, since our viral vectors are replication disabled, there'll be no new viral protein generation in the cells. So all that leaves are rare cases of "cross presentation" where the mechanism that normally presents newly-generated viral proteins (called MHC I) picks up and presents bits of the virus that just infected the cell. This is a very short-term presentation mechanism however, and may not occur at all. So if we immunosuppress the patient for a few weeks, we prevent their T cells from spotting the transformed cells during the brief time that they have foreign proteins on their cell surfaces.
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    I should correct myself. I stated above that gene therapy for diabetes has gone to Phase I studies- I suspect it's actually still in preclinical studies, though it may just be that I can't find the paper I thought I 'd read. :x
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    I am not sure.HIV is virtus?VIRTUS as a kind of microorgans will attack the cell of body
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  34. #33  
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    Quote Originally Posted by xumin123
    I am not sure.HIV is virtus?VIRTUS as a kind of microorgans will attack the cell of body
    Yes, HIV is a virus. It infects immune cells and kills them as it replicates.
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  35. #34  
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    The viral vaccine apparently is structured as a plasmid with a section of unmethylated CpG DNA to act as an adjuvant and a gene encoding proteins of the pathogen you are trying to vaccinate against. The hope for this vaccine is that it could induce a Th-1 response through activation of TLR-9, this could result in a more effective vaccine against intracellular pathogens.

    This can use attenuated adeno viruses to deliver the plasmid, or apparently just injecting huge amounts of the plasmids in a solution.

    Apparently, a vaccine for horses against west nile virus has been approved for use, but it doesn't seem to work very well in people yet.
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  36. #35  
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    Quote Originally Posted by i_feel_tiredsleepy
    The viral vaccine apparently is structured as a plasmid with a section of unmethylated CpG DNA to act as an adjuvant and a gene encoding proteins of the pathogen you are trying to vaccinate against. The hope for this vaccine is that it could induce a Th-1 response through activation of TLR-9, this could result in a more effective vaccine against intracellular pathogens.

    This can use attenuated adeno viruses to deliver the plasmid, or apparently just injecting huge amounts of the plasmids in a solution.

    Apparently, a vaccine for horses against west nile virus has been approved for use, but it doesn't seem to work very well in people yet.
    Since you're doing the transfection in vitro, there's probably not much call for using a viral vector. Electroporation with pure plasmids or maybe liposome complexing should be as efficient if not better, and it reduces the risk of viral contamination when you re-introduce the DCs.
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    Quote Originally Posted by TheBiologista
    Quote Originally Posted by i_feel_tiredsleepy
    The viral vaccine apparently is structured as a plasmid with a section of unmethylated CpG DNA to act as an adjuvant and a gene encoding proteins of the pathogen you are trying to vaccinate against. The hope for this vaccine is that it could induce a Th-1 response through activation of TLR-9, this could result in a more effective vaccine against intracellular pathogens.

    This can use attenuated adeno viruses to deliver the plasmid, or apparently just injecting huge amounts of the plasmids in a solution.

    Apparently, a vaccine for horses against west nile virus has been approved for use, but it doesn't seem to work very well in people yet.
    Since you're doing the transfection in vitro, there's probably not much call for using a viral vector. Electroporation with pure plasmids or maybe liposome complexing should be as efficient if not better, and it reduces the risk of viral contamination when you re-introduce the DCs.
    Would be incredibly expensive and time consuming for effective use in vaccinating the human population though.
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  38. #37  
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    Quote Originally Posted by i_feel_tiredsleepy
    Would be incredibly expensive and time consuming for effective use in vaccinating the human population though.
    Not if it's contagious.
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  39. #38  
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    Quote Originally Posted by Pong
    Quote Originally Posted by i_feel_tiredsleepy
    Would be incredibly expensive and time consuming for effective use in vaccinating the human population though.
    Not if it's contagious.
    Go down that route and you firstly lose control over your agent and secondly engage the immune system. Not to mention taking choice away from patients who may not want, need or be suitable for your therapy. That's a massive ethical no no.
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  40. #39  
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    Quote Originally Posted by i_feel_tiredsleepy
    Quote Originally Posted by TheBiologista
    Quote Originally Posted by i_feel_tiredsleepy
    The viral vaccine apparently is structured as a plasmid with a section of unmethylated CpG DNA to act as an adjuvant and a gene encoding proteins of the pathogen you are trying to vaccinate against. The hope for this vaccine is that it could induce a Th-1 response through activation of TLR-9, this could result in a more effective vaccine against intracellular pathogens.

    This can use attenuated adeno viruses to deliver the plasmid, or apparently just injecting huge amounts of the plasmids in a solution.

    Apparently, a vaccine for horses against west nile virus has been approved for use, but it doesn't seem to work very well in people yet.
    Since you're doing the transfection in vitro, there's probably not much call for using a viral vector. Electroporation with pure plasmids or maybe liposome complexing should be as efficient if not better, and it reduces the risk of viral contamination when you re-introduce the DCs.
    Would be incredibly expensive and time consuming for effective use in vaccinating the human population though.
    Ah, I think I misunderstood. When you talked about plasmids in solution, I assumed you were talking about an ex vivo style therapy. Certainly a replication-deficient viral vector delivered in vivo would be less time consuming for the patient, though I'm not sure how the expenses will match up. I suspect they won't be very different.
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  41. #40  
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    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    contagious.
    ethical no no.
    HIV bloodhound then - it spreads just as its quarry spreads, through the risk population.


    I was really just hoping to grow agents in vivo.
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  42. #41  
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    Quote Originally Posted by Pong
    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    contagious.
    ethical no no.
    HIV bloodhound then - it spreads just as its quarry spreads, through the risk population..
    How would that work? By what mechanism would the virus either fail to spread to those not infected by HIV or detect those who were? I know of no virus that is selective for hosts- they non-selectively enter a host and either successfully infect or they do not. Remember, viruses are not active or even alive in the conventional sense. They will not "seek" anything. They are blown in aerosol, or flow in in fluids etc... entirely passively. No active movement.

    You still have the consent issue anyway. You're not ethically permitted to medicate a HIV positive patient with conventional anti-retrovirals without consent, so a modified virus is definitely not going to fly with an ethics board.
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  43. #42  
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    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    contagious.
    ethical no no.
    HIV bloodhound then - it spreads just as its quarry spreads, through the risk population..
    How would that work? By what mechanism would the virus either fail to spread to those not infected by HIV or detect those who were? I know of no virus that is selective for hosts- they non-selectively enter a host and either successfully infect or they do not. Remember, viruses are not active or even alive in the conventional sense. They will not "seek" anything. They are blown in aerosol, or flow in in fluids etc... entirely passively. No active movement.

    You still have the consent issue anyway. You're not ethically permitted to medicate a HIV positive patient with conventional anti-retrovirals without consent, so a modified virus is definitely not going to fly with an ethics board.
    So we can't engineer a virus that only replicates in HIV infected cells? I guess we would if we could. I was thinking such a virus could be a sort of flag marking cells for treatment... well, like a hunting dog on "point".

    I understand the ethical problem of a treatment spread unwittingly through blood.

    I still think growing our treatments in vivo expedient, if it's the difference between a single injection in the field or hauling a patient through a lot of blood transfusions.
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  44. #43  
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    Quote Originally Posted by Pong
    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    Quote Originally Posted by TheBiologista
    Quote Originally Posted by Pong
    contagious.
    ethical no no.
    HIV bloodhound then - it spreads just as its quarry spreads, through the risk population..
    How would that work? By what mechanism would the virus either fail to spread to those not infected by HIV or detect those who were? I know of no virus that is selective for hosts- they non-selectively enter a host and either successfully infect or they do not. Remember, viruses are not active or even alive in the conventional sense. They will not "seek" anything. They are blown in aerosol, or flow in in fluids etc... entirely passively. No active movement.

    You still have the consent issue anyway. You're not ethically permitted to medicate a HIV positive patient with conventional anti-retrovirals without consent, so a modified virus is definitely not going to fly with an ethics board.
    So we can't engineer a virus that only replicates in HIV infected cells? I guess we would if we could. I was thinking such a virus could be a sort of flag marking cells for treatment... well, like a hunting dog on "point".

    I understand the ethical problem of a treatment spread unwittingly through blood.

    I still think growing our treatments in vivo expedient, if it's the difference between a single injection in the field or hauling a patient through a lot of blood transfusions.
    We can make a virus that only replicates in HIV infected cells, but we can't make a virus that wont attempt to infect everyone if we allow it to be replication competent.
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