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Mitochondrial Gene Therapy Via Nested Viral Vectors

Deliver mitochondrially-targeted bacteriophage by adenovirus vector
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Gene therapy is the mitigation of an undesirable phenotype by way of modifying the genetic information. For many years this held enormous promise, however, not much has happened in the clinic.

The reasons for this centre around delivery of the new genetic information. The best way of getting DNA into cells is by virus. Viruses are really fantastic at this job. The viruses that scientists tend to pick for this task fall into two main categories: those which infect and then express their DNA cargo directly, such as adenoviruses. Or those which infect and subsequently incorporate selected information into the host genome, such as lentivirus, which is a kind of retro virus.

Each have their own problems, adenovirus expression tends to be temporary, and repeated doses are far from a cheap/simple solution. Lentiviruses tend to incorporate themselves into the genome in an indiscriminate manner, where they may interrupt an important gene... doing more harm than good. Specifically, they may cause cancer.

Mitochondria have their own genome. They used to be bacteria in the dark history of evolution, before they were captured, and they now live as obligate intra cellular symbiotes. Most of their genome has been stolen away and now resides in the dusty library of the nucleus (carefully robbing them of their independence, naturally). However, a number of genes remain. Almost exclusively, these code for the proteins of the electron transport chain (ETC). A clever series of proteins that makes eukaryotes a whole lot more efficient. However, there are a number of defects associated with these proteins leading to very real human diseases.

The example I will use is Leber's hereditary optic neuropathy <link>. This disease is caused by mutations of the mitochondrial genome, all of which have been mapped, which prevent the normal operation of mitochondrial complex 1.

Now, there are viruses that infect bacteria; bacteriophage. There are even bacteriophage which infect bacteria and subsequently incorporate themselves into the genome <link>. This is great, it means that it may be possible to infect mitochondria with bacteriophage. Then, if you've been a good little molecular biologist, you can get your phage to incorporate itself into the mitochondrial genome and start expressing a FUNCTIONAL copy of the affected mitochondrial complex 1 sub unit.

Here's the first problem: The phage will not be able to infect or enter a eukaryotic cell. It will never reach the mitochondria. Worse, in a clinical setting it would just tool around in the bloodstream before getting destroyed by the immune system.

Here's the solution: create an adenovirus (more accurately an adenovirus associated vector; AAV) which encodes the phage genome. The phage will then be directly manufactured and constrained within individual human cells. The phage genomes will fit; AAV inserts go to about 5.5kb, phage genomes are rarely bigger than 4.5, and we may not need all of it.

Then, your AAV infects human cells, it's DNA starts being transcribed and replicated, the Phage then self- assembles inside the host cell, the functional phage infects the mitochondria, incorporates into the genome and then SOME of the complex 1 subunits begin to function normally.

There should be no problems with causing cancer: while there are MANY mitochondrial proteins associated with control of cell death etc. They are all encoded for by nuclear genes. There should be no problems with the AAV, you can give pretty massive doses of AAV which are replication-deficient. The phage should only ever be present INSIDE cells, so there should be no immune problems.

Potential problems: 1. There may be no phage capable of infecting mitochondria; it's been a while since they were free- living, and so their outer membrane may have become a little too specialized (would make a cool paper to infect mitos w/a micro-injected phage though).

2. The phage may not self-assemble in a eukaryotic cell: phage proteins may not be correctly processed and get stuck in the ER/fold incorrectly etc.

3. There may not be a way of controlling where the phage incorperates its DNA. You may fix complex1, but destroy complex 2. This may be mitigated by the multiple copies of mitochondrial DNA.... you should end up with a full complement of functional proteins, even if they're from diverse DNA molecules.

bs0u0155, May 08 2013

Leber's hereditary optic neuropathy http://en.wikipedia...ry_optic_neuropathy
[bs0u0155, May 08 2013]

Phage genome incorperation http://www.ncbi.nlm...articles/PMC277231/
[bs0u0155, May 08 2013]

Great though this idea is... Mitochondrial_20genome_20refurbishment
...it's practically identical to the one immediately below it. (Copy-number variation?) [Wrongfellow, May 11 2013]

[link]






       Viral evolution is pretty snappy. A free-living mitochondria-like bacterium would be a useful development model. Then, just culture it in an Intracellular-like medium and screen.
bs0u0155, May 08 2013
  

       No [beanangel] ?
normzone, May 08 2013
  

       Damn. Not to detract in any way from the idea, but I had this self same thought a while back, and it's still on my list of potential "cool shit" projects to try someday.   

       With regard to your three problems:   

       (1) finding a phage that can infect mitochondria. Easy, as long as you can find one that infects them to even a very limited extent. Just cook purified mitochondria and phage in a cytosol-like soup; spin down the mitochondria; extract the phage DNA; amplify and mutate; repeat for several cycles. A complication is that you probably want a phage that can be grown up on an easy bacterial host, as well as infecting mitochondria; but this is doable.   

       (2) self-assembly in eukaryotic cells. Again, in vitro selection should work; just purify any intact (packaged) phage from infected cells, mutate and repeat...   

       (3) This will be fixable; it's just Lego.   

         

       //No [beanangel] ?// If this were a [beanangel] idea, it would go something like:   

       "Research reported in WallMart Weekly suggests that mitochondrial oldening is a key factor at the ageing process. People could be made happylong by transmitting improved mitochondrial genome sequences, with extra sweetness molecules, via nanotube receivers using phased interference. Handyphones could be made to transmit the sequences, if two were used on opposite sides of the body. it would also be possible to discover the mitocodes of beatiful women an upload to less pretty people, possibly crowdsourced."   

       [+]
MaxwellBuchanan, May 08 2013
  

       Have you just bunned [beanangel]'s version of this idea, [MB]? He wouldn't've put any capitals into the title.   

       [+].
nineteenthly, May 09 2013
  

       No, I was bunning [bsetc]'s.
MaxwellBuchanan, May 09 2013
  

       It is an interesting question as to whether mitochondria suffer from phage. They might be pretty safe in there, all domesticated. Complacent.   

       If this nested vector thing works it could work for other genetic engineering projects too. The idea of sidestepping cancer by targeting the mitochondria is super slick.
bungston, May 09 2013
  

       Most of the proteins needed in eukaryotes are encoded in the nucleus. Messenger RNA is imported into the organelle where it is translated into the protein.
It seems to me that rather than trying to get DNA in there one might use the host cell machinery:
  

       1) Introduce DNA to the cell as a plasmid. If memory serves these sometimes and somehow end up in the nucleus and can be transcribed. The plasmid also carries both selection and counter-selection.
2) Plasmid encodes RNA tagged for transport to mitochondria: the mitochondrial genome patch, a reverse transcriptase and a recombinase.
3) All three RNAs are transferred. The reverse transcriptase and recombinase are translated to produce proteins.
4) The genome patch is reverse transcribed (it has a signal sequence to prime transcription, the other mRNAs do not).
5) The DNA patch is integrated into the Mt genome by the recombinase.
6) This process is kept up for long enough to convert most copies of the genome.
7)The cells are grown. The plasmid cannot replicate, so is lost from some cells.
8) These plasmid-free cells are selected by some counter-selection.
9) The cells are grown some more to create clonal lines
10)The cells are screened for successful integrants by PCR.
  

       The human mitochondrial genome is only around 17 kb. That's smaller than many eukaryotic genes. It might be easier to try for a complete replacement rather than patching. I can imagine other issues with that, though.
Loris, May 10 2013
  

       [Loris] I like the idea of using the intrinsic import machinery to get an RNA version into the mitochondrion - that's a neat idea. It depends on a bit more stuff going on in the mitochondrion in order to produce the DNA, but the simplification of the import process is a huge potential advantage.   

       But I'm not sure about your ideas for selection/ counterselection/ screening. Surely what we're after is whole-body, in situ mitochondrial genome relacement (or patching)? In which case screening or selection are pretty much out, unless you wanted to kill off a small minority of cells that hadn't received the mitochondrial upgrade.
MaxwellBuchanan, May 10 2013
  

       Heh, I usually work with bacteria. They're just easier.   

       I suppose one could try an RNA virus vector's packaging machinery for a one-shot conversion, or something like that. I'd be a bit wary of putting something into human cells which would be modifying the genome and wouldn't be eliminated afterwards.
Loris, May 10 2013
  

       I don't think it would matter if some part of the system replicated in vivo; in fact you might need the whole shebang to replicate in order to achieve high enough infection levels (not my area, though).   

       Whatever you do, it'll be easy enough (compared to everything else) to put an Achilles' heel into the carrier virus so you can zap it once its job's done. Or, obviously, the immune system would clear it - though hopefully not before it's done that thang it does.
MaxwellBuchanan, May 10 2013
  

       There is no way a eukaryotic nucleus is going to transcribe what is on a random plasmid. Bacteria sometimes make with that sort of anonymous bathhouse exchange and sometimes it works for them but eukaryotes have too much at risk.
bungston, May 10 2013
  

       //The human mitochondrial genome is only around 17 kb. That's smaller than many eukaryotic genes. It might be easier to try for a complete replacement rather than patching. I can imagine other issues with that//   

       I'm not trying to replace the whole mitochondrial genome, admittedly there are several phage that are big enough (T4), and there are hundreds of very large mammalian virus genomes (vaccinia), however that just steps up the difficulty. Working with Lambda/AAV is pretty trivial to the right people.   

       I'm looking for a replacement of a single defective protein (a single subiunit of C1 in my LHON example).
bs0u0155, May 10 2013
  

       incidentally, LHON was a good example, and the mitochondrial ETC is VERY amenable to screening.   

       Once you've made your custom phage, you can simply microinject them to LHON-HeLa or whatnot, and use the suite of crazily specific complex inhibitors.... 3NP for CII for example. If your cells live, your onto a winner.
bs0u0155, May 10 2013
  

       //There is no way a eukaryotic nucleus is going to transcribe what is on a random plasmid.//   

       Well, yeah. I don't think you understand my proposal. I think we can take it as read that this requires carefully engineered sequence to provide the functionality required, however it's done.   

       //I'm not trying to replace the whole mitochondrial genome ... I'm looking for a replacement of a single defective protein (a single subiunit of C1 in my LHON example).//   

       The bulk of my comment covered 'patching' the mitochondrial genome. I was just pointing out at the end that if you can perform a full replacement then you have a 'one size fits all' treatment. That is, there'd be no need to match the treatment to the particular mutation, it would just work for everybody.
The potential disadvantages I alluded to are that the larger RNA would be harder to generate reliably, and the larger the replacement sequence the greater the risk of introducing other mutations.
Loris, May 10 2013
  

       //requires carefully engineered sequence to provide the functionality required, however it's done.// Exactly. Vectors for transient expression in mammalian cells are well-known. That is not the difficult part.
MaxwellBuchanan, May 10 2013
  

       Wrongfellow: Thanks for the HB link. I did a quick search on this but didn't catch it. I think there is sufficient novelty here to differentiate the two ideas. The differences are more genOME therapy vs. gene therapy. In addition, the nested nature of dual independent virus vectors is new here. It gets around the delivery issues associated w/the straight up phage idea.
bs0u0155, May 20 2013
  

       Oooo! Oooo! How about this idea! Take out stem cells and put in whole new mitochondria. Just get them from other cells and plunk them in there. You might not even need to get rid of the defective ones first. Then transplant cells back! The new mitos will plug along shoulder to shoulder with the old, picking up their slack.   

       No need for dna jiggery-pokery! Just good old cell microsurgery.
bungston, May 20 2013
  

       An anno from the linked idea, by the formerly esteemed [Basepair]:   

       //I'd thought of the problem of getting a virus that'll get into both eukaryotic and prokaryotic- like cells. What we need is a Trojan Horse virus. basically, you make a phage genome but you package it in vitro with eukaryotic viral coat proteins. This infects the eukaryotic cell. The phage genome encodes the packaging proteins necessary to re-package it as an active phage to enter the mitochondria. With a nifty bit of coding, you could (hopefully) ensure that the phage-coat- encoding genes were inactive in the mitochondrion (by suitable choice of promotors perhaps). The phage would also encode, of course, a fully active mitochondrion.//
MaxwellBuchanan, May 20 2013
  

       Very nice, I thought that at about the same moment*. Mitochondria, when injected into cells do retain some functionality... they move and fuse as normal, in fact these experiments were among the first to demonstrate mitochondrial fusion. This was back in the days when we couldn't switch fluorophores on and off. (take a cell w/red mitochondria, remove mitos, put in cell w/green mitos observe as some mitos become red and green).   

       There's also research showing how stem cells can actually transfer mitos to other cells (not sure it's published, saw it at a conference).   

       So this could work in two ways: 1. By simply populating the body with cells derived from the healthy-mitochondria containing stem cells. 2. By actively transferring healthy mitos to certain target cells.   

       Some problems that are yet to be resolved: 1. We don't know if transplanted mitos won't be selectively degraded over time... i.e. is there and intracellular immunity governing self/not self decisions, and is that somehow based upon the mitochondrial genome. 2. Without selective pressure, would the donor mitochondrial DNA just be so dilute as to be negligible.   

       *lies, I thought that as soon as I read about 2 words of your comment ;-)
bs0u0155, May 20 2013
  

       You want the mitos to be self? Engineer your own exvivo. You could do it in fibroblast culture. Once you get the one you want, sequence it to make sure it has not acquired weirdness. Then back home it goes.
bungston, May 20 2013
  

       Yeah, I phrased that badly. I think it's pretty interesting as to whether there IS a form of intracellular immunity, there would have to be for non-self mitos to be an issue. As it is, mitos are pretty non-self. In crush/burn/necrosis injuries where a lot of mitochondrial content ends up in the circulation... you can end up dead from immune overreaction to mtDNA and protein fragments with formylmethionine on them.   

       This is either an Achillies' heel or great idea, depending upon which angle you look at it.   

       What could be interesting, is that several diseases are HUGELY potentiated when present on a background of certain subtypes of mitochondrial genome. So, you could ameliorate a few diseases simply by transplanting in the least-worst subtype.   

       Additionally, some mtDNA types are associated with different ATP generating efficiencies... i.e. some mitos are tightly coupled (many ATP) some are loosely coupled (some ATP, some heat). So, if you're planning some super cool Tour de France cheating, you could transplant in muscle-specific stem cells armed with central African mitos.
bs0u0155, May 20 2013
  

       Just thinking technical details, you could probably purge the cells of native mito with something like chloramphenicol. The cells would do glycolysis until the new mitos took. The new mitos would give a metabolic advantage (aerobic respiration) and allow succesfully mito transplanted cells to overgrow the non-mito cells.
bungston, May 21 2013
  

       That's one way, another would be antimycin A, it's non-competitive, so you could give a quick antimycin pulse, wash, then inject the new mitos and the old ones should be degraded.
bs0u0155, May 21 2013
  

       It would be very embarrassing if you purged all the native mitochondria, and then found that your replantation didn't work in all cell types. Brains don't really do glycolysis.   

       It would be safer to make the new mitochondria resistant to a drug which is toxic to normal mitochondria, so you could wait and see before burning your bridge over troubled water.
MaxwellBuchanan, May 21 2013
  

       Incidentally, cells regulate their numbers of mitochondria fairly tightly, so a few rounds of replacement would dilute out most of the native mitos.
MaxwellBuchanan, May 21 2013
  

       you're forgetting about the ongoing mitochondrial dynamics, fusion-fission is going to make chimeric hybrids out of all of them.
bs0u0155, May 21 2013
  

       //fusion-fission is going to make chimeric hybrids out of all of them.// Ah yes. Well, in that case just keep dosing with new genomes and dilute out the old ones.
MaxwellBuchanan, May 21 2013
  

       Ha. The Donor genome should code for a restriction enzyme. The donor genome should not be cut by the enzyme. The recipient genome should be.
bs0u0155, Oct 28 2014
  

       (obligatory quote every time something like this comes up)   

       Tyrell: The facts of life. To make an alteration in the evolvment of an organic life system is fatal. A coding sequence cannot be revised once it's been established.
Roy: Why not?
Tyrell: Because by the second day of incubation, any cells that have undergone reversion mutations give rise to revertant colonies like rats leaving a sinking ship. Then the ship sinks.
Roy: What about EMS recombination?
Tyrell: We've already tried it. Ethyl methane sulfonate as an alkylating agent a potent mutagen It created a virus so lethal the subject was dead before he left the table.
Roy: Then a repressive protein that blocks the operating cells -
Tyrell: Wouldn't obstruct replication, but it does give rise to an error in replication so that the newly formed DNA strand carries the mutation and you've got a virus again. But, uh, this-- all of this is academic. You were made as well as we could make you.
normzone, Oct 28 2014
  

       Nice quote but bollocks.
MaxwellBuchanan, Oct 28 2014
  
      
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