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Allotopic Expression & the "Mitochondrial Problem"


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#31 jaydfox

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Posted 05 April 2005 - 12:07 PM

Prometheus, I think you missed the other key aspect of the RHH. If there are a few mutant mitos in the cell, then yes, they would not be contributing much to the problem, since they are not producing ROS.

However, what is observed in the RHH is that in a very small minority (<1%) of cells, clonal expansion leads to cells where all or nearly all of the mitos have mutant mtDNA. In that case, there is almost no ROS production, and indeed almost no ATP production through the damaged complexes.

Sounds great, just a defective cell, right? The reason it's called the reductive hotspot hypothesis is that those cells actually are producing many times more ROS than healthy cells. How? The Plasma Membrane (NADH-)OxidoReductase system, which allows the cell to produce ATP by exporting electrons to the extracellular environment, where it oxidizes whatever is out there. The problem here is that, not only is the cell producing far more ROS than a healthy cell, but worse, it's exporting those ROS, affecting the entire system (if the oxidized products make it to the lymphatic or cardiovascular system).

Still just a hypothesis, but it helps explain the paradox of cells that should be producing very little ROS, and the correlation of the accumulation of those cells with the rate of oxidative damage observed in aging.

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Posted 06 April 2005 - 04:58 AM

The problem here is that, not only is the cell producing far more ROS than a healthy cell, but worse, it's exporting those ROS, affecting the entire system (if the oxidized products make it to the lymphatic or cardiovascular system).


Whilst I feel the downregulation of autophagy is a far more senescence inducing factor than putative RHH cells, it is a problem which would have a simple remedy: target a suicide gene containing vector under the control of an ROS specific promoter (will only express lethal gene if the ROS concentration is high enough). In this way all of these cells could be selectively ablated. A variation on the above theme: delivered gene expresses allogeneic MHC type thereby targeting cell for T-cell mediated killing.

#33 manofsan

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Posted 09 April 2005 - 05:23 PM

I'm curious, how does one at the molecular level actually specify sensitivity to ROS concentration levels, or concentration levels of anything? Sorry for the ignorance, but I'm unfamiliar with the basic toolset components used to regulate things in this way.

Because I took chem engineering rather than molecular biology (damn!), I guess I'd like to break these things down as "toolset components" which can be systematically reorganized and reconstituted for whatever purpose we see fit (eg. anti-aging)

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#34

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Posted 10 April 2005 - 02:38 AM

See the section on inducible promoters at NCBI bookshelf. An excellent reference and educational resource.

In theory one could couple virtually any intracellular condition to induce (or suppress) gene expression. In the case of high ROS presence, a ROS-sensitive molecule (i.e. whose conformation is able to change in the presence of a particular ROS concentration) could be used as an adapter between a specific promoter and the transcription factors that initiate transcription.

#35 Michael

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Posted 14 April 2005 - 07:42 PM

All:

prometheus:

Jay:
The problem here is that, not only is the cell producing far more ROS than a healthy cell, but worse, it's exporting those ROS, affecting the entire system (if the oxidized products make it to the lymphatic or cardiovascular system).

Whilst I feel the downregulation of autophagy is a far more senescence inducing factor than putative RHH cells, it is a problem which would have a simple remedy: target a suicide gene containing vector under the control of an ROS specific promoter (will only express lethal gene if the ROS concentration is high enough). In this way all of these cells could be selectively ablated. A variation on the above theme: delivered gene expresses allogeneic MHC type thereby targeting cell for T-cell mediated killing.

Aubrey did consider ablation early on, but rejected it (see Chapter 14 of de Grey, The Mitochondrial Free Radical Theory of Aging, on the grounds that one of the most important cell types that would be targeted are muscle fiber segments. Unfortunately, as Aiken's group have shown (1), the loss of even a single 1 mm muscle segment (in this case, from mt dysfunction) leads to intrafiber atrophy and fiber breakage, so that the entire 1" fiber is lost. This could rapidly lead to significant sarcopenia.

Also, even normal, healthy muscle segments will be exporting significant superoxide during acute exercise, so controlling a ROS-specific promoter could be technically difficult or even impossible.

-Michael

1: McKenzie D, Bua E, McKiernan S, Cao Z, Aiken JM; Jonathan Wanagat.
Mitochondrial DNA deletion mutations: a causal role in sarcopenia.
Eur J Biochem. 2002 Apr;269(8):2010-5. Review.
PMID: 11985577 [PubMed - indexed for MEDLINE]

#36

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Posted 15 April 2005 - 05:58 AM

controlling a ROS-specific promoter could be technically difficult or even impossible



Good point. One would not want to trigger suicide in a cell that only transiently experiences a ROS spike. Therefore the regulatory mechanism would have to be triggered by a specific threshold of ROS that is sustained over an extended period of time (commensurate to a strong probability of such ROS levels inducing irreversible cell damage).

#37 jaydfox

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Posted 19 April 2005 - 12:48 PM

Another point to bear in mind is that the rate of autophagy must be coupled by the rate of biogenesis (as Kevin pointed out). So if we accelerate the rate of autophagy beyond biogenesis capacity then very soon the cell would run out of mitochondria and ATP production would plummet. I would agree with you that overly frequent autophagy would be selected against due to the excess energy requirements.

Is there any way to mathematically model the lifespan of a cell, based on rates of lipofuscin accumulation, and using rate of autophagy as a variable parameter?


I would say yes and also factor in the number of healthy mitochondria versus zombies, the concentration of ROS and ATP production. I would also invite Jay to do the modeling having shown that he is quite adept at these things ;)

Thanks for the vote of confidence, although I am quite skeptical about modelling something like this with the available knowledge pool. In a way, this reminds me of Dave Gobel's idea of a computer simulation for aging, to allow us to test interventions. A great idea, but decades ahead of its time. Imagine the many wrong conclusions we could have come to 30 years ago if someone had modelled the vicious cycle version of the mitochondrial free radical theory of aging, and then used that to justify a quite wrong (and expensive) direction in research.

Sure, it probably wouldn't have taken them long to break the model, so in that respect, the model would have been useful for refining the existing knowledge base.

I feel the same today. I don't think the model would be useful for much besides making predictions that we will then break in the lab, allowing us to refine the underlying theory (which is good). In the meantime, however, I don't think the predictions will be useful beyond that, e.g., to allow us to predict the efficacy of therapies (which is bad).

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Posted 04 August 2006 - 09:55 AM

Of note, Bergamini et al (1) in the latest issye of RR, make make mention that "easy and safe ways to meet the goals of the SENS agenda are already available" in their article on inducing mitochondrial autophagy as a means of removing damaged mitochondria. This notion was of course first discussed as a SENS alternative (neoSENS) right here in this forum over 12 months ago. See: "The next important neoSENS and proposed SENS target is the rate of mitochondrial autophagy."



(1) Rejuvenation Research Vol 9, No 3, 2006

#39 John Schloendorn

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Posted 04 August 2006 - 12:35 PM

This is and always was great idea, and I guess has a chance of working, but not a high enough chance to discard the backup plan (allotopic expression), because of what Michael explained in detail: If it is true that rho0 and other damaged mitos expand due to an intrinsic bias of the autophagy apparatus against them, then upregulating mitochondrial autophagy should amplify this particular problem, not reduce it.

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#40

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Posted 04 August 2006 - 01:34 PM

This is and always was great idea, and I guess has a chance of working, but not a high enough chance to discard the backup plan (allotopic expression), because of what Michael explained in detail: If it is true that rho0 and other damaged mitos expand due to an intrinsic bias of the autophagy apparatus against them, then upregulating mitochondrial autophagy should amplify this particular problem, not reduce it.


If it is true then it would be a matter of fine tuning the autophagy mechanism by increasing sensitivity and/or triggers in detecting damaged mitochondria - much easier to do than trying to overcome the numerous technological hurdles of achieving allotopic expression.

#41 ag24

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Posted 04 August 2006 - 02:20 PM

Would that that were so... but no one has yet come up with a specific, detailed proposal for preferentially targeting OXPHOS-negative mitochondria for autophagy. Until someone does, a gut feeling that it's much easier than allotopic expression is not much use.

#42

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Posted 05 August 2006 - 01:18 AM

Which implies there is a specific, detailed proposal for allotopic expression? (at the moment your proposal ignores the oxidation-based regulation hypothesis)

Targetting a transmembrane protein which alters its external conformation according to the internal redox state of the mitochondrion to alert autophagic mechanisms would present a simpler solution.

#43 ag24

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Posted 05 August 2006 - 11:40 AM

aaaarrgh - Prometheus it is exceptionally exasperating that you persist in accusing me of ignoring things that I have painstakingly addressed in the very same thread. Would you please be so kind as to go back to the start of this thread and actually READ it before posting such accusations?

As to the alternative:

> Targetting a transmembrane protein

Any particular transmembrane protein? Any particular targeting mechanism?

> which alters its external conformation

By any particular mechanism?

> according to the internal redox state of the mitochondrion

Sensed by any particular means?

> to alert autophagic mechanisms

By any particular pathway?

> would present a simpler solution.

Simpler to state, maybe...

#44 John Schloendorn

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Posted 05 August 2006 - 01:15 PM

Well, one has to admit, a high-performance gene therapy capable of delivering therapeutic AE to long-lived cells has not exactly been proposed in detail. But this is something the yet-to-be-designed redox-sensing transmembrane signaling protein would require, too.

#45

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Posted 06 August 2006 - 05:08 AM

aaaarrgh - Prometheus it is exceptionally exasperating that you persist in accusing me of ignoring things that I have painstakingly addressed in the very same thread. Would you please be so kind as to go back to the start of this thread and actually READ it before posting such accusations?

Is it not incumbent upon those who seek to engineer paradigm shifts to undergo such tribulations? ;)

As to the alternative:

> Targetting a transmembrane protein

Any particular transmembrane protein?  Any particular targeting mechanism?

Intramitochondrially should be sensitive to [ROS] (or anything else that is undesirable) conferring a conformaitonal change on the extramitochondrial side which may then alert an autophagic mechanism. We could look for these properties in existing proteins and fuse respective functional domains. Further refinement could be achieved by combinatorial methods (ie phage libraries).

> which alters its external conformation

By any particular mechanism?

Phosphorylation is a typical mechanism.


> according to the internal redox state of the mitochondrion

Sensed by any particular means?

There exist protein domains sensitive to [ROS].


> to alert autophagic mechanisms

By any particular pathway?

Increased binding on extramitochondrial side for vacuole vesicle proteins; Beclin-1 upregulation?
Anyway, here is a nice recent open access review on Mitochondria and Autophagy to exolore this notion further:
http://www.landesbio...d0941d2a64357ad

> would present a simpler solution.

Simpler to state, maybe...

No, dear Aubrey, simpler and faster to implement. We are in a race to save as many lives as possible after all..

#46 ag24

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Posted 06 August 2006 - 11:22 AM

Putting all that together and making it work without unmanageable side-effects seems to me to be enormously less simple and fast to implement than AE, not least given how little we know about how selective autophagy works.

But you don't need to persuade me of this. AE has been considered the Holy Grail of therapy for mitochondriopathies for 20 years, and in those 20 years virtually no progress has been made in treating these progressive degenerative diseases, so any promising new idea will be given a hearing by anyone in the field who thinks it's even slightly promising. If you think the sort of approach you outline is practical, you should be writing to people whose main job is treating those diseases and persuading them to try it. I forget where in Australia you're based, but if in Melbourne you might want to contact David Thorburn (Thorburn DR in PubMed).

#47 olaf.larsson

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Posted 07 August 2006 - 02:02 PM

Are you familiar with this 'protofection' of mitos?

From Rafal Smigrodzki - via the Extropy chat list - excellent news on progress made by the research group he works with:

Today our team confirmed our previous preliminary data showing that we can achieve robust mitochondrial transfection and protein expression in mitochondria of live rats, after an injection of genetically engineered mitochondrial DNA complexed with our protofection transfection agent. A significant fraction of cells in the brain is transfected with this single injection even though we so far did not optimize the dose.
This achievement has important implications for medicine: protofection technology works in vivo, and should be capable of replacing damaged mitochondrial genomes.


For those new to mitochondrial research and its relationship with aging and rejuvenation science, you can find more on Rafal's work here at Fight Aging! and details on the importance of repairing mitochondrial DNA damage at Aubrey de Grey's Strategies for Engineered Negligible Senescence (SENS) website. In short, this merits celebration! We're going be hearing much more about the repair of damaged mitochondria in the years ahead, firstly to cure specific age-related disease, and then to tackle general age-related damage to the mitochondrial genome.


http://www.fightagin...ives/000539.php

Here is a nice mito paper by the way:

http://www.nature.co...df/embor145.pdf

#48 ag24

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Posted 07 August 2006 - 03:48 PM

Very familiar, yes - Rafal spoke at SENS2, among other things. It's a technique that Rafal thinks may make AE unnecessary, and I'm certainly keen to see it pursued.

#49 olaf.larsson

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Posted 08 August 2006 - 12:28 AM

One thing that surprises me is that you mito guys seem to talk about mitochondria as they are described in the biology books; as sausage shaped static organells containing genomes which could cause cellular havoc when mutated. The real picture looks a little different as you might know, the mitos is rather a network of dividing and in fact merging mitochondial network structures. Defect mitochondria seem in fact be digested. I have tried to find more information about how this defect mitos are identified and digested but I have found very little information. This dynamical processes are of fundamental importence for the understanding of what is acctually happening, so why do you seem so unintressted of them? If defect mitochondria including defect mtDNAare digested, maybee it would be a lot easy to do enhance the hypothetical(?) mitoptosis pathways rather then making something more challenging like AE.

#50 Athanasios

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Posted 08 August 2006 - 01:08 AM

Defect mitochondria seem in fact be digested. I have tried to find more information about how this defect mitos are identified and digested but I have found very little information. This dynamical processes are of fundamental importence for the understanding of what is acctually happening, so why do you seem so unintressted of them?


Have you read Aubery's "Mitochondrial Free Radical Theory of Aging"

I think it was in there, hmmm...or maybe in the first SENS discussion, where this is discussed. I believe a theory was put forward that explained a way in which the respiration defected ones would live, not be digested, but pump out free radicals.

As far as lysosome function, I thought this research was interesting, but I need more research/background to understand all the implications. Supposedly rupturing lysosomes degrade mitochondria as well, creating an amplifying loop:

Prevention of oxidant-induced cell death by lysosomotropic iron chelators.

        * Persson HL,
        * Yu Z,
        * Tirosh O,
        * Eaton JW,
        * Brunk UT.

    Division of Pathology II, Faculty of Health Sciences, University of Linkoping, Linkoping, Sweden. Lennart.Persson@lio.se

    Intralysosomal iron powerfully synergizes oxidant-induced cellular damage. The iron chelator, desferrioxamine (DFO), protects cultured cells against oxidant challenge but pharmacologically effective concentrations of this drug cannot readily be achieved in vivo. DFO localizes almost exclusively within the lysosomes following endocytic uptake, suggesting that truly lysosomotropic chelators might be even more effective. We hypothesized that an amine derivative of alpha-lipoamide (LM), 5-[1,2] dithiolan-3-yl-pentanoic acid (2-dimethylamino-ethyl)-amide (alpha-lipoic acid-plus [LAP]; pKa = 8.0), would concentrate via proton trapping within lysosomes, and that the vicinal thiols of the reduced form of this agent would interact with intralysosomal iron, preventing oxidant-mediated cell damage. Using a thiol-reactive fluorochrome, we find that reduced LAP does accumulate within the lysosomes of cultured J774 cells. Furthermore, LAP is approximately 1000 and 5000 times more effective than LM and DFO, respectively, in protecting lysosomes against oxidant-induced rupture and in preventing ensuing apoptotic cell death. Suppression of lysosomal accumulation of LAP (by ammonium-mediated lysosomal alkalinization) blocks these protective effects. Electron paramagnetic resonance reveals that the intracellular generation of hydroxyl radical following addition of hydrogen peroxide to J774 cells is totally eliminated by pretreatment with either DFO (1 mM) or LAP (0.2 microM) whereas LM (200 microM) is much less effective.

    PMID: 12726917 [PubMed - indexed for MEDLINE]



#51 Athanasios

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Posted 08 August 2006 - 01:33 AM

ok, here is a small excerpt of what i was talking about

a mtDNA mutation may occur that lowers the respiratory capability of its host mitochondrion. That mitochondrion's lower level of respiration results  in a smaller proton gradient across its inner membrane. That, in turn, will translate into a lower concentration of harmful LECs in its immediate environment. This will result in a slower accumulation of damage to its inner membrane than is occurring in properly respiring ones. Such a mitochondrion will, therefore, preferentially still be intact when many of the cell's non–mutant mitochondria have succumbed to the degradation process hypothesised above. Thus it will be preferentially replicated . Repetition of this cycle will rapidly divest the cell of all its properly respiring mitochondria. I have termed this process "survival of the slowest," or SOS.


feel free to delete if this isnt supposed to be here

#52 olaf.larsson

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Posted 08 August 2006 - 08:41 AM

The "survival of the slowest" is an intressting idea. But isn't it so that a mutation would rather produce more ROS then less?

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#53

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Posted 08 August 2006 - 08:54 AM

In humans there is cell type that can exist without a nucleus but still requires mitochondria. It is called a reticulocyte and goes on to become a red blood cell (RBC) in the bone marrow. These cells are also unusually RNA rich, to compensate for the lack of a nucleus and nuclear DNA (the genes that would have had need to be transcribed already exist in the form of mRNA).

If the mitochondria in these reticulocytes do not have DNA (vis a vis AE) how will this alter their function in RBC production? Interestingly, reticulocytes would possibly present the best cellular platform in which to engineering DNA-negative mitochondria since it would eliminate an experimental variable (that of the nucleus).

This then leads to an alternative form of AE (AAE): that of introducing the mitochondrial nDNA encoded genes via a plasmid or artificial chromosome in a cell which has had these sequences knocked out in nuclear nDNA as a partial proof of concept.

We must not forget what the aim of AE is: to maintain mitochondrial efficiency and decrease ROS mediated nDNA damage. One hypothetical solution is AE but that relies on the assumption that mtDNA does not serve any redox-sensing function. If indeed it does not (which I doubt) then there are other vehicles from which mtDNA can be delivered (such as AAE). Of note is that even if AE works as advertised (once again, doubtful), the problem of malfucntioning mitochondria will need to be addressed and that can only be via enhanced mitophagy either by overall greater turnover or increased sensitivity of altered redox conditions.

#54 olaf.larsson

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Posted 08 August 2006 - 02:18 PM

Considering "survival of the slowest". After some searching I found the following. :

http://www.blackwell...ct.asp?id=17636

Many kinds of apoptotic signals are amplified by mitochondria and mitochondria-generated reactive oxygen species (ROS). Moreover, such signals can be produced by mitochondria themselves, resulting in opening of the permeability transition pore (PTP) and in release of the “death proteins” hidden in the intermembrane space. PTP opening (i) can be a result of ROS production and (ii) can increase the ROS level due to exhaustion of mitochondrial anti-oxidants. Long-lived PTP entail “a suicide” of mitochondrion (mitoptosis), dead mitochondria being eliminated by autophagosomes. Alternative mechanism of mitoptosis was recently discovered in our group. After treating cell culture by uncoupler and respiratory chain inhibitor, mitochondria (which under such conditions cannot form ATP but can hydrolyze it) were found to be concentrated in some regions of cytosol, surrounded by a membrane and then exiled from the cell. To study collective apoptosis, our group has developed a new method of confronting cell cultures. It was found that staurosporine- or TNF-treated cells sent a death signal to the nontreated cells. The signal in question proved to be mitochondrially produced hydrogen peroxide since (i) the treated cells release hydrogen peroxide, maintaining its concentration in the medium at level of several micromoles, (ii) the cell-to-cell signal transmission was interrupted by added catalase or by mitochondria-targeted anti-oxidant MitoQ, which, according to M. Murphy, et al. blocks the hydrogen peroxide-induced apoptosis.

Edited by wolfram, 08 August 2006 - 02:34 PM.


#55 ag24

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Posted 08 August 2006 - 09:11 PM

Mitochondrial fusion was actually very new in 1997 and 1998 when I wrote the BioEssays paper and my book - it was first discovered by Karen Hales in fly spermatogenesis (Cell 90:121). There is no doubt that if it occurs a lot in vivo it would slow down clonal expansion of mutant mtDNA if that expansion occurs via the SOS mechanism (or a variety of alternative proposals). Axel Kowald very recently pointed out that, conversely, it would in fact rescue a very early proposal that I had thought was totally dead, viz. that mtDNA clonally expands when it has big deletions because the smaller molecule can be replicated faster -- Aging Cell 4:273. However, the bulk of evidence in my view says that fusion is very common in cell culture, i.e. in 20% oxygen, but much rarer in vivo. I've been trying for a while to get people to test this directly.

The idea that mutant mitochondria make more ROS is very deep-seated but it is almost certainly not true in typical cases, because the commonest mtDNA mutations are ones that delete a chunk of the DNA, and that always means a loss of at least one tRNA gene, thus eliminating synthesis of all 13 mtDNA- encoded proteins. This will certainly prevent assembly of the respiratory chain complexes that make the most ROS, so ROS production will fall. This has duly been verified in rho0 cells (Singh's group, Mutagenesis 18:497). There are some complications -- complex II and AKG dehydrogenase can both also make ROS in some circumstances (see my commentary in RR last year), and conceivably the nuclear-coded parts of Complex I could partly assemble in the matrix and make ROS -- but the direct evidence seems pretty clear.

SDkulachev's mitoptosis (and related) work probably doesn't have much to do with all this. Apoptosis is generally a good thing, remember.

Prometheus's reference to reticulocytes is interesting and might lead to a few useful experiments. However, let's not forget that, unlike WILT, there is no need to make AE work in all tissues, only the ones where mutant mtDNA accumulates and cell therapy is very tricky. That basically means muscle (skeletal and cardiac especially) and nerves. (I should probably make this point in print somewhere, yes...)

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Posted 09 August 2006 - 12:59 AM

let's not forget that, unlike WILT, there is no need to make AE work in all tissues, only the ones where mutant mtDNA accumulates and cell therapy is very tricky. That basically means muscle (skeletal and cardiac especially) and nerves.


What about the consequences of mtDNA mediated damage in other critical tissues essential to life (for instance thymus, liver, etc.)?

Given that the delivery of transgenes in the generally non-mitotic tissues (CNS neurons, cardiomyocytes and myocytes) you mention is critical to any SENS-type intervention is there an initiative for how this profoundly critical step this may be accomplished? For example, do you have in your sights a method for tissue specific targeting?

More importantly, it is eminently clear that a vast chasm exists in the implementation of genetic modifications that can be made on single celled embryos (which are then propagated to all derived tissues) versus adult organisms. Does SENS in its present incarnation discriminate against the adult population or it relying on the emergence of delivery technologies from non-SENS initiatives?

#57 jaydfox

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Posted 09 August 2006 - 05:47 AM

Given that the delivery of transgenes in the generally non-mitotic tissues (CNS neurons, cardiomyocytes and myocytes) you mention is critical to any SENS-type intervention is there an initiative for how this profoundly critical step this may be accomplished? For example, do you have in your sights a method for tissue specific targeting?

As I understand it, one of the main tenets of the SENS platform, at least in practice (not from a scientific standpoint), is that emphasis is best spent on areas that are critically needed, but not otherwise being pursued. Assuming Aubrey could get some 8-figure sums for SENS research, he could easily blow a large fraction of it on pursuing gene delivery mechanisms. His tens of millions would be added the hundreds of millions already being spent by others seeking this critical "holy grail" of the next couple decades in gene therapy.

If anything, Aubrey doesn't need so much a detailed plan of how to invent such delivery mechanisms, as he needs good timelines on when they'll be developed by the mainstream research pipeline.

Of course, I could be talking out of my ass. But it seems to me, anyway, that gene therapy is something that already has lots of attention on it, and is being actively pursued.

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Posted 09 August 2006 - 07:43 AM

Jay, a survey of cutting edge, government funded science will always reveal that research is largely conducted for the sake of pure science rather than directly for clinical applications. Where we see research into clinical interventions it is mainly conducted by corporations whose research is shrouded in IP protection strategies and usually involves pharmaceuticals which seek to treat specific diseases afflicting Western society. Initial activity into gene therapy took some major blows on account of a lack of control of where genes inserted into genomes (and resulted in inducing cancer). Stem cell research has been retarded for well known reasons. Furthermore aging has not yet been accepted by the scientific and medical community as a treatable condition.

Consequently, the gene delivery methods that SENS would rely on such as whole body or specific organ/tissue transgene targetting are not being aggressively investigated. Specifically, there are three issues: how does one control the delivery of a transgenic construct (this is the new gene/s and its regulatory mechanism/s) to a specific site; how does one control the gene dose (what if one cells gets multiple versions of the construct); and how does one ensure an ongoing expression of the gene throughout the lifetime of the cell and once the cell divides. These problems are not intractable and some have been solved in vitro as well as in vivo. But there is no imperative as there is with SENS to bring such technologies to the forefront of scientific research with definitive clinical endpoints.

Therefore, either SENS must consider this issue as an actual SENS challenge/imperative or else change its approach entirely.

#59 ag24

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Posted 09 August 2006 - 07:53 PM

> other critical tissues essential to life

Those tissues certainly matter, but, as you surely know, they are nearly all found to harbour far lower levels of OXPHOS-negative cells than postmitotic cell types do. The simplest explanation for this is that cells that are easier to replace are set to be more prone to apoptose when compromised. On top of that, tissues that are mitotically competent can be targeted by (relatively!) simple cell ablation of the rather rare OXPHOS-negative cells, following which cell replacement will happen automatically by division of an OXPHOS-positive cell, so achieving the desired net removal of mutant mitochondria.

As for gene therapy, Jay is basically right, but a key additional point that answers your concern is that SENS explicitly emphasises mice first. We are already immensely better at gene therapy in mice than in humans, mainly for safety reasons. I had a whole session on gene targeting at SENS 2, remember, and I have a whole page on delivery options at sens.org. The technology of greatest interest to me at present is bacteriophage integrases, which have all the key requirements - sequence-specificity of integration site, large cargo, destruction of integration site by integration (giving dosage control) and low levels of random integration because the phages are double-stranded circular DNA. Its most ideal use may be in combination with zinc finger nucleases that can tweak a good integration site into a perfect one. Tissue-specificity is relatively easy, because sending a construct to tissues where it's not wanted can be made harmless by putting the genes under the control of endogenous genes that are only expressed in the tissues where the construct *is* wanted. Also, gene therapy is mercifully recognised as having huge therapeutic potential, so the curiosity-vs-goal problem only minimally applies to it.

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Posted 26 August 2006 - 07:01 AM

A fascinating debate on the feasibility - or not - of allotopic expression based on the unique physiology of neurons and other reasons..
http://www.technolog...e.aspx?id=17146
(see "Comments" section below Estep's dissent, authored by an eminently patient and insightful Dave Whitlock)




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