mitochondrial DNA therapy for aging

Treon Verdery treon at u.washington.edu
Fri Oct 8 16:38:57 EST 1993


	*All comments are welcome!*  *Please write!*

A few months ago I had what seemed to be an insight about mitochondrial
DNA therapy, longevity, and cancer.  Other activities in life have kept
me from devoting as much time as I'd like to these ideas.  I post them
now in hopes that they will provide some useful stimulation to real
researchers.  All copyright is reserved, and I specifically claim
ownership of the ideas for patent or other purposes.  Distribution is
set to USA only, please maintain this distribution on any followups.

>> Dr. Patil/Chris - I'll be amending this document with lots of postnotes,
>> indicated by the double >>.  This file consists of three parts:
>> the unsophisticated write up produced a few hours after having the basic
>> ideas.  This section contains some *drastic* inaccuracies, but you will 
>> get the drift of it.  The second part is a letter with some additional 
>> concept refinement, and the third part is a patentistic description of 
>> most of the above, plus many new ideas.  

>> This file is huge, but the crux of it is in the first 2-3K of words
>> so don't despair at the length.

>> I hope you find it thought provoking, and I welcome (a polite way to 
>> say urge!) any comments.

------------------------------------------------------------------------------
anyway, here's the big idea... maybe a BIG idea, sort of section three, 
subparagraph two of the holy grail of Big Ideas...

        A new class of pharmaceutical therapy for keeping       
        mitochondria from accumulating deletions during replication, 

also known throughout Europe and America as...
  
        "Eternal Youth"

(Wow what Severe Hype!  I'm not sure Hype can get any more grandiose than 
that... seriously though, I've been looking though Medline this evening 
trying to pin the idea(s) down and offer real support for some vague 
points.  you will find *many* errors, but read on!)

here goes:

Have you heard of the mitochondrial theory of aging?
As I vaguely remember it goes quite a bit like this:

Mitochondria do not reproduce sexually.  As your cells
divide mitochondria replicate themselves.  In order to create a new cell, 
'about' 100 new mitochondria have to be replicated.   
        
Mitochondrial DNA  is kinda like a 
simple circular plasmid with a Start sequence, an End
sequence and in-between sequences encoding protein
production.  These '100' mitochondria, each with its own mtDNA, 
replicate as individuals.  Each of these '100'
are subject to point mutations and deletions during the life of the cell.  

Mitochondrial DNA  is unusually susceptible to deletion at specific weak 
points.  So much so that while about 100%
of a newborn's mitochondria will be doing their job, only 
20% of a 60 year old's mitochondria will be working.
Each cell has much less energy available for protein synthesis in general.  
This has been specifically linked with changes in tissue behavior and 
senesence.

Particularly exciting is the observation that damaged
(smaller/bearing deletions) mtDNA is replicated
*preferentially* 'simply' because there is less stuff between the Start and 
End sequences of the mtDNA!  
Nonfunctional mutations accumulate in that cell's line with each cell 
division, and with each generation of new cells there is a (modified) 
exponential rise in the number of dysfunctional mitochondria per cell.

AND NOW FOR THE PUNCHLINE

I'm pretty darned sure I've figured out a way to make mtDNA 
(mtDNA specifically) less sensitive to deletions and/or cause the replication 
process to avoid replicating mtDNA that is missing (specific) big chunks.
Perhaps in a pill no less -no viruses or even  nanotechnology ;-)

It'll seem easier to swallow (gulp!), if I describe a couple functioning 
concepts that are commercialized now, and operate using methods I'm going 
to borrow:

Component idea 1)

Zovirax is an anti-viral drug for herpes.  here's how it works:

Viruses that Zovirax targets like to make themselves
out of specific amino acid sequences.  during replication they find these 
sequences floating around and incorporate them into structure.  
People who take zovirax tablets are really taking pills filled with a 
specific amino acid-sequence that the virus wants. ALMOST.  The Zovirax 
sequence contains an amino-acid that is the L-isomer.  (er, maybe) The cell 
produces all the little virus copies, but none of them work - that is, none 
of them can infect a second generation of cells.   The drug
is 'safe' because the rest of the body has little interest in the 
tricked-out amino acid sequence, and because virii reproduce quickly and 
are the preferential uptakers. 

Component idea 2)

In the Bad Old Days there was a class of sedatives called Bromides.  
Essentially, people would take Bromine compounds (KBr) until some of their Cl
had been replaced by Br and the electrical potentials in their nerves had 
changed a bit.  Unfortunately, as the Br built up, eventually all sorts of 
stuff would go out of whack.

and here's component idea 3)

Cellular chemistry is traced with radioactive isotopes.
Scientists have recently taken specific note than some
tritiated proteins (tritium instead of H) diffuse across
mitochondrial membranes much more slowly(increased mass).  The cellular
chemistry that the radiolabelled compounds execute is, however, generally 
the same. At sufficient mass increases however, the chemical 
functionality radically changes.


*******Now, as best as I can explain it so far here's the idea:

Assemble amino acid sequences that have preferential
uptake for mtDNA replication. 

'weight' these sequences so that their total mass
is the same as a naturally occuring sequence, BUT
make some of the individual proteins out of Heavier, 
and also Lighter *stable* isotopes. ((**This is the drug.**))

an example:

normal original protein fragment: CGGACCTAGT
Heavy Gaunidine version: CggACCTAg

(lowercase = deuterium as substitute for H, or perhaps
C13 for C12, or perhaps some O isotope
let's pretend that the T's have some lighter stable isotope(N) so that the 
total mass of this sequence is the same as the normal original.)

I have to pause and add some helpful additional concepts:

One of the reasons mtDNA accumulates deletions is that
some places along the amino acid sequence are fragile, that is,
they like to drop out under replication.  Simplistically, there are some 
weak links in what is otherwise a strong chain.

(NOT! -my how learned this all sounds. free grains of salt: .. . ... )

Bacteria that live in deep sea thermal vents thrive at
temperatures above 212 F.  These bacteria have many of the same enzymes 
and protein chemistry cycles as cool cells, their proteins are almost 
exactly the same, however, some of their proteins are less 'ornate', 
less 'sticky'; with fewer opportunities to pick up thermal motions and 
cross-link.  The rapid folding-and unfolding of hot-cell proteins is 
also somewhat different...

Some scientists speculate that hot bacteria were first, and that 
proteins evolved stickier, more ornate, more-cross-linkable molecular
forms to adapt to cooler environments.  

Just as a floating concept: imagine a partially deuterated enzyme as much 
more sluggish at low temperatures...normal at higher temperatures...
obviously the things the enzyme reacts with would have trouble w/the 
higher temperatures though...

>> sudden thought: is there a dueterated PCR patent enzyme work around?
 
It strikes me that there are possible strategies for using light and heavy 
amino acids to 
        
        1) bog down/break the replication of specific   
            misbegotten (deletion-flagging) amino acid sequences

        2) protect weak spots from damage, but not from
            replication (w/a slight differential of charge 
            potential)

        3)  all kinds of other stuff that messes with RNA

A typical 1) strategy:
A Grungy strategy to bog down shortened mtDNAs is this: After dosing 
the organism with isotopically peculiar amino acid sequences the 
'normal nonmutant' mtDNA has a 'normal' mass and reactivity.  
Those mtDNA that have experienced typical deletions at the 'weak links'
have unusual isotope concentrations that affect the behavior of the 
protein replication:

ok-to-replicate version:
CgAgg + gCTCC = thermodynamically ok to replicate

but:

CgAgggggg + GCTCCCCCC = thermodynamically slow/prohibited as all the heavies 
cluster together and/or  are limited by diffusion speed/availability of 
bits with all those heavy ggggs.

I guess you could call this "putting the wrong size pins on
the zipper" - when you get the big pins, and the big zip
there isn't enough energy/room to pull it any farther - but if you have at 
least 1 small pin it will work.  (Iffy indeed, but I can think of an 
experiment to test it - hoping to find the components of the experiment 
on medline). 

An interesting alternative to "wrong size pins on the zipper" is 
"slower pins on the zipper reduce the risk of speedy accidents", or perhaps, 
"I'm only going to zipper you if your start sequence includes light/heavy 
isotopes - and deletion-prone versions versions lack this 
light or heavy sequence.

In fact, once you begin thinking about it, there are a lot of 
"ordinary" things that could happen to work on this problem - without 
any isotope naughtiness at all.

I guess what is most important from my perspective is the idea that stable 
isotopes, with some degree of clever manipulation, can be used as amino 
acid replication/synthesis modifiers.  
Now I have a lot of reading to do! (Did you notice that I 
didn't mention mRNA hardly at all?)

-Treon


Here are some medline references at the end for background and stuff.  
I should have copied more but they seemed long enough as it was.

you can search medline anytime you want to follow your own fancies - 
(you probably know this address) just:
telnet uwin.u.washington.edu

#1 is an example of Tritiated compounds doing the same chemistry,
but 3-9X slower (within mitochondria (but not reproduction) no less!)

#2-4 are lame, but exploratory examples of mtDNA
 accumulating damage at extreme exponential rates and how this affects 
cellular energy and the respiration cycle.
my info comes from a dimly remembered review article that was much clearer...

P.S.  BOO!!

be my friend!  write back!!
(you are eligible for a medal for reading this far...)

#1
Loss of tritium from a substrate is often used to estimate
                the rate of dehydrogenation.  However, loss of 3H may be much 
		slower than loss of H because of the tritium isotope effect.
                In order to assess the impact of the tritium isotope effect,
                loss of 3H from the C-5 position of proline during
                dehydrogenation by rat liver mitochondria and bacteroids from
                soybean (Glycine max [L.] Merrill) nodules was compared with
                appearance of 14C in products of [14C]proline
                dehydrogenation.  Incubations were carried out in the
                presence of o-aminobenzaldehyde (added to trap the initial
                product, delta 1-pyroline-5-carboxylate).  The fraction of
                total 14C products trapped by o-aminobenzaldehyde varied from
                0.07 to 0.75 depending upon experimental conditions.  With
                rat liver mitochondria, dehydrogenation of [14C]proline was
                between 3.27 and 9.25 times faster than dehydrogenation of 3H
                proline, depending upon assay conditions.  Soybean nodule
                bacteriods dehydrogenated [14C]proline about 5 times faster
                than [3H]proline.  We conclude the following: (i) the rate of 
		proline dehydrogenation may be greatly underestimated by the
                tritium assay because of the tritium isotope effect, and (ii)
                the 14C assay may underestimate the rate of proline
                dehydrogenation if it is assumed that o-aminobenzaldehyde
                quantitatively traps delta 1-pyrroline-5-carboxylate under
                all conditions.  The simplicity of the tritium assay makes it
                attractive for routine use.  However, its use requires
                determination of the tritium isotope effect, under the
                specific conditions of the assay, in order to correct the
                results.  The considerations discussed here have broad
                applicability to any dehydrogenase assay employing tritium
                loss.


#2

The analysis of human skeletal muscle mitochondria revealed aprogressive 
		decline in mitochondrial respiratory chain
                function with age.  The activities affected to the greatest
                extent were those of complexes I and IV which were decreased
                by 59% and 47% respectively between the ages of 20-30 years
                and 60-90 years of age.  Quantitation of the 5 kb 'common'
                deletion of mtDNA using PCR revealed a progressive
                accumulation with age, from approximately 1 in 100,000 at 21
                years to 1 in 10,000 at 56 years and 1 in 5000 at 78 years of
                age.  The low absolute levels of this mutation are unlikely
                to contribute significantly to the observed mitochondrial
                dysfunction.


 Previous theories of aging based on somatic mutation
                neglected mtDNA, which has a high propensity for mutational
                error.  Knowledge of yeast mtDNA mutations and their
                functional effects, and of human mtDNA mutations identified
                in the mitochondrial cytopathies, provides for a concept of
                aging based on the cumulative effect of mutations affecting
                human mtDNA.  An essential feature of this concept is
                heteroplasmy, representing mixtures of normal and mutant
                mtDNA at the cellular and mitochondrial level, resulting in a
                "tissue mosaic" of focal bioenergetic deficits.  Direct
                evidence for the concept is provided by (i) focal loss of
                staining for mitochondrially encoded enzymes, such as
                cytochrome c oxidase, in tissues of aged individuals (humans
                and rats) and (ii) an age-related increase in deletional
                mutations in mtDNA demonstrable by application of the
		polymerase chain reaction to DNA templates from individuals
                of different ages.

Some mutations in mitochondrial DNA (mtDNA) causing a number
                of neuromuscular diseases are suggested to arisespontaneously
		during the life of an individual.  To
                substantiate the extent and the rate of these somatic
                mutations, mtDNA specimens from post-mortem human heart
                muscles of subjects in differing age groups were hydrolyzed.
                8-Hydroxy-deoxyguanosine (8-OH-dG), a hydroxyl-radical adduct
                of deoxyguanosine, in mtDNA, was quantitatively determined
                using a micro high-performance liquid chromatography/mass
                spectrometry system.  In each specimen, the mtDNA with a 7.4
                kilo base-pair deletion was quantified by the kinetic
                polymerase chain reaction method.  In association with age,
                the 8-OH-dG content accumulated exponentially up to 1.5% with
                a correlative increase in the content of the deleted mtDNA up
                to 7%.  Clear correlation between the 8-OH-dG content in
                mtDNA and the population of mtDNA with a deletion (r = 0.93,
                P < 0.01) gives insight into the mechanism for the generation
                of a large deletion.  These results indicate thataccumulation 
		of somatically acquired oxygen damage together
                with age-associated mutations in mtDNA which lead to
                bioenergetic deficiency and the heart muscle weakness are
                inevitable in human life.
-------------------------------------------------------------------------------
End of the email extraction from the first letter.

Here is an extraction from a second letter that I wrote to a friend.  


Anyway, Hello!   Thanks for asking if you could pass the idea on
first - I never bothered to get it notarized so I'll do that today
so that you can pass it on to Peter say Thursday.  It is a building
thrill to have someone else interested and to hopefully add some
explorations and accuracies.  Makes me nervous too!  That version 
I sent you was written about 4 hours after the original inspiration
So, um, it'll read really dumb to someone who knows what they
are talking about.  I think it has been 6-8 weeks since I wrote that,
and have learned all sorts of shoring-up and eroding-down influences
to the central idea:


And now... some inferences from the literature:

Plus:
* There are specific common deletion sites in mtDNA that result in 
  large, unique amino acid sequences suitable as labels 

* the idea is obviously portable to other genetic and protein
 pharmacological things; RE aging, it also has direct application
 to other aging theories/aging markers notably the telomeric
 marker/tape leader theories and what I'd call the 
 psuedocaloric restriction methionine displacer theory

* A similar idea/set of ideas has never appeared in the abstracted
  literature/patent databses I've looked in

* Rats forced to live out their lives on 30% D2O are normal
  (an incredible puzzle there I'd say)

Minus:

* people with progeria, a very unusual rapid aging syndrome do not
  have mtDNA problems at all (secret plus: they do have shortened
  telomeres proportional to the shortening seen in people of their
  apparent physical age!)

* Cancer research suggests that that targeting 
   is very difficult; then again, maybe I've lucked out
   and thought of a harmless-unless-cancerous mechanism.  or even
   a cancer prophylactic of a different nature than antioxidants
   sort of like invisible chemotherapy.
>> -distributed stable isotopes lie in transparent wait until cancer/other
>> bioprocesses concentrate them and 1) retard them 2) signal them.
>> Advantage: minimal/no toxicity excaept at active concentration/comnination
>> sites

(Andrew... the minuses are turning into positive wild speculation!
 This is good - but I started out wanting to write a balanced treatment
 for You and Peter)

More Wild Speculation

* currently existing gene therapies could add another degree
  of 'facility' by influencing the residence times and
  stabilities of introduced genes, as well as marking sites for 
  secondary therapies. The whole idea of tagging, signalling, and modifying
  cellular stuff without active chemical groups just floats there...
  suggesting wild stuff like cellular early-early-early diagnoses
  dianoses based on excretion/assay of oddball Isotope-loaded amino acid
  sequences.  

  The various forms of matching and concentration
  that I know about in cells suggest statistical 'control'(?) of how much
  of what accumulates where.  Nucleic acid dynamics are so bizarre
  that there are many different sorts of active and inactive structures
  to tweak on...

  ...I guess I'm just really turned on by the idea of chemicals that
  in low concentrations mimic perfectly their normal isotope
  cousins, but at times of unusual concentration due to replication,
  or other cellular protein production can influence cellular processes.
------------------------------------------------------------------------------

End of text extraction from letter to Andrew Greenberg.  

Here are some notes, wrtten today, June 23, 1993 about the whole
thing...Ideas not stated here may have been created at an earlier date than 
today, but are in some other paper form I don't have access to right now.

A* Ok, so we've got a stable isotopes incorporated into nucleic acid sequences
or other, even *non-protein* polymers concept going.  I'm going to use the word
stable isotopes generally, but under the right conditions sufficiently 'cool' 
radioactive isotopes might be useful in producing the same kind of 
isotope-effects I'm expecting stable isotopes to be adequate for. 

These stable isotopes, lighter or heavier, or more homogenous-in-percentage 
than as found in a typical situation are instrumental in:

Causing rate of reaction changes 
Influencing thermal lability of proteins/polymers
Creating clines and/or thresholds of reactivity/bonding intensity/membrane 
    transport
Changing the physical spacing/volume dynamics of proteins/polymers
Causing absorption and emmisivity changes in response to light/radiation

These are very general, but combining them we get:

A0*: The facility of slowing/speeding up, stopping, forking, or breaking 
transcription, replication, or expression processes in relation to DNA and 
RNA, or other information containing polymers that are partially or completely 
composed of atypical stable isotopes.

This includes the possibility of creating proteins/polymers of the same 
molecular mass as a typical protein/polymer, but with both atypical heavy and 
light isotopes combining to give the typical mass.

and...

The specific use of A* + A0* to affect the way in which mitochondrial DNA is 
involved in replication, or for that matter nuclear DNA/RNA, esp. telomeres.  
I note here also the idea of atypical isotope biomolecules as differing 
structural elements so that they hold up differently over time - independent 
of replication.  

Isotopes might be introduced as a specific actor in response to an acute 
condition or a specific interest in action.  Alternatively, isotope loading 
could occur prior to an acute interest, perhaps as a baseline, or in the case 
of several paragraphs that follow, as a kind of prophylactic loading action
that would later be brought to the point of activity by significant grouping 
of the isotopes.

Specific uses of A0* to signal, label, or act upon DNA/RNA activities in a 
living organism that are of interest: 

	A disease, cancer, or other bioactivity is associated with the 
transcription, recombination, expression, or concentration of amino acids, 
proteins or amino acid sequences.  Could be a gene thing, or just a protein 
product.  As a result of this process, Numerically variable, 
significant-to-action 
quantities of isotopes are matched and/or otherwise grouped together, either 
within new molecules, or just as a nonbonded percentage. 

A1*: This togetherness or grouping:

	1)could cause a change in A* + A0* as therapy or create an intermediary
	 to to #2:
	
	2)could be noted as some sort of excretion product or other 
detectable/assayable concentration, and used as a label and/or signal as to 
the type or location of the disease/other bioactivity in progress.  These 
might, or might not, contain an element of signal coding to them to reveal 
specifics about the process that caused the grouping.
	
	3) could be used as a signal to an additional compound for binding or 
location information.  In addition to the straightforward case, here is one of 
several plausabilities: weaker bonding by the Isopically different amino acids 
on one side of a paired sequence might allow a different chemical to have 
special reaction/recognition possibilities with the isotopically normal side 
of the paired sequence because of changes in bond strength or shape. 
	
A2*: In a nongrouped, nonactive  situation, information could be noted, or 
accumulated as history in a DNA/RNA/protein product in vivo as a result of the 
isotope exchanges during the cellular process-history.  A plausibility is the 
embedment of data or compressed data for the use of introduced, or induced, 
cellular processes.  Because the coding involves isotope substitution and not 
active chemical groups, it should be essentially transparent to routine 
cellular chemistry.
In addition to activities that read this extra coding, the historical path of 
cellular disease might be specifically backtracked, or mechanisms might be 
used to encode previous in vivo states in the isotopic coding area.  


A3* an  example of isotopic substitution used in a viral therapy might be as 
follows:
	Let's write this part later huh?


B* The use of A* mechanisms in essentially nonbiological, ex vivo applications:

B1* Others have mentioned the use of amino acid sequences as masks and/or 
deposition surfaces or guides for making semiconductors and special micro-scale
machines - mechanical, chemical, electric, or photofunctional.  

By creating amino acid sequences or other polymers with areas of isotopic 
substitution, all of, or areas upon these masks/templates/sites might have an 
additional range of facility in processing that involves temperature, bonding,
or mass-sorting techniques like electrophoresis or centrifugal/acceleration 
effects that can be easily inferred from A* and even the loading mechanisms 
of A0*. Differences in spectral/charge properties as energized by 
light/radiation may be considered an additional area of facility also.  

B2* by creating a standard set of amino acid/polymer sequences with different 
areas of substitution, a kind of shape set is created. 

Example: standard sequence --> PCR for a plurality of identical seeds for 
next step (say 5 seeds for simplicity of example) -->amplification of 
standard sequence; seed one has special light/heavy aminos/mers on one end, 
seed two on the other end, seed three in the middle, seed 4 all heavy, light, 
whatever.  In polymers of width, angle patterning of isotopes might be 
constructed.     

Fabrication can be facilitated by:

Placing elements of this shape set together, most likely on a substrate, and 
then sorting/removing as necessary either the isotopically atypical portions 
or the other portions using the extra facility suggested by A* and  some A0* 
concepts.

B3*: Also, the possibility of using mass-sorting effects to, for example, spin
certain patterns by the facility of mass distribution, or electrophoretically 
draw with/patternize the amino acids/other polymers and create 
templates/masks/deposition sites so that desired designs are created.  

B4*: Photo effects might allow for selective absorption and [ablation or 
fixation] at atypical isotopic sites or molecules.  Photoeffects with 
emission-involving isotopic difference and feedbackto the photosource that 
causes special ablation or fixing may also be used to enhance patternmaking 
or fabrication of devices.

B5: Building up effects like arborization might take place, followed by 
removal or deposition of other active chemicals/semiconductors/etc. by the 
A* facility.

Ok, that's it for now.  It is 11:43 PM on 6/23/93 - Treon Verdery

>> B6: nanotech micromachines that make use of [matching/accumulation etc.]
>> or structural [from erosion to shaping to spacing] concepts, or 
>> photofunctional, or information carrying through isotopes, isotope 
>> laden polymers, or isotope layers, patterns, or tracks.

------------------------------------------------------------------------

Mr. Patil/Chris - thanks for reading all of that. I'd love a response by
email, but since the original text document is so large talking might
be more convenient.  If you like, call me at the University of Washington
at (206) 685-7831, and I'll call you back so it's on my dime.

What do you think?

-Treon




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