Issue 1

December 2007

Welcome to the first issue of the Methuselah Foundation’s newsletter! We will be

sending out this newsletter each quarter to keep you, our valued donors and supporters, up to
date with solid progress in the Foundation's efforts to advance the engineered reversal of the
molecular and cellular damage of aging - and also of the complementary efforts of scientists
from all over the world.

Donations Tripled Until End of 2007!

Thanks to the challenge pledges made by
PayPal co-founder Peter Thiel and 300 Member
Michael Cooper, donations to SENS research are
currently tripled in value – but only until the end of
2007. If you’ve been considering making a
donation, now is the time!
All donors giving at least $100 are also
eligible to receive a free, signed copy of Ending Aging, Dr. de Grey and Michael Rae’s
recent book on rejuvenation science.

SENS3: A World-Class Success

September 2007 saw the third SENS conference in Cambridge, England, which once
again featured world-class research from an outstanding panel of scientists. The SENS
conference series, although new, is now firmly established as the most exciting in the field,
and the pace of discovery is already increasing.
Videos of the sessions are now available at the Foundation’s website, alongside
material from previous SENS conferences and the Edmonton Aging Symposium, organised
by Foundation volunteer Kevin Perrott.

Upgrading the Methuselah Foundation Website

If you haven’t visited www.methuselahfoundation.org recently, you may be surprised
to see how much has changed – we welcome feedback and suggestions on our new look and
features. Among many other additions we now have an integrated Amazon store, all
purchases from which help to support the Foundation.

In This Issue
We are currently sponsoring research in two of the seven strands of the SENS
program; the preventing the harm caused by mitochondrial mutations (MitoSENS) and
degrading damaging long-lived cellular debris (LysoSENS). Although this work began only
recently, our teams have already seen interesting results and are moving forward rapidly.
Their work is detailed in the Progress Report section of the newsletter, accompanied by
highlights of related work from other groups.

Next, Michael Rae presents a selection of key advances in areas important to SENS,
accomplished by laboratories outside the Foundation. Dr. de Grey then describes a selection
of prospective projects for which the Foundation is now actively marshalling funds.

-1-
Issue 1
December 2007

Finally, Elliot Bergman provides us with an update on the Foundation’s first funding
program, the Mprize, whose total value is now well over $4 million.

Contents

3 Progress Report
3 MitoSENS Mark Hamalainen
4 MitoSENS: Several Promising Approaches Michael Rae
6 LysoSENS John Schloendorn
7 LysoSENS: Tackling Tau Michael Rae
8 SENS Around The World Michael Rae
9 Current Goals Aubrey de Grey
10 Mprize Status Elliot Bergman

We hope you enjoy the newsletter, and welcome any comments or suggestions for the
March issue!

Newsletter Editor: Ben Zealley (ben@sens.org)

-2-
Issue 1
December 2007

Progress Report
MitoSENS
Mark Hamalainen
Cambridge University / Quinze-Vingts National Center of Ophthalmology, Paris

The ultimate goal of MitoSENS is to transfer all 13 human mitochondrial protein-

encoding genes from their present home in the mitochondria to the protected cell nucleus.
This would represent a panacea for mitochondrial DNA related disease, including the gradual
loss of mitochondrial function that occurs during aging, by allowing the genes to function in
the nucleus even when damaged or destroyed in the mitochondria.

Plenty of examples exist in nature: intracellular gene transfer from the mitochondrial
DNA to the nucleus has occurred throughout the evolution of eukaryotes. Some
mitochondrial genes, such as subunit a (A6) of ATP synthase are rarely found in the nucleus,
however. One exception is found in the algae, Chlamydomonas reinhardtii, which in
evolutionary terms has only recently transferred the A6 gene to its nucleus.

In practice, the transfer of mitochondrial genes to the human nucleus (to produce what

-3-
Issue 1
December 2007

is called allotopic expression of the genes) has proved to be anything but straightforward.
We’ve recently found that targeting messenger RNA (mRNA) to ribosomes proximal to
mitochondria may be critical to achieving successful allotopic expression. Fibroblasts
carrying the NARP or LHON mutations were rescued by this strategy, demonstrated by
restoration of their ability to grow in galactose media. Experiments in 143B osteosarcoma
cells have failed to replicate this result. However, it is possible that cancer derived cell lines
are poor models for studying allotopic expression as mitochondrial dysfunction is thought to
be important in carcinogenesis.

Our current primary goal is to explore methods for reducing the toxic side effects of
allotopic expression, by reducing protein hydrophobicity and reducing or regulating
expression levels. Plasmids have been constructed containing a nuclear version of A6, each
including up to seventeen naturally occurring polymorphisms that reduce hydrophobicity. A
plasmid containing the human CF6 promoter, which drives expression of a mitochondrial
protein associated with Complex V of the respiratory chain, has been constructed and is
being tested.

The ability of these methods to eliminate toxicity will be tested by expressing the
constructs in cells that can be differentiated into a non-dividing state. The second objective
of our work is to achieve co-expression of multiple mitochondrial genes with the eventual
goal of complementing rho zero cells (which lack any mitochondrial DNA at all).

Further Reading
Holt IJ, Bokori-Brown M, Hamalainen M. “Allotopic expression: mitochondrial to nuclear gene transfer.”
Rejuvenation Res. 2007 Sep;10(Suppl1):S32(Abs53).

MitoSENS – Several Promising Approaches

Michael Rae
Research Assistant to Dr. de Grey

Excitingly, work on allotopic expression and related "engineering" solutions to

mitochondrial mutations is also taking place in other labs around the world, using a variety
of different biotechnological approaches. Several groups reported their progress in these
areas at the third SENS conference.

Dr. Marisol Corral-Debrinski, of Paris' Quinze-Vingts National Center of

Ophthalmology, presented her latest results in optimizing the import of allotopically-
expressed mitochondrial proteins by shifting their production site closer to the mitochondria
themselves. This technique is hoped to overcome a primary challenge in allotopic
expression: how do the proteins now produced by nuclear DNA get back to the mitochondria
where they are needed? She used this technique to insert an allotopic version of the defective
human gene that causes the mitochondrial disease Leber’s Hereditary Optic Neuropathy
(LHON) into mouse retinas, reproducing the same cell loss and abnormal lack of cell
communications branching that appears in the disease. She next hopes to take this to the next
level, and cure the disease in mice by introducing the healthy gene.

-4-
Issue 1
December 2007

Another way to ease the import of twisted-up (and thus hard to import back into
mitochondria) allotopically-expressed proteins is through the introduction of special 'bracing
bars' called inteins into them, to hold apart their snarling bends and kinks - an idea first
proposed by Dr. de Grey in a 2000 paper in Trends in Biotechnology. After some preliminary
work done by Japanese scientists, the University of Zaragoza's Dr. Antonio Enriquez has
now picked up the ball, and described his early work with mitochondrial protein inteins
during the meeting.

In addition to the work on allotopic expression, two presentations at SENS3 covered

progress on entirely novel ways of overcoming the problem of mitochondrial mutations. Dr.
Volkmar Weissig of Northeastern University reported the import of whole new mitochondria
into the cell, an effect that had actually previously been reported by Jerry Shay’s group in
1982 – and then forgotten! – but which Dr. Enriquez was able to confirm in his own lab.

Finally, Dr. Samit Adhya of the Division of Molecular and Human Genetics at the
Indian Institute of Chemical Biology is pursuing yet another innovative approach, in which
he proposes to dispense with the need for mitochondrial DNA altogether, by instead
providing the mitochondrial protein-making machinery directly with the "working
instructions" (messenger RNA) that it normally receives in the form of a transcribed copy of
the mitochondrial DNA.
Dr. Adhya’s work borrows a trick used by a single-celled organism called
Leishmania tropica to move messenger RNA into the mitochondria. He provided evidence
that RNA imported into the mitochondria of human cells using this technique works as it
should by introducing antisense RNA - RNA that is designed as a mirror-matched copy of
the original, to which it binds and which it thereby inactivates. Introducing this antisense
RNA exerted effects similar to those seen in people with defective copies of the genes that he
had effectively kept from functioning as sources of mitochondrial proteins, supporting their
functionality as silencing RNA.
As with Dr. Corral-Debrinski's work, the next step will be to introduce functional
RNA into animal models with dysfunctional mitochondrial genes, to see if it can restore
normal function.

Further Reading
S. Ellouze, C. Bonnet, S. Augustin, V. Kaltimbacher, V. Forster, M. Simonutti, J-A. Sahel, M. Corral-
Debrinski. “Allotopic mRNA localization to the mitochondrial surface: a tool for rescuing respiration
deficiencies.”
Rejuvenation Res. 2007 Sep;10(Suppl1):S24(Abs 23).

J.A. Enriquez. “Inteins and allotopic expression of mtDNA encoded proteins”

Rejuvenation Res. 2007 Sep;10(Suppl1):S28(Abs 36).

V. Weissig, E. Katrangi, S.V. Boddapati, G.G.M. D'Souza. “Manipulating (rejuvenating?) the mitochondrial
genome”
Rejuvenation Res. 2007 Sep;10(Suppl1):S50(Abs 124).

S. Mukherjee, B. Mahata, B. Mahato, S. Adhya. “Use of a parasite-derived protein complex to modulate the
function of mitochondria in human cells.”
Rejuvenation Res. 2007 Sep;10(Suppl1):S19(Abs 2)

-5-
Issue 1
December 2007

LysoSENS
John Schloendorn
Biodesign Institute, Arizona State University

As we age, our bodies produce many types of junk molecules as a side-effect of

normal functioning. For some of these molecules, no efficient removal system exists, and
their accumulation gives rise to deposits of intracellular junk. This leads to age-related
storage disease. The major diseases of this type are Alzheimer’s disease (beta-amyloid
plaques in the brain) atherosclerosis (7-ketocholesterol/7KC, a cholesterol derivative in the
artery wall), age-related macular degeneration (a compound called A2E in the eye), and
diabetes (AGEs, sugar-derived protein-modifications, throughout the body). Medical
Bioremediation is the field of research seeking environmental microorganisms that break
these molecules down, whose gene products can then be harnessed for therapy in humans.

For the past two and a half years, the Methuselah Foundation has been funding
research into Medical Bioremediation at Arizona State’s Biodesign Institute in Tempe,
Arizona and at Rice University, Austin, Texas. When these projects began in the summer of
2005, there was no strong evidence to suggest that any enzymes or organisms degrading
intracellular junk existed in nature. But this did not intimidate Methuselah Foundation
research volunteers Jacques Mathieu, Mark Hamalainen and John Schloendorn. With very
limited funding and compensation, they visited leading environmental resarchers Pedro
Alvarez, PhD and Bruce Rittmann, PhD at their labs and began culturing experiments. All
three reseach volunteers had successfully cultured 7KC degraders before the end of 2005.
This initial success laid the foundation for the scaling-up and professionalization of
these projects. In 2006, Mathieu and Schloendorn enrolled as PhD candidates at their
universities and won departmental support. Hamalainen has now enrolled at University in
Paris, France to pioneer Mito-SENS as a second Methuselah-Foundation funded effort.

2006 saw further characterization of the 7KC degraders and corroboration of the
results. In summer 2007, six undergraduate research assistants and one additional PhD
candidate joined the effort at Biodesign Institute. They helped with synthesizing additional
target compunds, such as A2E and CML (a major AGE). This veritable army of volunteers
also was able to culture six independent degraders of CML, and identify two enzymes which
break A2E in different ways.

Today, researchers at the Biodesign lab are working on identifying the enzyme
initiating the breakdown of 7KC and CML. They are also characterizing the A2E-degrading
enzymes further, and are preparing to move them into a cell model of age-related macular
degeneration for initial safety and efficacy testing. Various other projects at the Tempe lab
are at earlier stages. Novel targets include artificial lipofuscin and the infamous glucosepane
AGE-crosslink.
Meanwhile, Mathieu at Rice University employs a modern microarray-based
approach to identify genes that get expressed in the presence of 7KC, but not other nutrients.
Such enzymes are likely involved in the breakdown of 7KC. This has the potential to
characterize the complete genetics of 7KC degradation in one elegant experiment.

-6-
Issue 1
December 2007

Further Reading
Rittmann BE, Schloendorn J. “Engineering away lysosomal junk: medical bioremediation.”
Rejuvenation Res. 2007 Sep;10(3):359-65.

LysoSENS – Tackling Tau

Michael Rae
Research Assistant to Dr. de Grey

Neurofibrillary tangles (NFTs), aggregates of the protein tau, are a kind of molecular
damage that accumulates inside our brain and other nerve cells with aging, and are associated
with Alzheimer's and a variety of rare neurological diseases. There's good reason to think that
removing these tangles would help to rejuvenate the aging brain. The cell already has an
‘incineration and recycling center’, the lysosome, which is responsible for the removal of
damaged molecules within itself, so the age-related accumulation of NFTs in brain cells
suggests some essential weakness in lysosomal functioning - either a failure to engulf NFTs,
or a lack of the enzymes needed to shred them up into reusable parts once they have been
taken in.

This summer, scientists working in the New York University School of Medicine
under Dr. Einar Sigurdsson reported that the burden of NFTs could be significantly reduced,
and neurological function substantially preserved, in laboratory animals that normally suffer
neurological damage because of a genetic predisposition to form NFTs, by immunizing them
with a form of the tangles' precursors. This appeared to result from the antibodies'
shepherding the NFTs into the lysosome for disposal. The benefits were mild, but could
likely be enhanced by fortifying the lysosome with enzymes more suited to recycling the
NFTs - a projected project for the LysoSENS strand of the SENS platform.

Further Reading
Asuni AA, Boutajangout A, Quartermain D, Sigurdsson EM. “Immunotherapy targeting pathological tau
conformers in a tangle mouse model reduces brain pathology with associated functional improvements.”
J Neurosci. 2007 Aug 22;27(34):9115-29.

-7-
Issue 1
December 2007

SENS Around The World

Michael Rae
Research Assistant to Dr. de Grey

Toxic Cells
In January, Dr. Alan Saltiel and his colleagues at the Departments of Internal
Medicine and Physiology at the University of Michigan Medical School reported results that
will be central to the implementation of one of the SENS platform's planks: the removal of
one of the several classes of toxic cells that accumulate in the body over time. We've known
for some time that the systemic inflammation, insulin resistance, increased levels of blood
fats, and other metabolic disorders that progress with aging is mostly related to the
accumulation of body fat over the lifespan. Recently, however, we've come to understand
that the metabolic effects of being overweight are not the result of having more fat generally,
but are due to the attraction of inflammatory immune cells into the so-called "visceral" body
fat (the depot around the internal organs of the gut).

The removal of these cells would thus reverse the metabolic dysfunction that they
induce, restoring a more youthful systemic metabolism. But to do that requires a way of
selectively targeting such cells, while leaving healthy immune and fat cells alone.

Dr. Saltiel's lab has now reported that these immune cells have distinctive binding
sites and sugar-protein markers that distinguish them from their beneficial cousins elsewhere
in the body. This will allow us to develop interventions similar to vaccines and some of
today's targeted cancer therapies, removing the toxic cells with minimal collateral damage.

Further Reading
Lumeng CN, Bodzin JL, Saltiel AR. “Obesity induces a phenotypic switch in adipose tissue macrophage
polarization.”
J Clin Invest. 2007 Jan;117(1):175-84.

-8-
Issue 1
December 2007

Current Goals
Aubrey de Grey
Methuselah Foundation

A selection of projects within the SENS plan are ready to be launched as Foundation-
sponsored research programs, conditional only on the availability of sufficient funding. As
for MitoSENS and LysoSENS, these projects will start small (likely with only a single
researcher), with the aim of delivering high leverage in terms of the credibility of the
approach.

Amyloid in tissues other than the brain

Most people are aware of the amyloid deposits associated with Alzheimer's Disease:
they are the main constituent of senile plaques, the aggregates that accumulate in the spaces
between neurons as the disease progresses. Encouraging progress is being made in
stimulating the body's immune system to eliminate these deposits. However, amyloid
composed of different proteins also accumulates in other tissues during aging. Progress in
removing these other amyloids has been much less intensive thus far, even though they are, if
anything, more clearly linked to the progression of age-related illness than senile plaques are
to Alzheimer's. I have been in preliminary discussion with one of the leaders in this area,
with a view to initiating work as soon as possible.

Identifying genes essential for ALT

WILT, the anti-cancer therapy incorporated into SENS, entails (among other things)
the elimination of genes for the enzyme telomerase, which allows cancer cells to divide
indefinitely without losing material from the ends of their chromosomes. Unfortunately,
about 10% of cancers solve this problem in a different way, not using telomerase, via a
process known as ALT, for alternative lengthening of telomeres. Even more unfortunately,
ALT is still only very poorly understood. Recently, however, some intriguing observations in
two different organs have given good reason to suspect a hitherto unsuspected gene. A
relatively simple sequence of initial experiments could test this, and I am already in
discussions with a leading ALT researcher concerning the possibility of launching this
project.

-9-
Issue 1
December 2007

Mprize Status and Future Prospects

Elliot Bergman
Competitor Development Coordinator

The Mprize is designed to jumpstart scientific research into life-extending

biomedicine with the twin incentives of (1) a large cash award, and (2) a prestigious public
victory in a prominent research competition. The support of our generous donors is the key to
this strategy: the magnetic draw of the Prize grows with every dollar pledged to this program.
Therefore, we are pleased to report a substantial increase in the total amount of money in the
Mprize fund, which now stands at $4.6 million.

To broaden the field of competing research, a focused program was initiated this year
to actively recruit new competitors, expanding the “mouse race” for breakthroughs in
preventive and regenerative anti-aging biotechnology. Spearheading this campaign is Elliot
Bergman, Ph.D., of biotech consulting firm ChemLifeSciences, as the Foundation’s
Competitor Development Coordinator.

Elliot’s efforts to date have brought in four new competitors for the Prize, for a total
of eleven scientific teams independently racing to extend the lives of their furry subjects as of
this writing. The four most recent additions are Professor Andrzej Bartke of Southern Illinois
University; Professor Craig Cooney of the University of Arkansas for Medical Sciences;
Alan Cash, founder of Terra Biological LLC; and Elise Sacane, co-founder of Neural
Learning Systems. Each of these teams is testing a different anti-aging strategy, creating
exactly the kind of wide-open, multi-strategy competition needed to weed out ineffective
approaches and bring successful therapies into the spotlight.

Several other potential competitors have been identified and are being qualified for
participation. We hope to have a total of 13-15 competitors by the first quarter of 2008.

We are particularly delighted that Professor Andrzej Bartke a renowned, world-class

gerontologist has now announced that he will re-enter the fray in a second round of
competition. In 2004, his Growth Hormone Receptor Gene Knockout (GHR-KO 11C) mice
set the standard against which future competitors for the preventive (“Longevity”) Prize will
be judged: a previously unheard-of lifespan of 1819 days, or nearly five years – a remarkable
50% extension of lifespan compared to normal, healthy mice. Dr. Bartke has not yet
disclosed the new protocol that he will be testing in this second season of the anti-aging
challenge, but we can be certain that he will be a major factor in the race.
In addition to putting his own mice onto the gridiron, Professor Bartke is now also
leading the Foundation’s efforts to promote the wider use of mice as an experimental model
for anti-aging research. Today, the trend is increasingly toward lower cost studies using
cheaper, shorter-lived organisms such as roundworms, yeast, and fruit flies – organisms that
are progressively poorer proxies for human test subjects with every evolutionary step that
they take away from us. Success in this initiative would not only attract many more new
competitors to the Prize, but also broaden the range of interventions being tested within and
without the Prize structure. We aim to enhance the impact of mouse research in general, and
to the Methuselah Foundation in particular, while increasing the yield of results that are
likely translatable into human interventions.

- 10 -
Issue 1
December 2007

We have also secured access, through cooperation with the National Institute on
Aging (NIA) division of the National Institutes of Health (NIH), to a database of US-
government-funded projects on mouse aging research, invaluable as a source of potential
new competitors. It is notable that 3 of our 4 new competitors are supported, at least in part,
by NIA/NIH grants, while two are scientific entrepreneurs who have raised private capital to
fund part (Cash) or all (Sacane) of their Mprize research. We are scouring other external
sources – such as reports in the scientific literature, and grant applications submitted to other
non-governmental scientific organizations such as the Ellison Medical Foundation for
additional potential competitors. In addition we follow up with researchers testing new aging
treatments in rats, or in cultured cells or tissues, in an effort to identify new approaches
which should result in lifespan studies in mice.

Future prospects for the Mprize look bright, although there are some important
challenges that continue to discourage use of mice as test subjects for anti-aging
interventions. A major limiting issue is the relatively high cost of this research, which can
range into the hundreds of thousands of dollars per year, as well as the length of time
required to complete lifespan studies in an animal that normally lives for over three years. A
hopeful sign is that despite these disincentives, several well-funded start-up companies in the
new, important Nutritional Genomics field – such as Sirtris and IKARIA – are recognizing
the importance of testing their interventions in mammals, and will likely be using mice
extensively in their research.

We are assessing the possibility of reducing research costs by outsourcing mouse

trials to well-organized laboratories in Asia (mainly China, India, Japan, Taiwan, Thailand,
and South Korea), where strict scientific standards can be assured. If such an international
research facility becomes available, the scale of the investment required to perform mouse
studies will be reduced considerably, broadening the scope of mouse treatments that can be
tested and allowing a wider variety of new competitors to take up the gauntlet.

Another way of limiting the cost and time required to perform anti-aging studies in
mice is to encourage more researchers to compete in the Rejuvenation arm of the Mprize,
which requires researchers to test their interventions in 16-month-old mice (instead of
starting at weaning, as in the Longevity prize). Most of the Foundation’s donors already
favor the Rejuvenation Prize over the Longevity Prize by a very large margin, as measured
in completed pledges to the two Prize funds: $1.48 million vs. $0.16 million - a ratio of more
than 9 to 1. This preference is based on the urgent need to develop interventions that can
extend the healthy life spans of people who are already middle-aged, in hopes that people
alive today can still be rescued from a death by biological decay brought on by the aging
process.

- 11 -