Chief Science Officer, SENS Foundation Regenerative medicine for aging: are we reaching the knee of the exponential curve? Aubrey D.N.J. de Grey, Ph.D. Chief Science Officer, SENS Foundation aubrey@sens.org http://www.sens.org/

SENS Foundation SENS Foundation is a US-registered charity that works to develop, promote and enable widespread access to regenerative medicine solutions to the disabilities and diseases of… AGING

But surely <insert favourite knee-jerk reaction>? Treating aging is just preventative geriatrics Old people are people too, deserve health Is medicine good only so long as it doesn’t work very well? Was the Industrial Revolution a bad thing? How much did Jenner/Pasteur understand?

What is… Regenerative medicine? Aging?

What is regen med? Any intervention that seeks to restore the structure of a tissue/organ to its state before it suffered damage

What is aging? Metabolism ongoingly causes “damage”. Damage eventually causes pathology.

Options for intervention Gerontology Geriatrics Metabolism Damage Pathology

Problem: this is metabolism

Options for intervention Gerontology Maintenance Geriatrics Metabolism Damage Pathology Claim: unlike the others, the maintenance approach may achieve a big extension of human healthy lifespan quite soon.

No new type of damage confirmed since 1982! This is the damage Cell loss, cell atrophy Division-obsessed cells (cancer) Death-resistant cells Mitochondrial mutations Intracellular junk Extracellular junk Extracellular crosslinks Seven Deadly Things No new type of damage confirmed since 1982!

Robust Human Rejuvenation Giving the middle-aged 30 years of extra healthy life: Robust Human Rejuvenation Damage rising with age It or its effects reversible by Cell loss, cell atrophy Cell therapy, mainly Division-obsessed cells (i.e. cancer) Telomerase/ALT gene deletion plus periodic stem cell reseeding Death-resistant cells Suicide genes, immune stimulation Mitochondrial mutations Allotopic expression of 13 proteins Intracellular junk Transgenic microbial hydrolases Extracellular junk Phagocytosis by immune stimulation Extracellular crosslinks AGE-breaking molecules/enzymes

Robust Human Rejuvenation Some parts are in clinical trials Giving the middle-aged 30 years of extra healthy life: Robust Human Rejuvenation Some parts are in clinical trials Damage rising with age It or its effects reversible by Cell loss, cell atrophy Cell therapy, mainly Division-obsessed cells (i.e. cancer) Telomerase/ALT gene deletion plus periodic stem cell reseeding Death-resistant cells Suicide genes, immune stimulation Mitochondrial mutations Allotopic expression of 13 proteins Intracellular junk Transgenic microbial hydrolases Extracellular junk Phagocytosis by immune stimulation Extracellular crosslinks AGE-breaking molecules/enzymes

About those amyloid trials… Clinical endpoints not achieved Amyloid is removed Tau is not No one knows what causes what in AD Combination therapies = wings + engine

Robust Human Rejuvenation Other parts are making headlines Giving the middle-aged 30 years of extra healthy life: Robust Human Rejuvenation Other parts are making headlines Damage rising with age It or its effects reversible by Cell loss, cell atrophy Cell therapy, mainly Division-obsessed cells (i.e. cancer) Telomerase/ALT gene deletion plus periodic stem cell reseeding Death-resistant cells Suicide genes, immune stimulation Mitochondrial mutations Allotopic expression of 13 proteins Intracellular junk Transgenic microbial hydrolases Extracellular junk Phagocytosis by immune stimulation Extracellular crosslinks AGE-breaking molecules/enzymes

Robust Human Rejuvenation Others are early but exciting Giving the middle-aged 30 years of extra healthy life: Robust Human Rejuvenation Others are early but exciting Damage rising with age It or its effects reversible by Cell loss, cell atrophy Cell therapy, mainly Division-obsessed cells (i.e. cancer) Telomerase/ALT gene deletion plus periodic stem cell reseeding Death-resistant cells Suicide genes, immune stimulation Mitochondrial mutations Allotopic expression of 13 proteins Intracellular junk Transgenic microbial hydrolases Extracellular junk Phagocytosis by immune stimulation Extracellular crosslinks AGE-breaking molecules/enzymes

Intracellular junk in the artery Endothelial Cells Lipid-engorged Lysosome Foam Cell

Bioremediation: the concept Microbes, like all life, need an ecological niche. Some get it by brawn (growing very fast)… …some by brain (living off material that others can't). Any abundant, energy-rich organic material that is hard to degrade thus provides selective pressure to evolve the machinery to degrade it. That selective pressure works. Even TNT, PCBs…

Xenocatabolism: the concept Graveyards are abundant in human remains, accumulate bones (which are not energy-rich), do not accumulate oxysterols, tau etc., so, should harbour microbes that degrade them… …whose catabolic enzymes could be therapeutic

Environmental decontamination in vivo

7-ketocholesterol degradation a promising start 7KC over time in enrichment cultures days Biodegradation 2008; 19(6):807-813

Stable isotope labelling and LC/MS reveal 7-ketocholesterol metabolites in the culture supernatant 7-ketocholesterol M = 400, M13C = 401 Dione metabolite ? M = 398, M13C = 399 Hydroxylated dione ? M = 414, M13C = 415 Culture growing on 7-ketocholesterol Culture growing on 13C-labeled 7-ketocholesterol

COX/LAMP1 co-localizes with AO Acridine Orange EGFP Merge

pEGFP-COXL1 transfected cells are protected from 7KC-induced toxicity Mathieu et al., Biotechnol. Bioeng. 2012; 109(9):2409-2415

Age-related accumulation of fluorescent compounds in retinal pigment epithelium neural retina RPE As you can see here in this histological section through the retina and in human RPE cels that have been removed from the eye, this lipofuscin that accumulates in RPE has a golden yellow fluorescence and occupies a considerable volume of the eye. When a section through the retina is examined under the fluorescence microscope so that it is magnified many fold - this is a photograph of retina viewed under the microscope - this aging material can be seen as this golden yellow fluorescence And as you can see some of these retinal pigment epiuthelial cells that have been removed from the eye…the accumulation of this material is considerabble - it is estimated that by age 80, 20% of the cytoplasmic volume of the cell occupied . This granular material is the fluorescent pigment that accumulates with age

Fundus autofluorescence (= RPE lipofuscin) increases with age Exc.: 550 nm For instance, just as age is a risk factor for AMD, so lipofuscin increases with age These spectrophotometric measurements of fundus autofluorescence, and thus of RPE lipofuscin demonstrate that there is a linear increase in fluorescence with age - consistent with a linear increase in lipofuscin accumulation with age Fluorescence intensity at age 65 is 2-fold greater than at age 25 Positive correlation with age Fluorescence intensity declines after age 70 Have shown that there is a linear increase in fluorescence intensity with age This .. Is indicative of an increase in RPE lipofuscin with age and confirms what had already been known from histological studies Individually corrected for lens absorption Delori et al., IOVS 42:1855. 2001 MOD FA

A major constitutent of RPE lipofuscin is the fluorescent pigment A2E The best known, the best characterized of the lipofuscin fluorophores is a compound we call A2E It obtained its name from the fact that it can be synthesized in the laboratory from 2 vitamin A aldehyde molecules and 1 ethanolamine. Eldred and Lasky, 1993 Sakai et al., 1996 Parish et al., 1998

A2E pyridinium bisretinoid Sakai, Decatur, Nakanishi and Eldred, 1996 A2E was originally thought to have this structure - this structure would be N retinyulidene N retinyl-ethanolamine But this does not have the pyridinium ring - the pyrididinum ring is a signature feature of A2E The structure of A2E was determined by Nakanishi working with Eldred to be that of a pyridinium bisretinoid - a positively charged pyridine ring with two side arms each derived from a molecule of all-trans-retinal. Based on this structure and its precursors, Nakanishi coined the name A2E - 2 vitamin A aldehydes and 1 ethanolamine. A2E is often stated in the literature to be N-retinylidene-N-retinylethanolamine but the compound so named would be thus, a structure wihtout this unusual pyridinium ring - the pyridinium ring is a unique feature of A2E - a signature Sakai, Decatur, Nakanishi and Eldred, 1996

iso-A2E A2E Amphiphilic compound 2 hydrophobic side-arms cationic Cl - 2 hydrophobic side-arms cationic polar head Cl - From the outset we were interested in the tendency of A2E to perturb cell membranes because it has an amphiphilic structure - with a polar head and two hydrophobic side-arms. A2E is a cationic amphiphile but it exists as a salt. Its counterion, at least under physiological conditions, is probably chloride. The fact that it is an amphiphile, its bulky wedge-shape and the behaviour of its counterion are all factors that are important in terms of its interactions with membranes. Actually we would expect the photoisomer iso-A2E to penetrate the membrane more easily because it is more streamlined. so when introduced to the cells at moderate extracellular concentrations, A2E accumulates in the cells in a benign manner however, at high concentrations, a2E is damaging A1E(mw 352.7) is a nonphysiological single side armed pyridinium molecule that retains both hydrophobic and hydrophilic elements (ends) . A1E and A2E have the same hydrophilic head group (pyridinium ring) yet the structure of A1E is more typical of a detergent. Our finding that A1E penetrates and disrupts the cell membrane (as indicated by loss of viability) faster than A2E could well be an effect of shape rather than just size. That A2E enters more sluggishly is cnsistent with the view that A2E may traverse the plasmam membrane by endovesiculation. Since A2E becomes sequestered within the lysosomal compartment of the clel, these findings are important to our understanding of whether and how A2E may interact with lysosomal membranes. iso-A2E A2E

Steps to biomedical application Isolate competent strains; select by starvation. Identify the enzymes (mutagenesis, chemistry, genomics). Make lysosome-targeted transgenes; assay cell toxicity. Assay competence in vitro (more mutagenesis/selection). Construct transgenic mice; assay toxicity in vivo. Assay competence in disease mouse models. Test in humans as for lysosomal storage diseases.

A word about “MetaSENS” SENSF does research to implement SENS But is that enough? Three reasons why not: Is SENS complete? (Vijg lab): “prot pleio” Is SENS needed? (Reis lab): “weather” <cue interminable digression re primate CR> What will SENS cause? (Hughes group)

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www.sens.org SENS Foundation aubrey@sens.org SENS Foundation works to develop, promote and ensure widespread access to regenerative medicine solutions to the disabilities and diseases of aging. www.sens.org aubrey@sens.org