Removing toxic aggregates that our cells can’t break down Aubrey de Grey Department of Genetics, University of Cambridge

1. Removing toxic aggregates that our cells can’t break down Aubrey de Grey Department of Genetics, University of Cambridge Email: ag24@gen.cam.ac.uk Website: http://www.gen.cam.ac.uk/sens/

2. The lysosome: the cell’s garbage disposal machinery of last resort Amino acids P Lyso-some R ? M

3. The problem: the lysosome is not omnipotent Liver’s solution: exocytosis Lyso-some Lyso-some Not done elsewhere: kidneys would suffer?

4. All other cells’ “solution”: sequestration Obviously fails eventually Cellular function impaired Cell becomes toxic Cell dies

5. Lysosomal junk: The four major types Lipofuscin. Universal marker of postmitotic cell aging; reaches 10% of total cell mass in heart and motor neurons. No clear proof of pathogenesis, but in vitro evidence is strong

6. Lysosomal junk: The four major types 2) A2E -- weird molecule created by reaction of the membrane lipid phosphatidylethanolamine with the photoreceptor pigment retinal. More or less proven to be pathogenic: causes age-related macular degeneration.

7. Lysosomal junk: The four major types 3) Hyperphosphorylated tau in neurons. Widely believed to be a major cause of Alzheimer’s disease. Similar aggregates form in other neurodegenerative diseases and normal aging.

8. Lysosomal junk: The four major types 4) Oxidised (etc) cholesterol and cholesteryl esters (oxysterols) in arterial macrophages. Causes them to become “foam cells”, then fatty streaks, and eventually atherosclerotic plaques.

9. Why don’t graveyards fluoresce? de Grey 2002, Trends in Biotechnology 20:452-455 These (often fluorescent) materials do not accumulate in nature, even in areas rich in the remains of animals that produce them. Thus, something degrades them. They are energy-rich, so micro-organisms could live off them. Bacterial strains exist with astonishingly varied catabolic activities, and are being used by many groups for bioremediation purposes. They degrade rubber, TNT, PCBs, dioxin, ... Some fungi are similarly versatile. Can micro-organisms capable of degrading lysosomal junk be isolated from the soil???

12. Some FAQs Q: Aren’t foam cell lysosomes mainly native cholesterol? A: Yes - the major lysosomal component is not necessarily the culprit - the culprit may impair lysosomal trafficking of normally benign things Q: Won’t we need an awful lot of different enzymes? A: Probably not - degradation is synergistic - one step will often form a substrate for an enzyme we already have Q: What about immune rejection? A: Same problem/solutions as for normal gene therapy

13. 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 models (apoE-/- mice, etc.) Test in humans as for lysosomal storage diseases

14. Funding: the story so far 2001, 2002: Kronos deem it too “sciencey” :-( 2003: Archer wows NIA officials at IABG10 :-) 2004: “SENS 5” (July 26th, NIA offices, Bethesda) Jay Jerome: atherogenic junk Ralph Nixon: neurotoxic junk Janet Sparrow: ophthalmotoxic junk Ana Maria Cuervo: gene engineering for lysosomal targeting Roscoe Brady: protein engineering for lysosomal targeting Bruce Rittmann, Perry McCarty, Pedro Alvarez: biorem. Fingers crossed….