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SENS Foundation Research Center




  Matthew S. O’Connor, Ph.D.
SENS Foundation Research Center
       Mountain View, Ca
                                  sens foundation
                                  reimagine aging
SENS Research Foundation

• Rejuvenation Biotechnology
  – Repairing the Damage




                               sens foundation
                               reimagine aging
Lysosomes take out the trash




                    sens foundation
                    reimagine aging
Lipofuscin is resistant to Lysosomal Degradation




       Smooth Muscle                   Macrophages




                                 sens foundation
                                 reimagine aging
   Retinal Pigment Epithelia         Cardiac Muscle
RPE Lipofuscin = A2E




    pyridinium bisretinoid (A2E)
                                   sens foundation
                                   reimagine aging
A2E In Dry AMD




Normal   Dry               Ambati and Fowler, Neuron,
                           2012




                  sens foundation
                  reimagine aging
Enzyme Therapy For Lysosomal Aging
      Enzyme Therapy For Age Related Macular
               Degeneration (AMD)
• Leading cause of vision loss in Americans over age 60: dry and wet.

• AMD, 10 million; Cancer, 12 million; Alzheimer’s disease, 5 million.

• Affects the retina, the area in the back of the eye responsible for processing visual
  images.




                                                            sens foundation
                                                            reimagine aging
Peroxidases (MnP) Can Degrade A2E

    Manganese peroxidase: 2 Mn(II) + 2 H+ + H2O2                          2 M(III) + 2 H2O


                    6000

                    5000
(arbitrary untis)




                    4000
       A2E




                                                        *      *
                    3000

                    2000

                    1000

                       0
                           Control MnP mannosylated-   MnP mannosylated           No uptake into
                                        rMnP                 rMnP                   RPE cells
                                    4 hours              24 hours sens        foundation
                                                                   reimagine aging
A Candidate Human Lysosomal Enzyme (SENS20)
           Can Degrade A2E In Vitro

                                1600

                                1400

                                1200

                                1000
            (arbitrary untis)
                                800               *
                   A2E



                                600

                                400

                                200

                                      0
                                          A2E     A2E
                                                   +
                                                SENS20
                                                         sens foundation
                                                         reimagine aging
SENS20 Can Reduce Lysosomal A2E In RPE Cells
 Synthesis and               Uptake into         Analysis of lysosomal uptake
purification of A2E          RPE cells           after 2 days of recovery
                       A2E         Anti-SENS20                  MERGE
A2E Control
A2E + 3 µg/ml SENS20




                                                       sens foundation
                                                       reimagine aging
Future Studies


•Remodel the active site of SENS20
   • to specifically accommodate A2E

•Address uptake & safety of SENS20 in RPE cells of aged mouse
retinas.

•Address uptake, safety & efficacy of rSEN20 in a mouse model of
Stargardt’s macular dystrophy.

•Test in people, cure Stargardt’s disease, cure AMD



                                            sens foundation
                                            reimagine aging
Mitochondria




      Free radicals damage DNA

      Dysfunctional Mitochondria

                 sens foundation
                 reimagine aging
Mitochondrial Genes in the
             Nucleus
• 1000-1500 mitochondrial genes are expressed
  in the nucleus.


           DNA RNA
                         MTS-Protein




   However, 13 genes are still expressed ONLY in the mitochondria.

                                               sens foundation
                                               reimagine aging
The Lone 13
                                          ND1 mutations = LHON, PD
                         ND1              ND4 mutations = LHON
                         ND2              CyB mutations = Exercise intolerance
                         ND3              ATP6 mutations = Leigh syndrome
                         ND4
                         ND4L                                                        Co1
                         ND5                                                         Co2         ATP6
                         ND6                                   CyB                   Co3         ATP8




Figure modified from: http://www.nature.com/nrc/journal/v3/n3/box/nrc1013_BX1.html
                                                                                           sens foundation
                                                                                           reimagine aging
Co-Translational Import



     mts      3’ utr



Nuclear recoded mito
 gene with RNA and
 Protein targeting signals
                             sens foundation
                             reimagine aging
Previous research




               sens foundation
               reimagine aging
Goals

• To allotopically express these 13 genes in the
  nucleus
• To target the proteins to the mitochondria
• To have them effectively replace the loss of
  these 13 mito genes in aging and disease



                                  sens foundation
                                  reimagine aging
Mitochondrial localization


           mts-ND1-cox10 3’ utr




    Flag       Mitotracker        Merge



           Hek 293 stable cells

                                   sens foundation
                                   reimagine aging
Detection of Allotopic Proteins in
       Complexes I and V
                     IP Complex V
                             C
                          A6     6S
                UT       m     nA
                                                                            IP: Complex I
                                                                            CF             MF

     WB:                                                                        N   1C         1C 1S
                                                                                             N N
  anti-FLAG                                 ATP6-FLAG                      UT m          UT m m
                                                                WB:                                    ND1-FLAG
                                                             anti-FLAG                                 IgL


                                                                            Input for IP
                                                                           CF             MF
                                            ATP Synthase
                                            alpha subunit                      1C    1C 1S
                                                                          UT mN UT mN mN
   WB:                                                          WB:                                    GRIM-19
  anti-ATP                                                  anti-GRIM19
Synthase             Input for IP
alpha subunit
                                 C
                              A6       6S
                UT        m          nA

                                            ATP Synthase
                                            alpha subunit

                                                                          sens foundation
                                                                          reimagine aging
The Future

• Rescue patient lines and MitoDNA knockout
  (Rho 0) lines
• Express all 13 genes individually, then in
  groups, then all at once!
• Mouse gene therapy and/or mice transgenic
  for mitochondrial proteins
  – Make rho 0 mouse? Super mouse?
• Gene therapy in people
                                     sens foundation
                                     reimagine aging
OncoSENS: Stopping Alternative
       Lengthening of Telomeres
• All successful cancers
  immortal
• ~90% have telomerase
• ~10% use ALT. Osteosarcoma




                               sens foundation
                               reimagine aging
Workflow

•   Develop Assays
•   Test suspected genes
•   Look for more genes
•   Develop Therapies




                           sens foundation
                           reimagine aging
Assay Development




               sens foundation
               reimagine aging
Acknowledgements

SENS Foundation Research           Collaborators:
Center, Mountain View, CA          SRI
Aubrey de Grey, Mike Kope,         Steven Samuelsson
Tanya Jones, Daniel Kimbel, Anne   Rice University
                                   Pedro Alverez
Corwin, Heidi Ward, Thaddeus
                                   Jacques Mathieu
Campbell, David Halvorson, John
                                   UC Davis
Irving, Amutha Boominathan,        Gino Cortopassi
Jayanthi Vengalam, Lisa Fabiny,    Buck Institute
Maximus Peto, Gouri Yogalingum,    Kevin Perrott
Sarah Fazal, Alexandra Crampton,   Remi-Martin Laberge
Thomas Hunt,                       Inserm
Kelsey Moody,                      Marisol Corral-Debrinski
Gayathri Swaminathan               Stanford
                                   Kyle Kovary
                                     sens foundation
                                     reimagine aging

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Dr. Oki O'Connor at Health Extension Salon #4

  • 1. SENS Foundation Research Center Matthew S. O’Connor, Ph.D. SENS Foundation Research Center Mountain View, Ca sens foundation reimagine aging
  • 2. SENS Research Foundation • Rejuvenation Biotechnology – Repairing the Damage sens foundation reimagine aging
  • 3. Lysosomes take out the trash sens foundation reimagine aging
  • 4. Lipofuscin is resistant to Lysosomal Degradation Smooth Muscle Macrophages sens foundation reimagine aging Retinal Pigment Epithelia Cardiac Muscle
  • 5. RPE Lipofuscin = A2E pyridinium bisretinoid (A2E) sens foundation reimagine aging
  • 6. A2E In Dry AMD Normal Dry Ambati and Fowler, Neuron, 2012 sens foundation reimagine aging
  • 7. Enzyme Therapy For Lysosomal Aging Enzyme Therapy For Age Related Macular Degeneration (AMD) • Leading cause of vision loss in Americans over age 60: dry and wet. • AMD, 10 million; Cancer, 12 million; Alzheimer’s disease, 5 million. • Affects the retina, the area in the back of the eye responsible for processing visual images. sens foundation reimagine aging
  • 8. Peroxidases (MnP) Can Degrade A2E Manganese peroxidase: 2 Mn(II) + 2 H+ + H2O2 2 M(III) + 2 H2O 6000 5000 (arbitrary untis) 4000 A2E * * 3000 2000 1000 0 Control MnP mannosylated- MnP mannosylated No uptake into rMnP rMnP RPE cells 4 hours 24 hours sens foundation reimagine aging
  • 9. A Candidate Human Lysosomal Enzyme (SENS20) Can Degrade A2E In Vitro 1600 1400 1200 1000 (arbitrary untis) 800 * A2E 600 400 200 0 A2E A2E + SENS20 sens foundation reimagine aging
  • 10. SENS20 Can Reduce Lysosomal A2E In RPE Cells Synthesis and Uptake into Analysis of lysosomal uptake purification of A2E RPE cells after 2 days of recovery A2E Anti-SENS20 MERGE A2E Control A2E + 3 µg/ml SENS20 sens foundation reimagine aging
  • 11. Future Studies •Remodel the active site of SENS20 • to specifically accommodate A2E •Address uptake & safety of SENS20 in RPE cells of aged mouse retinas. •Address uptake, safety & efficacy of rSEN20 in a mouse model of Stargardt’s macular dystrophy. •Test in people, cure Stargardt’s disease, cure AMD sens foundation reimagine aging
  • 12. Mitochondria Free radicals damage DNA Dysfunctional Mitochondria sens foundation reimagine aging
  • 13. Mitochondrial Genes in the Nucleus • 1000-1500 mitochondrial genes are expressed in the nucleus. DNA RNA MTS-Protein However, 13 genes are still expressed ONLY in the mitochondria. sens foundation reimagine aging
  • 14. The Lone 13 ND1 mutations = LHON, PD ND1 ND4 mutations = LHON ND2 CyB mutations = Exercise intolerance ND3 ATP6 mutations = Leigh syndrome ND4 ND4L Co1 ND5 Co2 ATP6 ND6 CyB Co3 ATP8 Figure modified from: http://www.nature.com/nrc/journal/v3/n3/box/nrc1013_BX1.html sens foundation reimagine aging
  • 15. Co-Translational Import mts 3’ utr Nuclear recoded mito gene with RNA and Protein targeting signals sens foundation reimagine aging
  • 16. Previous research sens foundation reimagine aging
  • 17. Goals • To allotopically express these 13 genes in the nucleus • To target the proteins to the mitochondria • To have them effectively replace the loss of these 13 mito genes in aging and disease sens foundation reimagine aging
  • 18. Mitochondrial localization mts-ND1-cox10 3’ utr Flag Mitotracker Merge Hek 293 stable cells sens foundation reimagine aging
  • 19. Detection of Allotopic Proteins in Complexes I and V IP Complex V C A6 6S UT m nA IP: Complex I CF MF WB: N 1C 1C 1S N N anti-FLAG ATP6-FLAG UT m UT m m WB: ND1-FLAG anti-FLAG IgL Input for IP CF MF ATP Synthase alpha subunit 1C 1C 1S UT mN UT mN mN WB: WB: GRIM-19 anti-ATP anti-GRIM19 Synthase Input for IP alpha subunit C A6 6S UT m nA ATP Synthase alpha subunit sens foundation reimagine aging
  • 20. The Future • Rescue patient lines and MitoDNA knockout (Rho 0) lines • Express all 13 genes individually, then in groups, then all at once! • Mouse gene therapy and/or mice transgenic for mitochondrial proteins – Make rho 0 mouse? Super mouse? • Gene therapy in people sens foundation reimagine aging
  • 21. OncoSENS: Stopping Alternative Lengthening of Telomeres • All successful cancers immortal • ~90% have telomerase • ~10% use ALT. Osteosarcoma sens foundation reimagine aging
  • 22. Workflow • Develop Assays • Test suspected genes • Look for more genes • Develop Therapies sens foundation reimagine aging
  • 23. Assay Development sens foundation reimagine aging
  • 24. Acknowledgements SENS Foundation Research Collaborators: Center, Mountain View, CA SRI Aubrey de Grey, Mike Kope, Steven Samuelsson Tanya Jones, Daniel Kimbel, Anne Rice University Pedro Alverez Corwin, Heidi Ward, Thaddeus Jacques Mathieu Campbell, David Halvorson, John UC Davis Irving, Amutha Boominathan, Gino Cortopassi Jayanthi Vengalam, Lisa Fabiny, Buck Institute Maximus Peto, Gouri Yogalingum, Kevin Perrott Sarah Fazal, Alexandra Crampton, Remi-Martin Laberge Thomas Hunt, Inserm Kelsey Moody, Marisol Corral-Debrinski Gayathri Swaminathan Stanford Kyle Kovary sens foundation reimagine aging

Editor's Notes

  1. http://hyperphysics.phy-astr.gsu.edu/hbase/biology/lysosome.html
  2. RPE functions are: light absorption, epithelial transport, spatial ion buffering, visual cycle, phagocytosis, secretion and immune modulation. Wet macular degeneration is generally caused by abnormal blood vessels that leak fluid or blood into the region of the macula. Starts as dry amd.
  3. White rot fungi secrete MnP to aid lignin degradation. 3 Classes of enzymes: peroxidases, laccases, and carotenoid cleavage oxygenases
  4. http://hyperphysics.phy-astr.gsu.edu/hbase/biology/lysosome.html