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Please do not remove this page A Translational Approach to Enzyme-Mediated Retinal Lipofuscin Removal for Atrophic Age-Related Macular Degeneration and Stargardt's Disease Moody, Kelsey James https://scholarship.miami.edu/discovery/delivery/01UOML_INST:ResearchRepository/12355252850002976?l#13355471410002976 Moody, K. J. (2019). A Translational Approach to Enzyme-Mediated Retinal Lipofuscin Removal for Atrophic Age-Related Macular Degeneration and Stargardt’s Disease [University of Miami]. https://scholarship.miami.edu/discovery/fulldisplay/alma991031447210902976/01UOML_INST:ResearchR epository Open Downloaded On 2021/09/26 10:17:17 -0400 Please do not remove this page UNIVERSITY OF MIAMI A TRANSLATIONAL APPROACH TO ENZYME-MEDIATED RETINAL LIPOFUSCIN REMOVAL FOR ATROPHIC AGE-RELATED MACULAR DEGENERATION AND STARGARDT’S DISEASE By Kelsey James Moody A DISSERTATION Submitted to the Faculty of the University of Miami in partial fulfillment of the requirements for the degree of Doctor of Philosophy Coral Gables, Florida May 2019 ©2019 Kelsey James Moody All Rights Reserved UNIVERSITY OF MIAMI A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy A TRANSLATIONAL APPROACH TO ENZYME-MEDIATED RETINAL LIPOFUSCIN REMOVAL FOR ATROPHIC AGE-RELATED MACULAR DEGENERATION AND STARGARDT’S DISEASE Kelsey James Moody Approved: _______________________ _______________________ Zafar Nawaz, Ph.D. Feng Gong, Ph.D. Professor of Biochemistry and Associate Professor of Biochemistry Molecular Biology and Molecular Biology _______________________ _______________________ Michal Toborek, M.D., Ph.D. Sanjoy Bhattacharya, Ph.D. Professor of Biochemistry and Professor of Ophthalmology Molecular Biology _______________________ _______________________ Ralf Landgraft, Ph.D. Guillermo Prado, Ph.D. Associate Professor of Biochemistry Dean of the Graduate School and Molecular Biology _______________________ Gerold Feuer, Ph.D. Scientific Consultant, HuMurine MOODY, KELSEY JAMES A Translational Approach to Enzyme- (Ph.D., Biochemistry and Molecular Mediated Retinal Lipofuscin Removal for Biology) Atrophic Age-Related (May 2019) Macular Degeneration and Stargardt’s Disease Abstract of a dissertation at the University of Miami. Dissertation supervised by Professor Zafar Nawaz. No. of pages in text. (116) Despite significant resource expenditures and decades of focused research, chronic age- associated diseases have not been met with improvements in patient outcomes that reflect the successes against infectious disease over the last century. This observation calls into question contention approaches to drug discovery for diseases of aging, and more importantly, their molecular targets. Herein, the strategies for engineered negligible senescence (SENS) framework for drug discovery -- an approach to treating aging broadly based on damage mitigation -- is presented. A translational program for age- related macular degeneration is detailed as illustration of this approach. Collectively, this work validates the academic and commercial viability of the SENS damage repair platform and presents a novel therapeutic modality that may eventually treat the global leading cause of vision loss in the elderly. Dedication To all future generations that may take for granted the cures we strive to develop today. iii Acknowledgment This work would not have been possible without foundational training and steadfast support from my family, research team, mentors, and investors: Annette Deyo Bill Hunt Meegan Sleeper Adam Blanden Mark Yandell Brandon Moyer Aaron Wolfe Barry Moore Aubrey de Grey Kelly Moody Cornelis Wortel Mike Kope Miles Moody Nick LeClair Lisa Fabiny Doug Hagrman Kris Grohn John Schloendorn Eric Zluhan Stephanie Martens Nick LeClair Danique Wortel Nichole Fish David Gobel Donald Levine Zach Thomas Roger Bagg William Tooke Kathleen Kelly Leon Apel Tom Curle Anthony Bianchi Gerold Feuer William Faloon Scott Campbell Doyle Lab Tomas Burl David Reed Rohrer Lab For the work described in chapter 2, the authors would like to thank SENS Research Foundation, Center State CEO, and our private investors for their assistance in funding this work, and to SENS Research Foundation for the original conception of this approach via the LysoSENS program. For the work described in chapter 3, the authors are thankful to Maximus Peto and Aubrey de Grey for providing valuable feedback for this manuscript. This work was supported by grants from the Life Extension Foundation and Longecity (formerly Immortality Institute). For the work described in chapter 4, we are grateful to Gerold Feuer, Maximus Peto and Aubrey de Grey for providing valuable feedback for this manuscript, and to the staff at the Fayetteville Free Library for 3D printer training and facility access. This work was supported by grants from the Life Extension Foundation and Longecity (Immortality Institute). iv Disclosures For the work described in chapter 2, the authors declare the following competing financial interest(s): All authors are employees or interns of for-profit companies or affiliate companies that own and commercialize the described technology (LysoClear, Inc.) excepting R.D., B.R., E.O., and J.T. R.D. receives consistent grant support from LysoClear, Inc. and Ichor Therapeutics, Inc. to support his work on A2E and macular degeneration. A.R.B., A.J.W., and K.J.M. have a substantial equity positions in LysoClear, Inc. and Ichor Therapeutics, Inc., both of which may profit from the technology. For the work described in chapter 3, KJM holds equity positions in the following for-profit stem cell therapy companies; Ichor Therapeutics, Inc., ImmunePath, Inc., and Advanced Cell Technologies, Inc. EZ and DW hold equity positions in Ichor Therapeutics, Inc. GF holds equity positions in Humurine Technologies, Inc. and Ichor Therapeutics, Inc. The authors declare no further conflicts. For the work described in chapter 4, KM holds equity positions in several for- profit stem cell therapy companies, including Ichor Therapeutics, Inc., ImmunePath, Inc., and Advanced Cell Technologies, Inc. EZ and DW hold equity positions in Ichor Therapeutics, Inc. The authors declare no other disclosures. v TABLE OF CONTENTS Page LIST OF FIGURES ..................................................................................................... vii LIST OF TABLES ....................................................................................................... viii Chapter 1 INTRODUCTION ......................................................................................... 1 Strategies for Engineered Negligible Senescence (SENS)…………………… 2 A Brief Review of Age-Related Macular Degeneration………………………. 4 Narrative Introduction ...................................................................................... 10 2 RECOMBINANT MANGANESE PEROXIDASE REDUCES A2E BURDEN IN MOUSE MODEL OF STARGARDT’S DISEASE ...................................... 15 3 LEAN START-UP: A COMPREHENSIVE CASE STUDY IN THE ESTABLISHMENT OF LABORATORY INFRASTRUCTURE .................. 65 4 AUTOMATING HESC DIFFERENTIATION WITH 3D PRINTING AND LEGACY LIQUID HANDLING SOLUTIONS ............................................. 86 5 CONCLUSION ............................................................................................... 103 WORKS CITED…………… ...................................................................................... 108 vi LIST OF FIGURES Figure 1.1. Classes of age-associated damage ..................................................... 12 Figure 1.2. SENS damage-repair approach .......................................................... 13 Figure 1.3. Summary of the Visual Cycle ............................................................ 14 Figure 2.1. wtMnP and rMnP activity in vitro ..................................................... 48 Figure 2.2. rMnP activity in cell-free and cell culture models ............................ 50 Figure 2.3. rMnP activity in vivo ......................................................................... 52 Figure 2.S1. Monosaccharide chromatograms of rMnP performed by UC San Diego GlycoAnalytics Core ............................................................................................ 55 Figure 2.S2. DMP activity of wtMnP and rMnP ................................................. 56 Figure 2.S3. A2E degradation by rMnP in the presence and absence of oxygen 57 Figure 2.S4. Breakdown of A2E by (A) wtMnP and (B) rMnP by overnight incubation as a function of pH ............................................................................. 58 Figure 2.S5. Representative A2E breakdown kinetics with wtMnP and rMnP ... 59 Figure 2.S6. CD206 detection on (A) ARPE-19 and primary (B) RPE cells by flow cytometry ............................................................................................................. 60 Figure 2.S7. A2E concentration in Abca4(-/-) after 6 weekly doses of rMnP ..... 61 Figure 2.S8. Mouse weights for the efficacy study in 2.3B ................................. 62 Figure 2.S9. ERG recordings for the 12-week 56 µg x 6 dose toxicity study ..... 63 Figure 2.S10. Normalized HPLC chromatograms (left) and absorbance spectra of the indicated major peak (right) ................................................................................. 64 Figure 3.1. Business Model Canvas ....................................................................... 85 Figure 4.1. Custom cell scraper and filter box labware ......................................

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