ANIMAL MODELS OF PARKINSON S DISEASE: STATE OF THE FIELD & THE FUTURE! KULDIP DAVE 21 February 217
MJFF IS THE WORLD S LARGEST NONPROFIT FUNDER OF PD RESEARCH Our Mission To accelerate the development of improved therapies, and ultimately a cure, for people living with Parkinson s disease today. Vital Stats» Founded in 2 by actor Michael J. Fox» Public charity» Nearly 7, donors in 215 (individuals, corporations, nonprofits)» No chapters: team of 14 based in NYC» 3,3 grassroots fundraisers reaching 15, supporters worldwide in 215» 89 cents on every dollar spent goes directly into research» More than $65 million in research programs funded to date» $87.8 million in research programs funded in 215» Nearly 2,1 projects funded to date» 55 active grants in current portfolio» 33% of funded projects are led by researchers outside the United States» Fund academics, biotechs and pharma 2
MJFF RESEARCH PRIORITIES DISEASE MODIFYING SYMPTOMATIC FIELD-WIDE CHALLENGES LRRK2 Alphasynuclein Motor Symptoms Non-Motor Symptoms Biomarkers Clinical Trial Recruitment Emerging Targets GBA1 & Parkin Emerging Targets Research Tools We prioritize and pursue research in these areas, providing financial and intellectual support for projects from discovery through the clinic to ensure progress toward new therapies for PD patients. 3
State of the field & MJFF Strategy» Challenges Many toxin and genetic models exist, however, none have closely recapitulated all of PD-related pathology and symptomology. Outcome measures may or may not be relevant to human condition; limited translation of preclinical findings. Lack of standardization in outcome measures and methodologies for characterization; animal models on the market that have not been independently phenotyped. Inability to compare across laboratories, sheer lack of replication!. IP/patent issues, restrictions on use, huge label license fees lead to limited availability.» Strategy Identification: Prioritize field-enabling animal models that will fill gaps and advance research. Generation: Contract with expert CROs to generate models, and make basic characterization data available to the community. Characterization: Collaborate with relevant CROs to phenotype MJFF-generated animal models. Replication: Partner with CROs to independently characterize and replicate existing models. Distribution: Work with vendors to facilitate wide distribution to both for-profit and not for profit researchers. 4
MOUSE MODELS OVERVIEW Live Strains Description CRO Available Tg Mice line15 (MJFF) JAX Available asyn KO Mice (MJFF) JAX Available A53T asyn Mice (Lee) JAX Available A3P/A53T asyn Mice (Richfield) JAX Available WT Human asyn Mice (Nussbaum) JAX Available asyn Floxed Mice (Buchman) JAX Available A53T asyn Mice (Nussbaum) JAX Available asyn KO Mice (Buchman) JAX Available Tg WT LRRK2 Mice (MJFF) JAX Available LRRK2 KO Mice (MJFF) JAX Available LRRK2 T1348N Mice (MJFF) JAX Available LRRK2 D1994A Mice (MJFF) JAX Available LRRK2 G219S Mice (MJFF) JAX Available Tg WT LRRK2 Mice (Li) JAX Available LRRK2 G219S Mice (Yue) JAX Available LRRK2 G219S Mice (Li) JAX Available G219S KI Mice (MJFF) Taconic Q2 217 Parkin W42A KI Mice (MJFF) JAX Q2 217 GBA D49V KI Mice (MJFF) JAX Available GBA D49V x Mice (MJFF) JAX Q4 217 Cryopreserved Strains Description CRO Available Tg WT Human asyn Mice (MJFF) JAX Cryopreserved Tg Mice line12 (MJFF) JAX Cryopreserved LRRK2 KO Mice (Yao) JAX Cryopreserved WT LRRK2 Mice (Melrose) JAX Cryopreserved LRRK2 G219S Mice (Yue) JAX Cryopreserved LRRK2 A216T Mice (MJFF) JAX Cryopreserved LRRK2 R1441G Mice (MJFF) JAX Cryopreserved LRRK1 KO Mice (MJFF) JAX Cryopreserved LRRK1/2 dko Mice (MJFF) JAX Cryopreserved VPS35 D62N Mice (MJFF) JAX Cryopreserved Eif4g1 KO Mice (MJFF) JAX Cryopreserved Eif4g1 R127H Mice (MJFF) JAX Cryopreserved 5
RAT MODELS OVERVIEW Live Strains Description CRO Available asyn Humanized A53T KI Rat Horizon Q1 217 asyn KO Rat Horizon Q2 217 LRRK2 KO Rat Horizon Available Tg G219S LRRK2 Rat Taconic Available TH-GFP Rat (X-Linked) Taconic Available TH-GFP Rat (Non-X-Linked) Taconic Available Pink1 KO Rat Horizon Available DJ-1 KO Rat Horizon Available Parkin KO Rat Horizon Available» Notable accomplishments: Description CRO Available Tg WT Human asyn Rat Cryopreserved Strains Taconic Cryopreserved Tg A53T asyn Rat Taconic Cryopreserved Tg E46K asyn Rat Taconic Cryopreserved LRRK1 KO Rat Horizon Cryopreserved LRRK1/2 dko Rat Horizon Cryopreserved First ever alpha-synuclein KO rat will be available to the community in Q2 217. Animals have been distributed by Horizon, Taconic, and Jackson Labs beginning Q1 212. 29 animal models are live, many more available for cryorecovery. 7,849 animal models have been distributed to date (over four years). 6
ASYN MODELS FOR PD DRUG DISCOVERY Visanji et al., 216, Trends in Neurosciences 7
ALPHA-SYNUCLEIN PRECLINICAL MODELS Models Rodents NHP Mice Rats Viral Vector Transgenic Spread model Viral Vector 8
CHARACTERIZATION OF MOUSE MODELS Characterization asyn Comparison Study MJFF mouse phenotyping Nussbaum Vlee Masliah Elan Richfield Thy1 asyn WT BAC asyn WT asyn KO (Het) asyn KO (Hom) Phenotyping at 4, 8 and 12 months Rotarod, grip strength, gait and motor movement, catalepsy, rearing, pole test, muscle tone, open-field, extensor strength, footsplay Immunohistochemistry Tyrosine hydroxylase, alpha-synuclein, GFAP, IBA1, ubiquitin, stereology of Pars Compacta Striatal concentrations of dopamine, DOPAC, HVA, Serotonin and 5-HIAA DNA copy #, mrna levels, Protein expression (n=15) (n=9) (n=6) (n=4) 9
ROTAROD TIME TO FALL 6 4 Months 6 8 Months 6 12 Months Rotarod Time to Fall (seconds) 4 2 Rotarod Time to Fall (seconds) 4 2 Rotarod Time to Fall (seconds) 4 2 C57Bl/6 W T (Masliah) A3P/A53T asyn (Richfield) C 57B l/6 W T (Masliah) A3P/A53T asyn (Richfield) asyn KO asyn KO (Hets) C57Bl/6 W T (Masliah) A3P/A53T asyn (Richfield) Rotarod Time to Fall Overview Rotarod Time to Fall (seconds) 5 4 3 2 1 *** *** ** C57Bl/6 WT (Masliah) A3P/A53T asyn (Richfield) 4 8 12 Age of mice (months) 1
TH-POSITIVE CELLS IN SN # of TH +ve neurons in SNpc 2 15 1 5 C 57B l/6 W T 4 Months (Masliah) A3P/A53T asyn (Richfield) # of TH +ve neurons in SNpc 8 Months 2 15 1 5 C57Bl/6 W T (Masliah) A3P/A53T asyn (Richfield) asyn KO asyn KO (Hets) # of TH +ve neurons in SNpc 2 15 1 5 C 57B l/6 W T (Masliah) 12 Months A3P/A53T asyn (Richfield) Stereology Overview # of TH +ve neurons in SNpc 15 125 1 75 5 *** *** C57Bl/6 WT (Masliah) A3P/A53T asyn (Richfield) 4 8 12 Age of mice (months) Key to Statistical Comparisons Across Age * significant effect of age C57Bl/6 WT & significant effect of age # significant effect of age (Masliah) @ significant effect of age $ significant effect of age % significant effect of age! significant effect of age A3P/A53T asyn (Richfield) ^ significant effect of age (1=p<.5; 2=p<.1; 3=p<.1) 11
7 DOPAMINE TURNOVER 4 Months 8 Months 7 7 12 Months Dopamine Turnover (DOPAC+HVA)/DA 6 5 4 3 2 1 Dopamine Turnover (DOPAC+HVA)/DA 6 5 4 3 2 1 Dopamine Turnover (DOPAC+HVA)/DA 6 5 4 3 2 1 C57Bl/6 W T (Masliah) A3P/A53T asyn (Richfield) Dopamine Turnover Overview ## ** C 57B l/6 W T (Masliah) A3P/A53T asyn (Richfield) asyn KO asyn KO (Hets) C57Bl/6 W T (Masliah) A3P/A53T asyn (Richfield) 4 * && ### & ### *** C57Bl/6 WT Dopamine Turnover (DOPAC+HVA)/DA 3 2 1 (Masliah) A3P/A53T asyn (Richfield) 4 8 12 Age of mice (months) Key to Statistical Comparisons Across Age * significant effect of age C57Bl/6 WT & significant effect of age # significant effect of age (Masliah) @ significant effect of age $ significant effect of age % significant effect of age! significant effect of age A3P/A53T asyn (Richfield) 12
EFFICACY OF ACTIVE IMMUNIZATION IN ASYN TRANSGENIC MICE (MASLIAH) 13
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MJFF INDEPENDENTLY REPLICATED ASYN PFF MODEL 9 days 18 days Reported by Luk et al., 212 Replicated Number of TH/Nissl positive neurons 1 8 6 4 2 * C75BL6/C3H (18 dpi) Injected Uninjected ps129 α-syn pathology TH+ cells in SNpc (at 18 dpi) Striatal DA content Striatal TH intensity YES YES YES YES PFF PBS Treatment Groups Mono *p<.5 DAT in striatum Motor deficits (rotarod & wire hang) YES NO 15
EFFICACY OF PASSIVE IMMUNIZATION IN ASYN PFF SPREAD MODEL 16
POC FOR LRRK2 INHIBITION IN ASYN RAT VIRAL VECTOR MODEL 17
NHP MODEL OF ALPHA-SYNUCLEINOPATHY (KOPRICH ET AL., 216) 18
Animal Treatment Advantages Caveats/Considerations Mice Transgenic Published data Breed to get larger colonies Mice Spread/transfer Synucleinopathy with DA loss Regional control Rats Viral Vector Synucleinopathy with DA loss Regional control Monkeys Viral Vector Allow for better PK/PD modeling and dose selection for human trials Data mostly from single lab Slow/delayed and incomplete phenotype Label license costs and use restrictions Relevance to human pathology Supraphysiological concentrations used Supraphysiological concentrations used No behavioral output NHP models are still under development and need to be validated 19
MJFF FIELD-ENABLING TOOLS PORTFOLIO Cell Lines DNA Plasmids Assays Protein Viral Vectors Animal Models Antibodies https://www.michaeljfox.org/research/research-tools.html 2