Successfully reported this slideshow.
We use your LinkedIn profile and activity data to personalize ads and to show you more relevant ads. You can change your ad preferences anytime.

Zwiegers Thesis Defense

1,072 views

Published on

Presentation for oral defense Nov 2015

Published in: Science
  • Be the first to comment

  • Be the first to like this

Zwiegers Thesis Defense

  1. 1. Thesis Defense for the Degree of Master of Science in the Experimental Medicine Program November 17, 2015 Pierre Zwiegers Targeted Lentiviral-mediated Delivery of Progranulin cDNA in a Genetic Model of Amyotrophic Lateral Sclerosis Committee members: Dr. Christopher A. Shaw Dr. Shernaz X. Bamji Dr. Charles Krieger External Examiner: Dr. Doris Doudet Meeting Chair: Dr. Jason JS Barton
  2. 2.  Amyotrophic Lateral Sclerosis  Neurotrophic properties of Progranulin and links to ALS  Preliminary data for LV-mediated PGRN upregulation  Research Theme and Objectives  Experimental Methods  Overt Neurobehavioural Measures  End-stage Neuropathology  Decreased mSOD1 Copy Number  Concluding Thoughts & Future Directions Outline  Background  Results  Summary of Outcomes  Discussion  mSOD1 Copy Number Variation  ALS Models & Pre-clinial Translation
  3. 3. Servier medical art modified under a Creative Commons Attribution 3.0 Unported License. Amyotrophic Lateral Sclerosis Background • Age-related and fatally progressive neurodegenerative condition of upper and lower motor neurons • World-wide prevalence of 4-7/100 000 population (Chiò et al. 2013) • Intractable to therapeutic intervention; Riluzole remains the only clinically-approved drug (Miller et al. 2012) Healthy ALS Upper MNs Healthy ALS Lower MNs
  4. 4. Apparently sporadic ALS 90-95% Familial ALS 5-10% Turner et al. The Lancet Neurology, Volume 12, Issue 3, 2013, 310 - 322 Amyotrophic Lateral Sclerosis • More than 180 mutations in the SOD1locus have been linked to ALS (ALSoD, 2015) • Point mutations primarily result in a toxic gain-of-function property that gives rise to an ALS phenotype (Harms & Baloh, 2013) Background Zwiegers and Shaw. Journal of Con. Biomed Res., Volume 12, Issue 1, 2015, 4-22 • Most widely tested paradigm utilized to model disease and test pre-clinical efficacy
  5. 5. Progranulin and ALS • PGRN mutations have not been causal to ALS (Petkau & Leavitt, 2014) • PGRN polymorphisms can result in an earlier onset and a more progressive ALS phenotype (Sleegers et al, 2008) Background ▪ Temporal ALS Phenotype Progression ▪ Increased Progranulin Expression Transgenic mSOD1 Mouse Servier medical art modified under a Creative Commons Attribution 3.0 Unported License. • Early stages of ALS, PGRN levels in the CSF and plasma do not differ from control patients (Philips et al 2010) • Post-mortem IHC indicates increased neuronal and microglial expression within areas of neurodegeneration (Irwin et al 2009) Early stage Late stage Neuron Microglia Progranulin Philips et al. J Neuropathol and Exp Neurol, Volume 69, Issue 12, 2010, 1191-1200 • PGRN has been shown to be protective in models of Parkinsonism, Alzheimer’s disease, and arthritis (Minami et al 2014; Van Kampen et al, 2014;Van Kampen & Kay, 2011, & Tang et al, 2011)
  6. 6. Preliminary data for PGRN upregulation Late-stage targeting of motor neurons in an mSOD1 model preserves neuronal viability Preliminary Data Wild-Type G37R PGRN WT G37R G37R/PGRN GFP Background Provided by Drs. CA Shaw, Denis G. Kay, J Van Kampen & G Lee
  7. 7. Hypothesis Research Theme & Objectives Degree of ALS phenotype severity No aberrant phenotype Severe paralysis Neuron Microglia Astrocyte Early stage PGRN intervention Can neuronal targeting of exogenous PGRN cDNA at an early stage of the disease cascade: Severe paralysis  lessen the severity of the expressed behavioural phenotype?  attenuate motor neuron loss?  diminish the severity of astrogliosis and microgliosis?
  8. 8. Lentiviral –mediated transgene delivery Servier medical art modified under a Creative Commons Attribution 3.0 Unported License. Experimental Methods G37R (line 29)/ WT mice at 3.5-4 months of age Transgene integration; under control of CMV promoter Histological Assays • Microgliosis (Iba-1) • Astrogliosis (GFAP) • Neuronal Viability & Morphology (Cresyl Violet, ChAT) Monitoring/Neurobehavioural Assays • Leg Extension Reflex Test • Latency to fall from an elevated grid • Weight 5x2µL injections @ 4.0x108 TU/mL VSV-G pseudotyped lentiviral vector encoding GFP/PGRN into gastrocnemius muscles 1 week 2 weeks 4 weeks
  9. 9. Overt Neurobehavioural Measures Results Weight • Male G37R animals presented with a delay in phenotype-related weight loss • PGRN administration did not attenuate the progressive loss of body mass .. observed in Tg mSOD1 animals Leg Extension & Wire Hang Test • Early-stage PGRN delivery did not ameliorate outcomes in either measure • Male animals showcased a more severe deficit at earlier time points e ap
  10. 10. Overt Neurobehavioural Measures Results Onset & Survival • PGRN cDNA delivery did not delay disease onset or prolong survival • Male Tg animals showed evidence of an attenuated disease progression Median Onset (d) GFP: 344 PGRN: 315 Median Survival (d) GFP: 609 PGRN: 607 Median Onset (d) GFP: 287 PGRN: 294 Median Survival (d) GFP: 582 PGRN: 573 Male Female
  11. 11. End-stage Neuropathology Results Exogenous transgene cDNA • no qPCR signal for PGRN/GFP in formalin-fixed L3-L5 tissue specimens • no immunohistological evidence for GFP expression in region L3-L5 Neuronal Morphology • Tg male animals presented with neurons of a smaller diameter • Early-stage PGRN intervention had no effect on neuronal morphology † Healthy ǂ Atrophying Atrophying neurons Cumulative %Distribution Healthy neurons Diameter (µm) ǂ ǂ ǂ ǂ ǂ † † Diameter (µm)
  12. 12. End-stage Neuropathology Results Exogenous transgene cDNA • no qPCR signal for PGRN/GFP in formalin-fixed L3-L5 tissue specimens • no immunohistological evidence for GFP expression in region L3-L5 Neuronal Morphology • Tg male animals presented with neurons of a smaller diameter • Early-stage PGRN intervention had no effect on neuronal morphology Neuronal Viability • Male mSOD1 animals showed reduced neuronal viability • PGRN administration did not diminish neuronal loss in the targeted L3-L5 region αnti-Choline acetyltransferaseCresyl Violet Assay Lumbar Spinal Cord Level
  13. 13. End-stage Neuropathology Results Neuroinflammation • Tg mSOD1 animals showed a significant increase in neuroinflammatory processess • Early-stage PGRN cDNA delivery did not attenuate microglial/astrocytic activation Astrogliosis Microgliosis
  14. 14. LV-mediated Transgene Delivery No aberrant phenotype Severe paralysis Early stage PGRN intervention Late stage PGRN intervention - No effect on disease onset or duration - No mitigation of neuronal loss/neuroinflammation - Preserved ChAT positive motor neurons Summary of Outcomes How do we reconcile these paradoxical outcomes? Δ time • Transgene presence not confirmed at end point - early targeting may have altered expression levels within neuronal populations more susceptible to the neurotoxic insult
  15. 15. LV-mediated Transgene Delivery Summary of Outcomes How do we reconcile these paradoxical outcomes? • Transgene presence not confirmed at end point - early targeting may have altered expression levels within neuronal populations more susceptible to the neurotoxic insult • Use of deficient mSOD1 model animals Disease Severity No aberrant phenotype Severe paralysis Early stage PGRN intervention Late stage PGRN intervention - No effect on disease onset or duration - No mitigation of neuronal loss/neuroinflammation - Preserved ChAT positive motor neurons
  16. 16. Changes in mSOD1copy number Results Age (d)
  17. 17. Changes in mSOD1copy number Results Age (d) • G37R animals generated for this study showed a 46% lifespan increase • Intra-sex comparisons showed a significant colony effect (p<0.0001) • Intra-colony comparisons indicated a significant difference in the relative abundance of the mutant locus • Commercially-derived animals showed a more uniform distribution in ΔCT values; with a near 3-fold reduc- tion in mSOD1 copy number *** *** ***** Near 3-fold reduction Colony Source: Collaborator Commercial
  18. 18. Murine SOD1 ALS Model Systems Fertilized Egg Integrated mutant human SOD1 locus Pseudopregnant mouse Established transgenic lines ▪ Degree of transgene integration ▪ Phenotypic disease severity Rare recombination events Varied disease presentation Transgene level variation Discussion Zwiegers et al. J. of Negative Results in Biomedicne, Volume 13, Issue 1, 2014 Servier medical art modified under a Creative Commons Attribution 3.0 Unported License.
  19. 19. Discussion Original Founder Line Line Deposited at Repository Animals Generated for Breeding Progeny Generated for Study Deposited with an uncharacterized drop in copy number Changes in mSOD1copy number Data provided by the Jackson Laboratory Servier medical art modified under a Creative Commons Attribution 3.0 Unported License. Historical Controls
  20. 20. • With > 50 RCTs, positive pre-clinical findings have not been recapitulated in the clinic (Mitsumoto et al. 2014) Pre-clinical mSOD1 Model Patient Population Putative therapeutics Pre-clinical Translational Studies Discussion
  21. 21. • With > 50 RCTs, positive pre-clinical findings have not been recapitulated in the clinic (Mitsumoto et al. 2014) Pre-clinical mSOD1 Model Patient Population Putative therapeutics • mSOD1models do not serve as a predictor of clinical success Pre-clinical Translational Studies Discussion
  22. 22. - models a small % of ALS cases; logistical considerations for clinical studies Why are mSOD1models a poor predictor of clinical success? Zwiegers and Shaw. Journal of Con. Biomed Res., Volume 12, Issue 1, 2015, 4-22 Pre-clinical Translational Studies Discussion
  23. 23. - models a small % of ALS cases; logistical considerations for clinical studies - lack of replicative studies prior to clinical translation Why are mSOD1models a poor predictor of clinical success? Turner et al. The Lancet Neurology, Volume 12, Issue 3, 2013, 310 - 322 Pre-clinical Translational Studies Discussion
  24. 24. - models a small % of ALS cases; logistical considerations for clinical studies - lack of replicative studies prior to clinical translation - pre-clinical outcomes may just be a measurement of experimental noise (Scott et al, 2008) Why are mSOD1models a poor predictor of clinical success? Turner et al. The Lancet Neurology, Volume 12, Issue 3, 2013, 310 - 322 Pre-clinical Translational Studies Discussion Zwiegers et al. J. of Negative Results in Biomedicne, Volume 13, Issue 1, 2014 mSOD1 Copy Number Lifespan Phenotype Severity Increasing transgene dependent severity of ALS SOD1 low-copy number SOD1 high-copy number ↓ likelihood of (+)ve outcome↑ likelihood of (+)ve outcome
  25. 25. Conclusion Servier medical art modified under a Creative Commons Attribution 3.0 Unported License. • Must adopt a multi-modal approach to test pre-clinical efficacy prior to translational efforts Putative therapeutics ALS Patient PopulationPre-clinical ALS Models Invertebrate models Patient-derived Nerve Cells • Critical need to publish negative data so that non-productive research efforts can be identified/addressed • Pre-clinical ALS studies need to publish findings in the context of loci copy number • Research community needs to incentivize independent replicative studies prior to any clinical translation
  26. 26. References ALSoD, 2015. ALS Online Genetics Database. [online] Available at: http://alsod.iop.kcl.ac.uk/Index.aspx. Available at: http://alsod.iop.kcl.ac.uk/. Chiò, a. et al., 2013. Global epidemiology of amyotrophic lateral sclerosis: A systematic review of the published literature. Neuroepidemiology, 41(2), pp.118–130. Harms, M.B. & Baloh, R.H., 2013. Clinical Neurogenetics: Amyotrophic Lateral Sclerosis. Neurologic Clinics, 31(4), pp.929–950 Irwin, D., Lippa, C.F. & Rosso, a., 2009. Progranulin (PGRN) expression in ALS: An immunohistochemical study. Journal of the Neurological Sciences, 276(1-2), pp.9–13. Available at: http://dx.doi.org/10.1016/j.jns.2008.08.024. Miller RG, JD M, Moore DH. Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND). Cochrane Database of Syst Rev 2012. Mitsumoto, H., Brooks, B.R. & Silani, V., 2014. Clinical trials in amyotrophic lateral sclerosis: why so many negative trials and how can trials be improved? The Lancet Neurology, 13(11), pp.1127–1138. Available at: http://linkinghub.elsevier.com/retrieve/pii/S1474442214701292. Petkau, T.L. & Leavitt, B.R., 2014. Progranulin in neurodegenerative disease. Trends in Neurosciences, 37(7), pp.388–398. Available at: http://dx.doi.org/10.1016/j.tins.2014.04.003. Philips, T. et al., 2010. Microglial upregulation of progranulin as a marker of motor neuron degeneration. Journal of neuropathology and experimental neurology, 69(12), pp.1191–1200. Ryan, C.L. et al., 2009. Progranulin is expressed within motor neurons and promotes neuronal cell survival. BMC neuroscience, 10, p.130. Scott, S. et al., 2008. Design, power, and interpretation of studies in the standard murine model of ALS. Amyotrophic lateral sclerosis : official publication of the World Federation of Neurology Research Group on Motor Neuron Diseases, 9(1), pp.4–15. Sleegers, K. et al., 2008. Progranulin genetic variability contributes to amyotrophic lateral sclerosis. Neurology, 71, pp.253–259. Tang, W. et al., 2011. The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice. Science (New York, N.Y.), 332(6028), pp.478– 484 Turner, M.R. et al., 2013. Mechanisms, models and biomarkers in amyotrophic lateral sclerosis. Amyotrophic lateral sclerosis & frontotemporal degeneration, 14 Suppl 1, pp.19– 32. Available at: http://www.ncbi.nlm.nih.gov/pubmed/23678877. Van Kampen, J. & Kay, D., 2011. Progranulin gene therapy prevents plaque formation and synapse loss in a rodent model of AD. Alzheimer’s & Dementia, 7(4), pp.e7–e8. Available at: http://dx.doi.org/10.1016/j.jalz.2011.09.021. Van Kampen, J.M., Baranowski, D. & Kay, D.G., 2014. Progranulin gene delivery protects dopaminergic neurons in a mouse model of Parkinson’s disease. PLoS ONE, 9(5). Zwiegers, P., Lee, G. & Shaw, C. a, 2014. Reduction in hSOD1 copy number significantly impacts ALS phenotype presentation in G37R (line 29) mice: implications for the assessment of putative therapeutic agents. Journal of Negative Results in BioMedicine, 13(1), p.14. Available at: http://www.jnrbm.com/content/13/1/14. Zwiegers, P. & Shaw, C.A., 2015. Disparity of outcomes : the limits of modeling amyotrophic lateral sclerosis in murine models and translating results clinically. Journal of Controversies in Biomedical Research, 1(1), pp.4–22.
  27. 27. Supplementary Slides Methods
  28. 28. Methods • 10μm-thick, 100μm apart tissue sections were compared to a spinal cord atlas and organized accordingly Spinal Cord Organization L1 L2 L3 L4 L5 L6 Ventral Horn Cuneate & Gracile fasiculus Dorsal Horn
  29. 29. Methods Neuronal Viability Small motor neurons: Area = 130-240um2 VH CC Large motor neurons: Area = 240-950 um2 • The VH of cresyl violet stained tissues sections were captured at 10x • Colour inverted, contrast between objects enhanced • Image analysed by CellProfiler software; based on size criteria • Experimenter manually confirmed that identified objects were not artefacts Add details re:comparison of MN sizes; Friese et al paper incl
  30. 30. Methods Neuronal Morphology • Targeted the VH of cresyl violet stained tissues sections captured at 40x • Cell diameter estimated by bisecting cell of interest by two lines; size measured with ImageJ • “Healthy” neurons: - prominent nucleolus; definitive neuron-like morphology - 1-5 processes present within the plane of sectioning • “Atrophying” neurons: - neuronal shrinkage - hyperchromatic appearance ǂ ǂ ǂ ǂ † † ǂ Atrophying MN † Apparently Healthy MN
  31. 31. Methods Neuroinflammation • 6X6 superimposed counting grid; 40x images restricted to the VH Astrocytes
  32. 32. Methods Neuroinflammation Need to explain how WT lost GFP expression over time LV vs AAV
  33. 33. Methods Viral Comparisons AMS Biotechnology
  34. 34. Supplementary Slides Results/ Prelim data
  35. 35. Supplementary Slides Nakajima, Uchida, Kobayashi & Inukai. Target muscles for retrograde gene delivery to specific spinal cord segments. (2008)
  36. 36. Supplementary Slides Friese, A. et al. Gamma and alpha motor neurons distinguished by expression of transcription factor Err3. Proceedings of the National Academy of Sciences 106, 13588–13593 (2009).
  37. 37. Supplementary Slides Preliminary In Vitro results Produced in conjunction with R Cruz-Aguado [MPTP] µM
  38. 38. Supplementary Slides LV-mediated cDNA Expression A 20x D 20x B 40x E 40x F 100x C 100x 100x LV-PGRN 100x LV-SCRB 20x LV-SCRB 20x LV-PGRN Produced by MS Petrik & R Cruz-Aguado GFP and ChAT immunolabeling following gastroc-mediated LV injection show viral delivery to MN LV-SCRB LV-PGRN
  39. 39. Supplementary Slides • No significant difference in mSOD1 copy number between treatment groups mSOD1 Transgene Presence
  40. 40. Supplementary Slides
  41. 41. Jan 2012 Grad program start Dec 2010- Mar 2011 Establish a breeding colony & generate a cohort of experimental animals. May 2011 Weekly baseline assessment at 2-2.5 months of age Jun + Aug 2011 Bi-lateral LV injections at 3-3.5 months of age Jun 2011 – Jan 2013 Continue acquisition of behavioral data: Late 2012 – Mar 2013 euthanize animals exhibiting signs of severe ALS Servier medical art modified under a Creative Commons Attribution 3.0 Unported License. Behavioural Measures Histological Processing & Measurements EXPERIMENTAL TIMELINE Aug 2013 – Apr 2015 Tissue processing and post-study histological assays. Antal et al 2007 • Injection Sites Weight Leg Extension Reflex Score Wire Hang Neuronal Viability & Morphology Astrogliosis Microgliosis Experimental Timeline
  42. 42. Results Male Female • PGRN administration in Tg mSOD1 animals did not attenuate the disease-related weight loss • Male Tg animals appeared to exhibit a delayed phenotypic progression compared to females 9 10 11 12 13 18 18 19 21 23 53 57 61 64 65 67 69 71 73 75 78 79 81 83 86 87 89 9 10 11 12 13 18 18 19 21 23 53 57 61 64 65 67 69 71 73 75 78 79 81 83 86 87 89 Average Age (weeks) Average Age (weeks) Weight (Mean +/- SEM)
  43. 43. • PGRN administration did not mediate a positive effect in the LE reflex score • Tg mSOD1 animals started to show deficits at later time points 9 10 11 12 13 18 18 19 21 23 57 61 64 65 67 69 71 73 75 78 79 81 83 86 87 89 Average Age (weeks) 25 27 29 31 33 35 37 40 41 43 45 47 49 51 53 Average Age (weeks) Male Female ResultsLeg Extension reflex (Mean +/- SEM) 9 10 11 12 13 18 18 19 21 23 57 61 64 65 67 69 71 73 75 78 79 81 83 86 87 8925 27 29 31 33 35 37 40 41 43 45 47 49 51 53
  44. 44. • Male Tg animals appeared to exhibit an earlier deficit in comparison to female counterparts • PGRN administration in Tg mSOD1 animals mediated no positive effect on the measure Results 9 10 11 12 13 18 18 19 21 23 57 61 64 65 67 69 71 73 75 78 79 81 83 86 87 89 Average Age (weeks) 25 27 29 31 33 35 37 40 41 43 45 47 49 51 53 9 10 11 12 13 18 18 19 21 23 57 61 64 65 67 69 71 73 75 78 79 81 83 86 87 89 Average Age (weeks) 25 27 29 31 33 35 37 40 41 43 45 47 49 51 53 Male Female Wire Hang Test (Mean +/- SEM)
  45. 45. Neuronal Viability & Morphology Results Atrophying neurons Cumulative %Distribution Healthy neurons Diameter (µm) • In Tg mSOD1 animals, PGRN administration did not affect the size distribution of motor neuron cell bodies Mean Neuronal Size (Mean +/- SD)
  46. 46. Neuronal Viability & Morphology (Mean +/- SD) Results Small motor neurons: Area = 130-240um2 VH CC Large motor neurons: Area = 240-950 um2 • Early-stage PGRN administration mediated no significant effect on end-stage neuronal viability throughout the lumbar spinal cord
  47. 47. Neuronal Viability & Neuroinflammation (Mean +/- SD) Results • Tg male mSOD1 animals showed significantly reduced MN numbers in the targeted L3-L5 region as well as an increase in neuroinflammatory cells • PGRN administration at an early stage did not ameliorate MN loss or gliosis
  48. 48. Mean signal 33.47 34.01 12.6 12.27 SD 11.55 10.91 2.535 6.122 n 12 6 24 23 • Tg animals that were generated from Jax experienced a drop in copy number prior to being deposited at the repository • Decreased copy number resulted in an attenuated disease presentation, with the end point more than 15 months following lentiviral administration, compared to a colony generated from a local collaborator

×