Zwiegers Thesis Defense

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

 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

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

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

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
• 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)

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

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?

Lentiviral –mediated transgene delivery
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

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

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

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)

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

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

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

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

Changes in mSOD1copy number
Results
Age (d)

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

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

Discussion
Original Founder Line
Line Deposited at Repository
Animals Generated for Breeding
Progeny Generated for Study
Deposited with an uncharacterized
drop in copy number
Data provided by the Jackson Laboratory
Historical Controls

• 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

• 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
Discussion

- 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
Discussion

- lack of replicative studies prior to clinical translation
Discussion

- lack of replicative studies prior to clinical translation
- pre-clinical outcomes may just be a measurement of experimental noise
(Scott et al, 2008)
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

Conclusion
• 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

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.

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

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

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

Methods
Neuroinflammation
• 6X6 superimposed counting grid; 40x images restricted to the VH
Astrocytes

Methods
Neuroinflammation
Need to explain how WT lost GFP expression over time
LV vs AAV

Methods
Viral Comparisons
AMS Biotechnology

Supplementary Slides
Results/ Prelim data

Nakajima, Uchida, Kobayashi & Inukai. Target muscles for retrograde gene delivery to specific
spinal cord segments. (2008)

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).

Preliminary In Vitro results
Produced in conjunction with R Cruz-Aguado
[MPTP] µM

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

• No significant difference in mSOD1 copy number between treatment groups
mSOD1 Transgene Presence

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
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

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)

• 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

• 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)

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)

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

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

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

Zwiegers Thesis Defense

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