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 SclerosisCommittee 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 SclerosisBackground

• 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

http://www.servier.com/Powerpoint-image-bank

http://creativecommons.org/licenses/by/3.0/

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

muscles1 week 2 weeks 4 weeks

Overt Neurobehavioural MeasuresResults

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 MeasuresResults

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 NeuropathologyResults

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

Cu

mu

lati

ve

% D

istr

ibu

tio

n

Healthy neurons

Diameter (µm)

ǂ

ǂǂ

ǂ

ǂ

†

†

Diameter (µm)


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 acetyltransferase Cresyl Violet Assay

Lumbar Spinal Cord

Level


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 numberResults

Age (d)

Changes in mSOD1copy numberResults

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

Changes in mSOD1copy 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 StudiesDiscussion

• 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


- 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



- lack of replicative studies prior to clinical translation





- lack of replicative studies prior to clinical translation

- pre-clinical outcomes may just be a measurement of experimental noise

(Scott et al, 2008)




Zwiegers et al. J. of Negative Results in Biomedicne, Volume 13, Issue 1, 2014

mSOD1 Copy Number

Lif

esp

an

Ph

en

oty

pe

Severi

ty

Increasing transgene dependent severity of ALSSOD1 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.

http://dx.doi.org/10.1016/j.tins.2014.04.003

http://www.ncbi.nlm.nih.gov/pubmed/23678877

http://dx.doi.org/10.1016/j.jalz.2011.09.021

Supplementary Slides

Methods

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

MethodsNeuronal 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

MethodsNeuronal 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

MethodsNeuroinflammation

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

Astrocytes

MethodsNeuroinflammation

Need to explain how WT lost GFP expression over time

LV vs AAV

MethodsViral Comparisons

AMS Biotechnology


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).

Supplementary SlidesPreliminary In Vitro results

Produced in conjunction with R Cruz-Aguado

[MPTP] µM

Supplementary SlidesLV-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 2012Grad 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 2013euthanize animals exhibiting signs of severe ALS


Behavioural MeasuresHistological 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 89Average 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


25 27 29 31 33 35 37 40 41 43 45 47 49 51 53


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

Cu

mu

lati

ve%

Dis

trib

uti

on

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