Reflections on genomic editing in nonhuman primates

Marina E. Emborg, M.D. Ph.D.

ILAR 12-07-2015

The animal model and how it is used impact clinical translation

Genomic Editing (GE) in non human primates (and other species)

Applications of GE

MODELS

THERAPIES

GE somatic
GE germline

GE somatic
GE germline

Before applying Genomic Editing in nonhuman primates.

Why do we do research in
nonhuman primates?
To understand disease
To test first-in-class and invasive therapies

Monkeys vs. rodents - Behavioral skills

UC Davis - NPRC

Monkey models provide outcome measures that have clinical impact

Monkeys vs. rodents Brain complexity

Rhesus Monkey

Mouse

Monkey models allow development of invasive

methods and can provide post mortem evidence of
success or failure

Parkinsons disease (PD)

PD is typically described as a progressive neurodegenerative movement disorder

associated to the loss of DA neurons in the SNpc and the presence of LBs and LNs
Mainstay of therapy: DA replacement.

RN

CC

SN

Aging

Goedert et al, 2001

Vermilyea and Emborg,, 2015

PD

PD affects multiple CNS and PNS areas

Cortex
Locus Coeruleus
Substantia Nigra
Nucleus Basalis of Meynert
Dorsal Motor Nucleus of the Vagus
Serotonergic Raphe Nuclei
Pedunculopontine Nucleus
Reticular Formation of
the Pons and Midbrain

Intermediolateral Cell Column

of the Spinal Cord

Fitzpatrick et al, Antiox. Red. 2009

Edinger-Westphal Nucleus
Paranigral Nucleus
Hypothalamus
Olfactory Bulb

Peripheral Sympathetic Ganglia

PD symptoms reflect the complexity of the disease

Swanson and Emborg, 2009

What causes PD?

Hirsch et al, 2013

What causes PD?

themagicshop.com.au

We do not know what triggers

PD

Alpha syn
protein
aggregation

Risk factors identified:

Aging
Environment
Genetics

Genes robustly associated with PD/Parkinsonism

Autosomal dominant

Autosomal recessive

SNCA (PARK1, PARK4)

PARKIN (PARK2)

LRRK2 (PARK8)

PINK1 (PARK6)

VPS35

DJ1 (PARK7)

EIF4G1

Puschman, 2013

The LRRK2 protein is a large (2527 aminoacids) GTP-regulated serine/threonine kinase

that contains several protein-protein interaction domains. It is present in the brain in areas
key to PD. Its role is not clear.

Since its discovery in 2004, LRRK2 mutations have been identified as the most common
genetic cause of familial and non familial PD (1-30% of all PD cases depending on
population studied).

LRRK2G2019S is the most frequent mutation. Patients carrying this mutation show a clinical
and neuropathological profile indistinguishable from sporadic PD.

PD monkey models currently available

MODEL

PD
etiology Neuroprotective Restorative

LID

Non motor
signs

Aging
Alpha synuclein gene
transfer

1--2

1--2

1--2

1--2

6-OHDA intrastriatal

1--2

1--2

1--2

MPTP systemic acute

MPTP systemic
intermediate

1--2

MPTP intracarotid

1--2

MPTP intracarotid + iv

MPTP systemic chronic

Model rating: 1- most suitable, 5-inadequate

Cardiac dysautonomia model in PD by systemic 6-OHDA

Emborg, ILAR 2007

Limitations of current models

Animal models

PD

Neurochemical focus

DA

DA++, but also NA, 5HT, Ach

Neuroanatomical focus

SNc

SNc, SNr and other CNS and PNS systems

cell loss

cell loss, lewy bodies

Pathogenesis

toxic

unknown toxic x genetic interaction

Time course

acute

slow progressive

Cellular pathology

Recovery
Other

significant (unless lesion complete) progressive once compensation exhausted

BBB opening associated to treatment,
glial scars in injection sites
****

Compared to other species NHP transgenesis has lagged

Lentiviral-based:

GFP rhesus (Chan et al., 2001; Wolfgang

et al., 2001; Niu et al, 2010) marmoset
(Sasaki et al., 2009).
Huntingtons disease GFP+CAG rhesus
huntingtin repeat (Yang et al, 2008)
PD human A53T-syn cynomolgous (Niu et
al., 2015).

CRISPR-based:

Ppar-g and Rag1 (Niu et al, 2014)

Duchenne syndrome (Chen et al, 2015)
X-linked adrenal hypoplasia congenita
(AHC) and hypogonadotropic
hypogonadism (HH), Dax1 knockout
cynomolgous fetus (Kang et al, 2016)

progress,
Mapping human and non human genome,
Identifying disease-associated genes,
Limitations of current models and therapies, are fueling change

Genes robustly associated with PD/Parkinsonism

Autosomal dominant

Autosomal recessive

SNCA (PARK1, PARK4)

PARKIN (PARK2)

LRRK2 (PARK8)

PINK1 (PARK6)

VPS35

DJ1 (PARK7)

EIF4G1

Puschman, 2013

With exception of SNCA trisomy, PD has been linked to single point mutations.
This suggests that the classic approach of transgenic overexpression of a mutated
protein may be less effective at producing the human PD phenotype than introducing the
identical allelic variant found in the human disease-associated allele.
GE can provide a solution.

Genomic Editing (GE) in non human primates (and other species)

Applications of GE

MODELS

THERAPIES

GE somatic
GE germline

GE somatic
GE germline

What gene is relevant to the disease?

Autosomal dominant

Autosomal recessive

SNCA (PARK1, PARK4)

PARKIN (PARK2)

LRRK2 (PARK8)

PINK1 (PARK6)

VPS35

DJ1 (PARK7)

EIF4G1

Puschman, 2013

What species?

Rhesus at day 15

Schultz-Darken et al, 2015

Marmoset at day 15

Transgenic Marmosets for Translational Stem Cell Research

A CRISPR approach

Genomic edited
offspring
LRRK2 G2019S

Zygotic cytoplasmic injection

IVF/ICSI
Embryo
Transfer
recipient

iPSC

Therapeutic
cells

Common marmoset monkeys developmental milestones

Schultz-Darken et al., Dev Psych. 2015

Do nonmotor signs predict PD?

Norepinephrine loss & increase in toxic metabolites
reduced VMAT2 expression
Neocortex

Locus coeruleus

CNS: impaired cognitive functions, depression,

sleep disturbance, anxiety
Detection of cognitive signs & other disorders at age range
of greatest PD risk? Imaging of impaired executive
function? Detection of toxic metabolite in periphery?

Cardiac/autonomic: orthostatic hypotension, fatigue,

dysrhythmias
Detection of autonomic, cardiac, or GI dysfunction at age range of
greatest PD risk? Imaging impaired uptake of NE analogue in cardiac
tissue? Detection of toxic metabolite in periphery?

Initiate pharmacological or lifestyle strategies to attenuate further dopamine

loss & reduce toxic metabolites
reduced VMAT2 expression
Salvatore, Disabrow and Emborg, J Neurochem. 2014

Genomic Editing (GE) in non human primates (and other species)

Applications of GE

MODELS

THERAPIES

GE somatic
GE germline

GE somatic
GE germline

Putamen

Nigra

GFP

CD68

CD3

CD8

HLA-DR
GFAP
(no nissle)
(no
(nonissle)
Nissl counterstaining)

Cell replacement is more than playing a puzzle

Bing images

Host and donor have a role in graft efficacy

Fitzpatrick et al, Antiox. Red. 2009

Cell replacement in a GE PD model provide an opportunity to assess the impact of

mutations in cell survival
GE Cell grafts can also allow to assess alternatives for personalized medicine
GE for DREADD approach to modulate IPSCs-derived dopamine neurons activity

Genomic Editing (GE) in non human primates (and other species)

Applications of GE

MODELS

THERAPIES

GE somatic
GE germline

GE somatic
GE germline

.of humans and non human primates

http://www.buildabear.com

Considerations BEFORE applying GE for NHP disease modeling

Disease-related issues: mutation,
age of onset, symptoms
Species: genome, lifespan,
developmental patterns,
housing needs, breeding demands,
impact of individual genetic
background (outbred vs. inbred)
Testing: specific to disease, age,
species
GE-related issues: methods for plasmid
inoculation, uniplex vs. multiplex,
random insertions/deletions, off
target effects, mosaicism

Vermilyea and Emborg, J Neurosci Meth 2015

Keep in mind what if scenario and be ready with clinical

primate veterinarian support.

Practices that help establish ethical basis for initiating first-in-human trials

Concern

Practices

Internal validity

A priori hypothesis stated

A priori power calculations
Randomization
Blinded treatment allocation
Blinded outcome assessments
Discussion of missing data

External validity

Explanation for selection of animal model

Explanation of selection of outcome measures
Correspondence of trial with preclinical
conditions

Optimism and

Critical review of preclinical data

publication bias

Full publication of preclinical studies

Kimmelman et al, Mov Dis. 2009

Special Thanks
UW - Madison:
Scott Vermilyea
Scott Guthrie
Kat Braun

NIH:
Mark Cookson
Carneggie Mellon
Alex London

Ted Golos
Jenna Kropp
Greg Wiepz
Michael Meyer

MacGill University:
Jonathan Kimmelman

Su-Chun Zhang
Jamie Thomson
Nancy Schultz-Darken
Jon Levine
Buddy Capuano

This research was supported by grants from the UW ICTR,

UW VCRGE and the NIH

Questions? emborg@primate.wisc.edu