Presented at the 3rd Synthetic Biology Congress in London. To find out more, visit: www.global-engage.com Mark Isalan, Reader in Gene Network Engineering at Imperial College London, presents results of applying lessons learnt from synthetic biology to the treatment of Huntington’s disease. Watch the full presentation at: blog.globalengage.co.uk/synthetic-biology-applications-huntingtons-disease

1. Mark Isalan
m.isalan@imperial.ac.uk
Gene Network Engineering Group
Imperial College London
Synthetic biology towards gene therapy:
synthetic repressors in Huntington's disease

2. Synthetic biology
for brain gene therapy?
Agustín-Pavón C, Isalan M.
Synthetic biology and
therapeutic strategies for the
degenerating brain. Bioessays.
Aug 6. doi:
10.1002/bies.201400094 (2014).

3. Huntington’s Disease
• >1 in 10,000 people (up to 1 in 400 in elderly)
• Autosomal dominant
• Typical onset: 35 to 45 yrs
• Abnormal movements, loss of cognitive function, dementia and death
•Pathology: specific neuronal cell death in striatum and frontal cortex
•Only palliative treatments, although RNAi and antisense are promising

4. The HD mutation and huntingtin protein
HD Protein (huntingtin)
Huntingtin:
• expressed throughout development and knock-out
mice die during embryonic development
• expressed in all tissues
180 kb DNA
66 67321
CAG CAG CAG….
> 40
> ~70
unaffected
adult onset HD
juvenile onset HD
36-39
6-35
incomplete penetrance
Glutamines
QQQ…….

5. Shutting off mutant Htt reverses the disease phenotype
“artificial” inducible system provided the rationale for “Htt-
lowering” strategies

6. Engineering zinc fingers to
bind new DNA sequences

7. The zinc finger code
e.g. R:g, Q:a, T:t, D:c
5’-GCA-3’

8. Zinc fingers to bind poly-CAG
•Huntington's disease: expanded poly-CAG repeats
•Zinc fingers to bind GCA, GCT (ie CAG)
PNAS 109:E3136 (2012)

9. • STHdh cells: Q7 or Q111
• retroviral delivery (pRetroX )
• extract protein and RNA after 20 days
Stable long-term ZF expression and repression in cells
PNAS 109:E3136 (2012)

10. Short CAG-repeat and neighbouring genes unaffected by ZF
7kb 188kb

11. From in vitro to in vivo
- R6/2 HD mouse model
- Age of onset of HD symptoms: 9 to 11 weeks
- Life expectancy: 12 to 17 weeks

12. Striatal injection of AAV2/1-GFP (CAG-WPRE)
one
hemisphere
both
hemispheres

13. ZF11xHunt-KoxI reduces mut HTT mRNA
in a dose-dependent manner
AAV
GFP
AAV
ZF11xH
KoxI
Treatment
with AAV2/1
ZF11-Kox-1
(2 weeks)
PNAS 109:E3136 (2012)

14. striatumcortex
untreated ZF11xHunt-KoxI
R6/2 brain IHC with anti-HttAAV
GFP
AAV
ZF11xH
KoxI
40%
Reduction
p=0.03
ZF11xHunt-KoxI reduces HTT aggregates
in the injected hemisphere
Treatment
with AAV2/1
ZF11-Kox-1
(2 weeks)
PNAS 109:E3136 (2012)

15. R6/2 phenotype: clasping and rotarod
2 4 6
Weeks after injection ZF expression transient at this stage

16. The deimmunization of synthetic zinc finger repressors
reduces toxicity in gene therapy for Huntington’s disease

17. Iba1+ staining – microglial activation
Injected Contralateral
ZF-KOX1mZF-KRABGFPPBS
Iba1, 6 weeks
B. B’.
D. D’.
F. F’.
H. H’.
Injected Contralateral
ZF-KOX1mZF-KRABGFPPBS
GFAP, 6 weeks
B. B’.
D. D’.
F. F’.
H. H’.
GFAP staining – reactive astrocytes

18. Neuronal cell death – Neu-N
Injected Contralateral
ZF-KOX1mZF-KRABGFPPBS
Neu-N, 6 weeks
B. B’.
D. D’.
F. F’.
H. H’.

19. Long term in vivo repression?

20. Repression of mutant HTT ~25% in whole brain after 24 weeks!
Other genes (incl. mouse endogenous WT Htt) unaffected
pNSE-mZF-KRAB constructs mediate long-term repression in
whole brain samples after single intraventricular injections.
Agustín-Pavón C et al. Molecular Neurodegeneration 11(1):64 (2016).

21. • peptide host-adaptation reduces toxicity
• promoter design allows long term expression in vivo (>6 months)
• mutant huntingtin repression in the range 25-77% is achieved in the
whole brain over this period
• WT alleles are unaffected
Conclusions
Agustín-Pavón C, Mielcarek M, Garriga-
Canut M & Isalan M. Deimmunization for
gene therapy: host matching of synthetic
zinc finger constructs enables long-term
mutant Huntingtin repression in mice.
Molecular Neurodegeneration 11(1):64
(2016).

22. Therapeutic strategies in HD
The HD mutation
DNA
The message
Zinc finger
gene silencing
RNA
silencing
The mutated
huntingtin protein
Anti-aggregation approach
Proteasome modifiers
Epigenetic inhibitors
PATHOLOGY

23. Thanks
Imperial
College
London
Richard
Amaee
Marc
Sturrock
Marta
Ciechonska
Vivek Raj
Senthivel
Diego
Barcena
Natalie
Scholes
Andreas
Broedel
Michal
Mielcarek
Alice
Grob
Alicia
Broto
Masue
Marbiah
Mireia
Garriga-Canut
Carmen
Agustin-Pavon