Slide 1

Mark Isalan m.isalan@imperial.ac.uk

Gene Network Engineering GroupImperial College London Synthetic biology towards gene therapy: synthetic repressors in Huntington's disease

1

48

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

2

Huntingtons 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

3

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

6667321CAG CAG CAG.

> 40> ~70unaffectedadult onset HDjuvenile onset HD36-396-35incomplete penetrance

GlutaminesQQQ.

4

Shutting off mutant Htt reverses the disease phenotype

artificial inducible system provided the rationale for Htt-lowering strategies

5

Engineering zinc fingers to bind new DNA sequences

6

The zinc finger code

e.g. R:g, Q:a, T:t, D:c5-GCA-3

7

48

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)

8

STHdh cells: Q7 or Q111

retroviral delivery (pRetroX )

extract protein and RNA after 20 daysStable long-term ZF expression and repression in cellsPNAS 109:E3136 (2012)

9STHdh cells30 are an established neuronal progenitor cell line from E14 striatal primordia, derived from wt mice (STHdhQ7/HdhQ7), or knock-ins, where the first exon of the mouse HTT gene has been replaced by a human exon with 111 CAG repeats (STHdhQ111/HdhQ111).

Short CAG-repeat and neighbouring genes unaffected by ZF

7kb 188kb

10The two adjacent genes, G protein-coupled receptor kinase 4 (~7 kb upstream) and G-protein signaling 12 (~188 kb downstream), were both unchanged

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

11

Striatal injection of AAV2/1-GFP (CAG-WPRE)

one hemisphereboth hemispheres

12

ZF11xHunt-KoxI reduces mut HTT mRNA in a dose-dependent manner

AAVGFPAAVZF11xHKoxITreatmentwith AAV2/1 ZF11-Kox-1(2 weeks)

PNAS 109:E3136 (2012)

No striatal volume or cell density in the striatum changes, showing that reduction in mutant HTT aggregates is not due to ZF toxicity13

striatumcortexuntreatedZF11xHunt-KoxIR6/2 brain IHC with anti-Htt

AAVGFPAAVZF11xHKoxI40%Reductionp=0.03ZF11xHunt-KoxI reduces HTT aggregates in the injected hemisphereTreatmentwith AAV2/1 ZF11-Kox-1(2 weeks)

PNAS 109:E3136 (2012)

No striatal volume or cell density in the striatum changes, showing that reduction in mutant HTT agrefgates is not due to ZF toxicity14

R6/2 phenotype: clasping and rotarod

2 4 6 Weeks after injection ZF expression transient at this stage

15

The deimmunization of synthetic zinc finger repressors reduces toxicity in gene therapy for Huntingtons disease

16

Iba1+ staining microglial activation

InjectedContralateralZF-KOX1mZF-KRABGFPPBSIba1, 6 weeksB.B.D.D.F.F.H.H.

InjectedContralateralZF-KOX1mZF-KRABGFPPBSGFAP, 6 weeksB.B.D.D.F.F.H.H.GFAP staining reactive astrocytes

17

Neuronal cell death Neu-N

InjectedContralateralZF-KOX1mZF-KRABGFPPBSNeu-N, 6 weeksB.B.D.D.F.F.H.H.

18

Long term in vivo repression?

19

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. Agustn-Pavn C et al. Molecular Neurodegeneration 11(1):64 (2016).

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

Agustn-Pavn 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).

21

Therapeutic strategies in HD

The HD mutationDNA

The message

Zinc finger gene silencing RNA silencing The mutated huntingtin proteinAnti-aggregation approachProteasome modifiers Epigenetic inhibitors

PATHOLOGY

ThanksImperialCollegeLondon

RichardAmaeeMarcSturrock

MartaCiechonskaVivek RajSenthivel Diego BarcenaNatalie Scholes

AndreasBroedelMichalMielcarek AliceGrobAlicia Broto Masue Marbiah

MireiaGarriga-CanutCarmenAgustin-Pavon

23