AAV5-miHTT gene therapy demonstrates sustained

huntingtin lowering and functional improvement in

Huntington disease mouse models

Lisa Spronck1, Astrid Valles-Sanchez1, Taneli Heikkinen2, Martin de Haan1, Harald Petry1, Pavlina Konstantinova1

and Melvin Evers1,#

1Department of Research & Development, uniQure biopharma B.V., Amsterdam, the Netherlands, 2Charles River Discovery Research Services, Kuopio, Finland

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a CAG trinucleotide

repeat expansion in the first exon of the HTT gene. This CAG repeat expansion results in an expanded

polyglutamine repeat in the huntingtin protein, causing toxic gain-of-function and affecting numerous cellular

processes.

Our approach involves lowering huntingtin using gene transfer of a cassette encoding an engineered microRNA

targeting human HTT, delivered via adeno-associated viral vector serotype 5 (AAV5-miHTT). We previously

showed strong reduction of huntingtin in the brain of humanized HD mice (Miniarikova et al., 2016) and prevention of

neuronal dysfunction in lentiviral HD rats after single intracranial injection of AAV5-miHTT (Miniarikova et al., 2017).

AAV5-miHTT treatment translates to strong and sustained huntingtin lowering with reduction of

huntingtin aggregates and improvement of motor coordination and survival in HD rodents.

Study design Q175 KI mice

Sustained reduction of soluble mutant huntingtin protein

in Q175 KI mice after AAV-miHTT treatment

Contact: m.evers@uniQure.com

Dose dependent vector DNA and miHTT expression in cortex of Q175

KI mice 12 months post intra-striatal injection of AAV-miHTT

Quantitative PCR showed dose-dependent vector DNA levels (SYBR green) and mature

microRNA expression (custom-made taqman RT-PCR) in the cortex of Q175 KI mice 12

months after bilateral intra-striatal injection of the AAV5-miHTT vector.

(gc = genome copies; dashed line represents the LLOQ)

Strong reduction of mutant huntingtin aggregates by AAV5-miHTT

in Q175 KI mice

Soluble mutant huntingtin-

specific mesoscale

discovery assay showed a

significant average

reduction of mutant

huntingtin protein of up to

39% in the striatum and up

to 13% in the cortex of

Q175 KI, 12 months after

intra-striatal injection of

AAV5-miHTT.

A single stereotactic administration of AAV5-miHTT at one

of three doses into the striatum of Q175 KI mice and wild

type littermates, followed for 12 months.

Behavioural tests were performed to assess motor skills at

several time points. In this study the Q175 KI mice did not

develop a behavioural phenotype. Brain biopsies were

taken for biomolecular analysis and huntingtin aggregate

staining for the Q175 KI mice.

A one-time bilateral intra-

striatal injection of AAV5-

miHTT resulted in a significant

increase in median survival

(149 days) compared with

vehicle-treated R6/2 HD mice

(120 days) (p=0.0270)

A significant improvement in

motor coordination on the

rotarod at 12 weeks of age with

a significant increase of 21

seconds in latency time was

observed in AAV-miHTT treated

R6/2 HD mice.

1. Upon parenchymal injection AAV5-miHTT binds to neuronal cell-surface receptors and is internalized

2. Transport to the nucleus and uncoating of the miHTT transgene which remains mostly episomal

3. Expression and processing of miHTT transgene by the endogenous RNA interference machinery

4. Hairpin structured precursor is transported to the cytoplasm and further processed to mature miHTT

5. Mature miHTT is loaded in the RNA-induced silencing complex and binds HTT mRNA

6. HTT mRNA is cleaved and degraded, resulting in lowering of huntingtin protein translation

Here, we have investigated long term distribution, efficacy and tolerability of AAV5-miHTT

treatment in the Q175 Knock In (KI) (heterozygous) (Menalled et al., 2012) and the R6/2 HD

mice (Mangiarini et al., 1996).

Data were evaluated using ordinary one-way ANOVA and

post-hoc Dunnett's test * p<0.05, ** p<0.01, ***p<0.001

Data were evaluated using ordinary one-way ANOVA and

post-hoc Dunnett's test * p<0.05

Data were evaluated using ordinary one-way

ANOVA and post-hoc Dunnett’s test * p<0.05

Group and strain

N=8

Treatment AAV-miHTT

1 – wild type Vehicle 0

2 – wild type AAV-miHTT High

3 – Q175 HET KI Vehicle 0

4 – Q175 HET KI AAV-miHTT Low 1x

5 – Q175 HET KI AAV-miHTT Mid 5x

6 – Q175 HET KI AAV-miHTT High 25x

Study design R6/2 HD mice

Staining of brain regions for huntingtin aggregates using

the EM48 antibody showed a dose-dependent reduction

of huntingtin aggregates in the striatum and cortex of

Q175 KI mice, 12 months after intra-striatal injection of

AAV-miHTT

Soluble mutant huntingtin protein

Four week increase in median survival of R6/2 mice

after AAV5-miHTT treatment

The time until touching

hindlegs and until clasping

was recorded at 12 weeks of

age and a trend of increasing

time to clasp/touch was

observed in AAV-miHTT

treated animals.

R6/2

vehic

le

R6/2

hig

h d

ose

AA

V-m

iHT

T

= behavioural testAAV5-miHTT Necropsy

Age (months): 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Mutant huntingtin aggregates

Clasping

p=0.12

Functional improvement in R6/2 mice upon AAV5-miHTT treatment

p=0.12

AAV5-miHTT Survival

Age (weeks): 0 4 8 12 16 20 24 The R6/2 mice (and wild type littermates) were injected

into the striatum bilaterally with a single stereotactic

administration of AAV5-miHTT at one of three doses and

followed for 5 months.

Behavioural tests showed a strong phenotype and survival

appeared to be an important outcome measure.

= behavioural test

Group and strain

N=20

Treatment AAV-miHTT

1 – wild type Vehicle 0

2 – R6/2 Vehicle 0

3 – R6/2 AAV-miHTT Low 1x

4 – R6/2 AAV-miHTT Mid 5x

5 – R6/2 AAV-miHTT High 25x

Data were evaluated using log rank and Gehan-Breslow-

Wilcoxon test * p<0.05

Striatum Cortex

Veh

icle

Mid

do

seH

igh

do

se