Allele-selective suppression of mutant huntingtin in primary
human myeloid cells
James RC Miller1, Edith Pfister2, Wanzhao Liu2, Lori Kennington2, Ralph Andre1, Gary Ostroff3, Neil Aronin2 and Sarah J Tabrizi1
1UCL Institute of Neurology, Dept. of Neurodegenerative Disease, London, UK.
2University of Massachusetts Medical School, Dept. of Medicine, Division Endocrinology & Metabolism, Worcester, MA, USA.
3University of Massachusetts Medical School, Program in Molecular Medicine, Worcester, MA, USA.
Introduction
Huntington’s disease (HD) is a fatal, inherited neurodegenerative condition caused by a CAG repeat expansion in the huntingtin (HTT) gene. A
repeat length of more than 40 is fully penetrant and results in the disease. Expression of the mutant protein (mHTT) is associated with disease
pathology, so lowering cellular HTT levels is a promising therapeutic strategy. We have previously demonstrated non-selective suppression of
wild-type and mHTT in primary human myeloid cells using small interfering RNA (siRNA). However, wild-type huntingtin has a number of
important roles in cellular function and an ideal therapy would selectively lower mHTT levels while preserving wild-type. In this study we aim to
selectively suppress mHTT in primary human HD cells using siRNA targeted to single nucleotide polymorphisms (SNPs) in the HTT gene.
Rationale
Targeting the CAG repeat with siRNA is technically challenging – SNPs
provide a promising alternative for discriminating between wild-type and
mHTT. We have chosen to target three SNPs located in exon 50, exon 57
and the 3’ UTR of the HTT gene. This approach will be applicable to
approximately 75% of HD patients (Pfister et al. Current Biology 2009).
Results
A
**
Figure
2.
Allele-selective
knockdown of HTT in primary
human myeloid cells. (A) HTT
mRNA levels following treatment
with siRNA targeting rs362331
(exon 50), n=8. (B) HTT mRNA
levels following treatment with
siRNA targeting rs362273 (exon
57), n=4. (C) Dose-response
analysis of siRNA targeting the A
allele of rs362273 (exon 57), n=2.
Data shown as mean +/- SEM,
paired t-test. *p<0.05, **p<0.01,
***p<0.001.
***
Patients are initially genotyped to determine heterozygosity at SNPs of
interest. SNP identities are then linked to the wild-type and mutant alleles
using previously described methodology (Figure 1).
A
B
Sample
Number
Exon 50
rs362331
Exon 57
rs362273
3' UTR
rs362307
Mutant
Wild-type Mutant
Wild-type Mutant
Wild-type
41159
C
T
G
A
C
C
41261
T
T
A
A
T
C
41275
T
T
A
A
T
C
41566
C
T
G
A
T
C
41568
T
T
A
A
T
C
41690
C
T
G
A
T
C
43046
C
T
G
A
T
C
43200
T
T
A
A
T
C
Figure 1. SNP linkage by circularization (SLiC). (A) The SLiC strategy.
cDNA is circularised to bring the target SNP into close proximity with the CAG
repeat in exon 1 of the HTT gene. This allows a relatively short section of
cDNA to be sequenced to determine SNP linkage with either the wild-type or
mutant allele (reproduced with permission from Liu et al. Nat Methods 2008).
(B) Representative SLiC results showing SNP linkage in individual subjects.
Methods
Human HD myeloid cells are hyper-reactive in response to LPS – we have
previously demonstrated that this phenotype is reversible following total HTTlowering by gene silencing. The innate immune system provides a convenient
model for testing potential therapies for HD due to this clear phenotype and
the ready availability of peripheral blood from patient volunteers.
Monocytes are isolated from human peripheral blood using anti-CD14 MACS
beads and cultured in RPMI media containing 20 ng/ml GM-CSF to
differentiate them into macrophages.
Traditional transfection methods are difficult in primary myeloid cells and only
low transfection rates can be achieved (~10%). Yeast-derived glucanencapsulated siRNA particles (GeRPs) take advantage of the phagocytic
properties of myeloid cells to achieve transfection. The siRNA is packaged
into a glucan shell which is engulfed by the myeloid cell, leading to siRNA
uptake and subsequent mRNA knockdown (Aouadi et al. Nature 2009).
Transfection efficiencies as high as 90% can be achieved using this method.
B
C
*
*
Conclusions
Analysis of mRNA expression has revealed allele-selective knockdown of
target SNPs in primary human myeloid cells. The SNPs can then be linked to
wild-type and mHTT by SLiC.
Selectivity of knockdown varies depending on the SNP – siRNA targeting
rs362331 offers greater discrimination than siRNA targeting rs362273.
Selectivity of knockdown may be optimised by varying the dose of siRNA.
These findings represent the first steps in evaluating siRNA-mediated alleleselective suppression in primary human HD cells, while demonstrating the
feasibility of tailoring treatments to an individual genotype.
Future work
RNA data will be validated using TR-FRET to measure total and mHTT
protein levels following allele-selective knockdown.
siRNA dose ranging will be further evaluated to optimise allele-selectivity.
Cells are harvested 72 hours after GeRP treatment and mRNA levels are
measured by qPCR using TaqMan probes specific for each SNP allele.
We would also like to thank UCLH, UCL
Biomedical Research Centre and NS
38194 (NA) for their support.
We will extend these findings by developing allele-selective silencing targeting
rs362307 in the 3’ UTR of the HTT gene.
These siRNAs will be used to investigate the effects of selectively knocking
down mHTT on the hyper-reactive phenotype seen in HD myeloid cells.