114P Queen Elizabeth II Conference Centre London
BPS Winter Meeting 2011

 

 

Therapeutic siRNA – Detecting Immunostimulatory Sequences

Jill Darton1, Ashley Broom3, Jill Coates1, Joel Parry3, Douglas Ball2, Stephen Hughes1, Kenneth Clark1, Mark Edbrooke1. 1GlaxoSmithKline, siRNA DPU, Stevenage/Herts/SG1 2NY, UK, 2GlaxoSmithKline, Allergic Inflammation DPU, Stevenage/Herts/SG1 2NY, UK, 3GlaxoSmithKline, IPT Molecular Pathology & Toxicology, Ware/Herts/SG12 0DP, UK.

 

Immunostimulation, as a consequence of Toll-like receptor (TLR) activation (Agrawal et.al., 2004), is a potential safety risk and a complicating factor when assessing the effects of nucleic acids in preclinical efficacy models. Since we are interested in exploring the therapeutic potential of inhaled short-interfering RNAs (siRNAs), the aim of this work was to put in place a series of in-vivo studies to screen novel siRNAs for possible immunostimulatory effects and to identify a suitable immunologically silent control siRNA for use in future studies.

Experiments were undertaken to investigate two non-targeting siRNAs, termed Universal Controls 2 and 3 (UC2 and 3).

siRNA sequence UC2
5’ U U A A U A G G U A U G U U A U A U G T T 3’ (S)
3’ U U A A U U A U C C A U A C A A U A U A C 5’ (AS)

 

siRNA sequence UC3
5’ G U A U G A C C G A C U A C G C G U A T T 3’ (S)
3’ U U C A U A C U G G C U G A U G C G C A U 5’ (AS)

 

Each non-targeting siRNA was administered as a single bolus intratracheal instillation (i.t.) instillation (10mg/kg) into the airway of the rat. Polyinosine:polycytidine (poly I:C) (1mg/kg), a TLR3 agonist, was administered to serve as a positive control. Bronchoalveolar lavage (BAL) was performed 24h post-dose to assess leukocyte infiltration into the lung, a marker of inflammation. Immediately after completion of lavage, samples of rat lung were removed from treated animals and snap frozen into liquid nitrogen for subsequent mRNA analysis by QRT_PCR.

Results obtained from both the lavage and transcriptional assessments indicated that UC3 induced an inflammatory response in the airway of the rat. This was exemplified by an increase in the numbers of infiltrating leukocytes into the lung, notably neutrophil induction, 0.39x10-6 (PBS) vs 22.58x10-6 (UC3) and by elevation of pro-inflammatory marker genes. Interferon-induced GTP-binding protein MX1, CXCL10 and oligoadenylate synthetase-like (OASL) protein were the most responsive genes in terms of fold change in expression ((34 (17-41), 54 (24-70) and 43 (19-63), respectively)). The magnitude of induction of these genes in response to UC3 was similar to that evoked by the positive control poly I:C. In contrast, UC2 displayed minimal immunostimulatory effects in the rat airway compared to those animals treated with UC3.

In conclusion, the use of transcriptional analysis coupled to assessment of BALf cellularity provides robust analysis of the immunostimulatory potential following intratracheal dosing of siRNAs to rats and is a useful model for screening novel siRNAs for undesired effects. This model was successfully used to identify unexpected immunostimulatory activity in a control siRNA and to select an alternative control sequence which avoids this liability.

 

Agrawal S. et. al., (2004) Nature Biotechnology, 22(12):1533-1537.