UCL Great Ormond Street Institute of Child Health


Great Ormond Street Institute of Child Health


Gene silencing in Cryopyrin-associated periodic syndrome (CAPS)

Supervisors: Professor Paul Brogan, Professor Despina Eleftheriou, Dr Ying Hong

Cryopyrin-associated periodic syndrome (CAPS) is a monogenic autoinflammatory disease associated with gain-of-function mutations in the NLRP3 gene encoding cryopyrin.  Cryopyrin nucleates an NLRP3 inflammasome, which regulates the activation and cleavage of caspase 1, which in turn, cleaves the proinflammatory cytokines interleukin-1β (IL-1β) and IL-18. Other functions attributed to the NLRP3 inflammasome include NF-κB activation and pyroptotic cell death.

Clinically CAPS encompasses a disease severity spectrum including familial cold autoinflammatory syndrome (FCAS) with episodes of fever and rash after exposure to cold, Muckle Wells syndrome (MWS) characterized by urticaria-like rash, amyloidosis and deafness and the most severe subtype, neonatal-onset multisystem inflammatory disease (NOMID), also known as chronic infantile neurologic, cutaneous and articular syndrome (CINCA). In addition, overlapping clinical phenotypes MWS/FCAS and MWS/ NOMID are increasingly recognized. Across all phenotypes, excessive IL-1β release results in debilitating symptoms of systemic inflammation, while severe organ inflammation and irreversible damage primarily occur in the moderate to severe CAPS phenotypes.

Inhibition of IL-1 β with Canakinumab, a fully human monoclonal anti-IL-1β antibody has been shown in clinical trials to effectively control CAPS disease activity (>90% remission rates), reduce systemic and organ inflammation and prevent organ damage. Based on these studies Canakinumab has now become the standard NHS treatment for CAPS in the UK. However, studies of real-life effectiveness of canakinumab in CAPS in daily clinical practice, outside of clinical trials have suggested that effectiveness was 53% with standard dose and rose to approximately 72% after dose increase suggesting that an estimated 1/4 of patients still failed to respond to this therapy. These studies have suggested that it is mainly the children with severe CAPS that were less likely to reach complete response and remained at significant risk of neurological deterioration and permanent hearing loss. Thus there is no good lifelong standard of care treatment for patients with severe CAPS, and the outlook for these patients is bleak.

RNA targeting interventions such as antisense oligonucleotides (AON), and small interfering RNAs (siRNAs) are a prominent therapeutic approach for specific manipulation of gene expression and have been successfully used in other genetic diseases, for instance: spinal muscular atrophy; and hereditary amyloidosis. Gene silencing with AON/siRNAs has never been used in treating genetic types of autoinflammation, but holds particular promise for CAPS since the disease is caused by gain of function NLRP3 mutation, and thus can be logically silenced using this approach. 

The aim of this preclinical project is therefore to establish the feasibility of suppressing the specific genetic drive of inflammation in CAPS using established RNA targeting techniques, already developed in our institution for other genetic diseases. 

This project will be conducted in the following steps using the below outlined methods: 

Step 1: Design and validate AON/siRNAs to target NLPR3 expression in mutant human dermal fibroblast cells (HDFC), and peripheral blood mononuclear cells (PBMC) from patients with CAPS. Gene expression (PCR) and protein expression for NLRP3 (blot) will be examined. 
Step 2: Evaluate the effects of AON/siRNAs on downregulating NLRP3 activity assessed through caspase 1 activation (flow cytometry), cytokine production for IL1b and IL18 (ELISA) in HDFC, and PBMC from patients with CAPS.
Step 3: Evaluate the effects of AO/siRNAs on downregulating NF-κB activation (assessed by p65 phosphorylation by flow cytometry and IL6/TNF release by ELISA) in patient HDFC/PBMC. Cell death will be also assessed. 
Step 4: Examine the biodistribution of the designed AO/siRNAs in wild type mice to confirm effective organ distribution using fluorescence and NLRP3 gene expression.

Months 0-6 Design AON/siRNAs
Months 6-24 Evaluate effects on NLRP3 activity and NF-κB activation, cell death
Months 12-18 Upgrade 
Months 24-30 Examine the biodistribution in wild type mice 
Months 30-36 Complete all experiments and write up

1.    Real-life effectiveness of canakinumab in cryopyrin-associated periodic syndrome 
 Kuemmerle-Deschner et al. Real-life effectiveness of canakinumab in cryopyrin-associated periodic syndrome. Rheumatology (Oxford), 2016, 689–696.
2.    Russo et al. Efficacy and safety of canakinumab therapy in paediatric patients with cryopyrin-associated periodic syndrome: a single-centre, real-world experience. Rheumatology (Oxford) 2014 Apr;53(4):665-70.
3.    J B Kuemmerle-Deschner et al. Clinical and Molecular Phenotypes of Low-Penetrance Variants of NLRP3: Diagnostic and Therapeutic Challenges. Arthritis Rheumatol
       2017 Nov;69(11):2233-2240.