Close
Title: Why the drugs don’t work: using nanopore sequencing to decode immune cells in immunocompromised babies with severe RSV bronchiolitis to find new therapies – 34 - III
Supervisors: Claire Smith, Samiran Ray
Project Description:
Background
Respiratory viral infections are common in children, usually causing mild symptoms. However, children with rare genetic immune deficiencies face severe risks from these infections, including prolonged and potentially fatal outcomes, even from common cold viruses. Additionally, children who have undergone transplants are at increased risk of severe outcomes from these infections. Unfortunately, there are currently no clinically approved treatments available.
Our research group has developed methods to study the impact of respiratory syncytial virus (RSV) infection on the airway and the early (innate) immune response. Neutrophils and other peripheral blood mononuclear cells (PBMCs) are crucial components of the immune system and are among the first responders to RSV infection. Despite their importance, the detailed molecular mechanisms and gene expression profiles of PBMCs during RSV infection are still not well understood.
In this project we will leverage the advanced capabilities of nanopore sequencing technology to decode the transcriptome of PBMCs from in immunocompetent and immunocompromised RSV-infected babies from GOSH. We will also collect samples from very sick babies undergoing therapy with novel anti-viral treatments. This approach offers unparalleled insights into immune cell responses, potentially leading to new therapeutic strategies for vulnerable children.
Aims/Objectives
*Collect tracheal aspirates and matched PBMCs from RSV-infected babies.
*Use nanopore technology to examine the transcriptome and differential gene expression profiles of PBMCs from RSV-infected children compared to healthy controls.
*Identify key genes and pathways involved in the immune response to RSV.
*Test the effect of novel and repurposed antivirals on identified biomarkers using an in vitro airway-immune cell migration model.
Methods
*Extract RNA from isolated PBMCs and prepare RNA libraries for nanopore sequencing.
*Perform sequencing using the Oxford Nanopore Technologies (ONT) platform to capture the complete transcriptome.
*Conduct differential gene expression analysis to compare transcriptomic profiles of PBMCs from RSV-infected patients and healthy controls using bioinformatics tools like DESeq2 or EdgeR.
*Utilize pathway analysis tools (e.g., GO, KEGG) to identify key genes and pathways in the immune response to RSV.
*Test novel and repurposed antivirals in our in vitro model of immune cell transepithelial migration in response to RSV infection.
Timeline
Year 1:
Months 1-4: Secure ethical approval and undergo training in lab practices.
Months 5-8: Prepare RNA libraries and conduct nanopore sequencing from existing samples.
Months 9-12: Isolate PBMCs and prepare RNA libraries from fresh samples.
Year 2:
Months 12-18: Analyze sequencing data and perform differential gene expression analysis.
Months 18-24: Develop and refine in vitro models of immune cell transepithelial migration.
Year 3:
Months 24-30: Test novel and repurposed antivirals in the in vitro model.
Months 30-36: Prepare findings for publication and write the PhD thesis.
References
Roy F. 2014 Clin Infect Dis. Nov 15;59(Suppl 5): S344–S35.
Nair H,. Lancet. 2010 May 1;375(9725):1545- 55
Robinson E, 2023. Thorax 10.1136/THORAX-2023-220081
Contact Information:
Dr Claire Smith