Identification of Host Factors of Norovirus Infections in Mini-Gut Model
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|ClinicalTrials.gov Identifier: NCT03342547|
Recruitment Status : Recruiting
First Posted : November 17, 2017
Last Update Posted : September 20, 2018
The primary objective in this study is to establish a list of host cellular proteins that mediate norovirus infection.
Norovirus is one of the most common pathogens attributed to diarrheal diseases from unsafe food. It is also the primary cause of mortality among young children and adults in foodborne infections. Norovirus is not just a foodborne burden. In a recent meta-analysis, norovirus accounts for nearly one-fifth of all causes of (including person-to-person transmission) acute gastroenteritis in both sporadic and outbreak settings and affects all age groups. Undoubtedly, norovirus is of paramount public health concern in both developed and developing countries. Research efforts to better understand norovirus pathobiology will be necessary for targeted intervention.
From Middle East respiratory syndrome coronavirus to Zika virus, efforts to identify host factors important for mediating virus infection has always been a research priority. Such information will shed light on potential therapeutic targets in antiviral intervention. Norovirus virus-host interaction studies have been hampered by the lack of a robust cell culture model in the past 20 years. In 2016, norovirus has finally been successfully cultivated in a stem cell-derived three-dimensional human gut-like structure called enteroid or mini-gut.
In this study, intestinal stem cells will be isolated from duodenal biopsies collected from participants, followed by differentiation into mini-guts. Genome-wide genetic screening for host essential and restrictive factors will be performed on infected mini-guts by knockout CRISPR and gain-of-function CRISPR SAM, respectively. Shortlisted candidates will undergo preliminary functional validation in cell lines. These data will provide insights into potential therapeutic targets against norovirus infection.
|Condition or disease||Intervention/treatment||Phase|
|Gastrointestinal Infection||Procedure: Duodenal biopsy Procedure: Saliva||Not Applicable|
Study design and overview - This is a laboratory-based study. The investigators plan to establish a list of host cellular proteins regulating norovirus infection. Methods will include establishment of patient-specific stem cell-derived human intestinal enteroids as an infection model and genome-wide CRISPR and CRISPR SAM genetic screens upon infections of two norovirus genotypes.
***Establishment of human intestinal enteroid model***
- Duodenal biopsies and saliva collection - After obtaining IRB-approved informed consent, two duodenal biopsy samples will be obtained from each participant undergoing upper gastrointestinal endoscopy without contraindications for biopsy sampling, such as participants on anti-coagulation or with other causes of bleeding diathesis. Biopsy samples will be immersed in ice-cold 1xPBS and transported immediately to the laboratory within 30 minutes for further processing. In addition, saliva (1-2 mL) will be collected from each participant for secretor status testing by ELISA.
- Isolation of crypt stem cells and differentiation - Upon arrival, biopsy samples will first be chopped into smaller pieces. Intestinal crypt cells will be isolated by repeated washing and incubation with 1x Complete Chelating solution (1xCCS) and EDTA buffer. Supernatant containing crypt cells will then be grown in Matrigel containing complete medium with growth factors (CMGF+) (Wnt 3 and Rspo-1 conditioned media, Noggin, A83, B27, EGF, N2, n-acetylcysteine, gastrin, nicotinamide, and SB202190). Budding crypts will then be incubated in differentiation medium (CMGF+, excluding Wnt 3 conditioned medium, SB202190 and nicotinamide as well as having reduced amount of Rspo-1 conditioned medium and Noggin). Formation of three-dimensional gut-like enteroids will be monitored daily.
|Study Type :||Interventional (Clinical Trial)|
|Estimated Enrollment :||20 participants|
|Intervention Model:||Single Group Assignment|
|Masking:||None (Open Label)|
|Primary Purpose:||Basic Science|
|Official Title:||Genome-wide CRISPR Screen for Host Factors Associated With Norovirus Infections in Stem Cell-derived Human Intestinal Enteroid Model|
|Actual Study Start Date :||April 18, 2018|
|Estimated Primary Completion Date :||December 31, 2020|
|Estimated Study Completion Date :||December 31, 2020|
Experimental: Intestinal stem cell-derived enteroids
Duodenal biopsy samples and saliva will be collected from participants and then processed in laboratory to generate intestinal stem cell-derived enteroids.
Procedure: Duodenal biopsy
Two duodenal biopsy samples will be collected from each participant
Saliva (1-2 mL) will be collected from each participant for secretor status testing
- Establishment of human intestinal stem cell-derived enteroids [ Time Frame: An average of three months ]Viability of enteroids as determined by microscopy
To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.
Please refer to this study by its ClinicalTrials.gov identifier (NCT number): NCT03342547
|Contact: Chi-Wai Chan, PhD||+852 firstname.lastname@example.org|
|Endoscopy Centre, Prince of Wales Hospital||Recruiting|
|Hong Kong, China|
|Contact: Chi-Wai Chan, PhD +852 35051523 email@example.com|
|Principal Investigator: Chi-Wai Chan, PhD|
|Sub-Investigator: Kay-Sheung Chan, MD|
|Sub-Investigator: Hei Wong, MBChB; DPhil|
|Principal Investigator:||Chi-Wai Chan, PhD||Chinese University of Hong Kong|