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Identification of Host Factors of Norovirus Infections in Mini-Gut Model

The safety and scientific validity of this study is the responsibility of the study sponsor and investigators. Listing a study does not mean it has been evaluated by the U.S. Federal Government. Read our disclaimer for details. Identifier: NCT03342547
Recruitment Status : Unknown
Verified September 2018 by CHAN CHI WAI, Chinese University of Hong Kong.
Recruitment status was:  Recruiting
First Posted : November 17, 2017
Last Update Posted : September 20, 2018
Information provided by (Responsible Party):
CHAN CHI WAI, Chinese University of Hong Kong

Tracking Information
First Submitted Date  ICMJE November 9, 2017
First Posted Date  ICMJE November 17, 2017
Last Update Posted Date September 20, 2018
Actual Study Start Date  ICMJE April 18, 2018
Estimated Primary Completion Date December 31, 2020   (Final data collection date for primary outcome measure)
Current Primary Outcome Measures  ICMJE
 (submitted: November 13, 2017)
Establishment of human intestinal stem cell-derived enteroids [ Time Frame: An average of three months ]
Viability of enteroids as determined by microscopy
Original Primary Outcome Measures  ICMJE Same as current
Change History
Current Secondary Outcome Measures  ICMJE Not Provided
Original Secondary Outcome Measures  ICMJE Not Provided
Current Other Pre-specified Outcome Measures Not Provided
Original Other Pre-specified Outcome Measures Not Provided
Descriptive Information
Brief Title  ICMJE Identification of Host Factors of Norovirus Infections in Mini-Gut Model
Official Title  ICMJE Genome-wide CRISPR Screen for Host Factors Associated With Norovirus Infections in Stem Cell-derived Human Intestinal Enteroid Model
Brief Summary

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.

Detailed Description

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***

  1. 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.
  2. 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  ICMJE Interventional
Study Phase  ICMJE Not Applicable
Study Design  ICMJE Allocation: N/A
Intervention Model: Single Group Assignment
Masking: None (Open Label)
Primary Purpose: Basic Science
Condition  ICMJE Gastrointestinal Infection
Intervention  ICMJE
  • Procedure: Duodenal biopsy
    Two duodenal biopsy samples will be collected from each participant
  • Procedure: Saliva
    Saliva (1-2 mL) will be collected from each participant for secretor status testing
Study Arms  ICMJE 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
  • Procedure: Saliva
Publications * Not Provided

*   Includes publications given by the data provider as well as publications identified by Identifier (NCT Number) in Medline.
Recruitment Information
Recruitment Status  ICMJE Unknown status
Estimated Enrollment  ICMJE
 (submitted: November 13, 2017)
Original Estimated Enrollment  ICMJE Same as current
Estimated Study Completion Date  ICMJE December 31, 2020
Estimated Primary Completion Date December 31, 2020   (Final data collection date for primary outcome measure)
Eligibility Criteria  ICMJE

Inclusion Criteria:

  • Adults aged 18 years or above
  • Prospective outpatients undergoing gastrointestinal endoscopy for symptoms of dyspepsia in the Endoscopy Unit of the Prince of Wales Hospital, Shatin, Hong Kong, China
  • Able and willing to provide informed written consent

Exclusion Criteria:

  • Use of anti-coagulants and/or aspirin that may have increased risk of bleeding
  • History of bleeding diathesis
  • Contraindications for biopsy sampling
Sex/Gender  ICMJE
Sexes Eligible for Study: All
Ages  ICMJE 18 Years and older   (Adult, Older Adult)
Accepts Healthy Volunteers  ICMJE Yes
Contacts  ICMJE Contact information is only displayed when the study is recruiting subjects
Listed Location Countries  ICMJE China
Removed Location Countries  
Administrative Information
NCT Number  ICMJE NCT03342547
Other Study ID Numbers  ICMJE MIC_CHAN_GRF_17_18_v1_20160930
Has Data Monitoring Committee No
U.S. FDA-regulated Product
Studies a U.S. FDA-regulated Drug Product: No
Studies a U.S. FDA-regulated Device Product: No
IPD Sharing Statement  ICMJE
Plan to Share IPD: Undecided
Responsible Party CHAN CHI WAI, Chinese University of Hong Kong
Study Sponsor  ICMJE Chinese University of Hong Kong
Collaborators  ICMJE Not Provided
Investigators  ICMJE
Principal Investigator: Chi-Wai Chan, PhD Chinese University of Hong Kong
PRS Account Chinese University of Hong Kong
Verification Date September 2018

ICMJE     Data element required by the International Committee of Medical Journal Editors and the World Health Organization ICTRP