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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

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.

Condition or disease Intervention/treatment Phase
Gastrointestinal Infection Procedure: Duodenal biopsy Procedure: Saliva Not Applicable

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.

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Study Type : Interventional  (Clinical Trial)
Estimated Enrollment : 20 participants
Allocation: N/A
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

Resource links provided by the National Library of Medicine

Arm Intervention/treatment
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

Procedure: Saliva
Saliva (1-2 mL) will be collected from each participant for secretor status testing

Primary Outcome Measures :
  1. Establishment of human intestinal stem cell-derived enteroids [ Time Frame: An average of three months ]
    Viability of enteroids as determined by microscopy

Information from the National Library of Medicine

Choosing to participate in a study is an important personal decision. Talk with your doctor and family members or friends about deciding to join a study. To learn more about this study, you or your doctor may contact the study research staff using the contacts provided below. For general information, Learn About Clinical Studies.

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Ages Eligible for Study:   18 Years and older   (Adult, Older Adult)
Sexes Eligible for Study:   All
Accepts Healthy Volunteers:   Yes

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

Information from the National Library of Medicine

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 identifier (NCT number): NCT03342547

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Contact: Chi-Wai Chan, PhD +852 35051523

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Endoscopy Centre, Prince of Wales Hospital Recruiting
Hong Kong, China
Contact: Chi-Wai Chan, PhD    +852 35051523   
Principal Investigator: Chi-Wai Chan, PhD         
Sub-Investigator: Kay-Sheung Chan, MD         
Sub-Investigator: Hei Wong, MBChB; DPhil         
Sponsors and Collaborators
Chinese University of Hong Kong
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Principal Investigator: Chi-Wai Chan, PhD Chinese University of Hong Kong
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Responsible Party: CHAN CHI WAI, Assistant Professor, Chinese University of Hong Kong Identifier: NCT03342547    
Other Study ID Numbers: MIC_CHAN_GRF_17_18_v1_20160930
First Posted: November 17, 2017    Key Record Dates
Last Update Posted: September 20, 2018
Last Verified: September 2018
Individual Participant Data (IPD) Sharing Statement:
Plan to Share IPD: Undecided

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Studies a U.S. FDA-regulated Drug Product: No
Studies a U.S. FDA-regulated Device Product: No
Keywords provided by CHAN CHI WAI, Chinese University of Hong Kong:
Additional relevant MeSH terms:
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Communicable Diseases
Caliciviridae Infections
RNA Virus Infections
Virus Diseases