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

Gastrointestinal Infection Clinical Trial

Official title:

Genome-wide CRISPR Screen for Host Factors Associated With Norovirus Infections in Stem Cell-derived Human Intestinal Enteroid Model

Clinical Trial Details — Status: Recruiting

Administrative data

• NCT number: NCT03342547
• Other study ID #: MIC_CHAN_GRF_17_18_v1_20160930
• Status: Recruiting
• Phase: N/A
• Start date: April 18, 2018
• Est. completion date: December 31, 2020

Study information

• Verified date: September 2018
• Source: Chinese University of Hong Kong
• Contact: Chi-Wai Chan, PhD
• Phone: +852 35051523
• Email: martin.chan[@]cuhk.edu.hk
• Is FDA regulated: No
• Study type: Interventional

Clinical Trial 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.

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.

Recruitment information / eligibility

• Status: Recruiting
• Enrollment: 20
• Est. completion date: December 31, 2020
• Est. primary completion date: December 31, 2020
• Accepts healthy volunteers: Accepts Healthy Volunteers
• Gender: All
• Age group: 18 Years and older

Eligibility

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

Related Conditions & MeSH terms

• Communicable Diseases
• Gastrointestinal Infection
• Infection

Intervention

Procedure:
• Duodenal biopsy
Two duodenal biopsy samples will be collected from each participant
• Saliva
Saliva (1-2 mL) will be collected from each participant for secretor status testing

Locations

Country	Name	City	State
China	Endoscopy Centre, Prince of Wales Hospital	Hong Kong

Sponsors (1)

Lead Sponsor	Collaborator
Chinese University of Hong Kong

Country where clinical trial is conducted

China, 

Outcome

Type	Measure	Description	Time frame	Safety issue
Primary	Establishment of human intestinal stem cell-derived enteroids	Viability of enteroids as determined by microscopy	An average of three months