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Project: Delivery of CRISPR Ribonucleoproteins to Airway Epithelia Using Novel Amphiphilic Peptides

PI:   Paul B McCray, Jr, MD  NIH Report

The proposed research is relevant to the public health because genetic and acquired diseases affecting the airways pose major disease and economic burdens. By advancing the delivery of gene editing tools, it may be possible to therapeutically modify the cells lining the airways. This novel strategy has implications for the treatment of both monogenetic and acquired lungs disease, and may have applications for other somatic cell therapies.
Summary of data submissions:
  • Data for 4  experiments were submitted on 2020-11-02 SCGE ID:1004
  • Data for 2  experiments were submitted on 2020-11-02 SCGE ID:1013
  • 1 of 6 experiments have been validated

Submissions Details

SCGE ID:1004 - Submission Date: 2020-11-02 
Experiment Name Type Description
Testing Shuttle Peptides ability to deliver GFP-NLS to airway epithelia.

In Vivo Delivery of GFP via shuttle peptides to mouse airway epithelium via nasal instilation. Delivery efficiency was quantified in large and small airways by counting the number of GFP positive cells divided by the number of DAPI cells.
Gene editing in vitro by various peptide variants delivering Cas12a in NK cells.

In Vitro In Vitro shuttle peptide delivery of Cas12a RNP targeting human NKG2A gene in human NK cells. Gene editing was assessed after 48hr by T7E1 digestion.
Gene editing in vitro by various peptide variants delivering Cas RNPs to primary Human airway epithelia cells.

In Vitro In Vitro shuttle peptide delivery of Cas12a RNPs targeting human CFTR and HPRT genes in human primary airway epithelia. Editing efficiency was assessed after 72hrs by sanger sequencing.
Shuttle peptides enable in vivo gene editing with Cas9 and Cas12a RNP in mouse airway epithelia

In Vivo In vivo shuttle peptide delivery of Cas9 and Cas12a RNPs in mouse airway epithelia. Gene editing was quantified by the GFP+ cells in large and small airways following 1 delivery of GFP protein by GFP positive cells compared to DAPI stained cells.
SCGE ID:1013 - Submission Date: 2020-11-02 
Experiment Name Type Description
Peptide Shuttle optimization to deliver Cas9 RNP to human airway epithelia cells

 In Vitro Delivery of Cas9 RNP targeting human CFTR in Primary human epithelia cells. Gene editing efficiency was determined by percentage of NGS reads that showed an indel
Peptide Shuttle optimization to deliver Cas12a RNP to human airway epithelia cells

 In Vitro Delivery of Cas12a RNP targeting human CFTR in Primary human epithelia cells. Gene editing efficiency was determined by percentage of NGS reads that showed an indel