CRISPR/Cas9-Mediated In Situ Correction of LAMB3 Gene in Keratinocytes Derived from a Junctional Epidermolysis Bullosa Patient.

Abstract

Deficiency of basement membrane heterotrimeric laminin 332 component, coded by LAMA3, LAMB3, and LAMC2 genes, causes junctional epidermolysis bullosa (JEB), a severe skin adhesion defect. Herein, we report the first application of CRISPR/Cas9-mediated homology direct repair (HDR) to in situ restore LAMB3 expression in JEB keratinocytes in vitro and in immunodeficient mice transplanted with genetically corrected skin equivalents. We packaged an adenovector carrying Cas9/guide RNA (gRNA) tailored to the intron 2 of LAMB3 gene and an integration defective lentiviral vector bearing a promoterless quasi-complete LAMB3 cDNA downstream a splice acceptor site and flanked by homology arms. Upon genuine HDR, we exploited the in vitro adhesion advantage of laminin 332 production to positively select LAMB3-expressing keratinocytes. HDR and restored laminin 332 expression were evaluated at single-cell level. Notably, monoallelic-targeted integration of LAMB3 cDNA was sufficient to in vitro recapitulate the adhesive property, the colony formation typical of normal keratinocytes, as well as their cell growth. Grafting of genetically corrected skin equivalents onto immunodeficient mice showed a completely restored dermal-epidermal junction. This study provides evidence for efficient CRISPR/Cas9-mediated in situ restoration of LAMB3 expression, paving the way for ex vivo clinical application of this strategy to laminin 332 deficiency.

• Authors: Daniela Benati, Francesca Miselli, Fabienne Cocchiarella, Clarissa Patrizi, Marta Carretero, Samantha Baldassarri, Virginia Ammendola, Cristina Has, Stefano Colloca, Marcela Del Rio, Fernando Larcher, Alessandra Recchia
• Journal: Molecular therapy : the journal of the American Society of Gene Therapy (Mol Ther) Vol. 26 Issue 11 Pg. 2592-2603 (11 07 2018) ISSN: 1525-0024 [Electronic] United States
• PMID: 30122422 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.

Chemical References

• Cell Adhesion Molecules
• DNA, Complementary
• Laminin
• kalinin

Topics

• Animals
• Basement Membrane (pathology)
• CRISPR-Cas Systems (genetics)
• Cell Adhesion Molecules (antagonists & inhibitors, deficiency, genetics)
• DNA Repair (genetics)
• DNA, Complementary (genetics)
• Epidermolysis Bullosa, Junctional (genetics, pathology, therapy)
• Gene Expression Regulation
• Genetic Therapy
• Humans
• Introns (genetics)
• Keratinocytes (metabolism, pathology)
• Laminin (genetics)
• Lentivirus (genetics)
• Mice
• Mutation
• RNA Editing (genetics)