Lentiviral Vector-Mediated Correction of a Mouse Model of Leukocyte Adhesion Deficiency Type I.

Diego Leon-Rico,Siobhan O. Burns,Julien Record,Adrian J. Thrasher,Giorgia Santilli,Elena Almarza,Cristina Mesa-Nuñez,Raquel Sanchez-Baltasar,Montserrat Aldea,Juan A. Bueren

doi:10.1089/hum.2016.016

Abstract

Leukocyte adhesion deficiency type I (LAD-I) is a primary immunodeficiency caused by mutations in the ITGB2 gene and is characterized by recurrent and life-threatening bacterial infections. These mutations lead to defective or absent expression of β2 integrins on the leukocyte surface, compromising adhesion and extravasation at sites of infection. Three different lentiviral vectors (LVs) conferring ubiquitous or preferential expression of CD18 in myeloid cells were constructed and tested in human and mouse LAD-I cells. All three hCD18-LVs restored CD18 and CD11a membrane expression in LAD-I patient-derived lymphoblastoid cells. Corrected cells recovered the ability to aggregate and bind to sICAM-1 after stimulation. All vectors induced stable hCD18 expression in hematopoietic cells from mice with a hypomorphic Itgb2 mutation (CD18HYP), both in vitro and in vivo after transplantation of corrected cells into primary and secondary CD18HYP recipients. hCD18+ hematopoietic cells from transplanted CD18HYP mice also showed restoration of mCD11a surface co-expression. The analysis of in vivo neutrophil migration in CD18HYP mice subjected to two different inflammation models demonstrated that the LV-mediated gene therapy completely restored neutrophil extravasation in response to inflammatory stimuli. Finally, these vectors were able to correct the phenotype of human myeloid cells derived from CD34+ progenitors defective in ITGB2 expression. These results support for the first time the use of hCD18-LVs for the treatment of LAD-I patients in clinical trials.

Highlights

LEUKOCYTE ADHESION DEFICIENCY TYPE I (LAD-I) is an autosomal recessive primary immunodeficiency caused by deficient cell surface expression of b2 integrins
In a first set of experiments we investigated the efficacy of the hCD18-lentiviral vectors (LVs) vectors to confer hCD18 expression in human LAD-I lymphoblastic cells (LCs), with no detectable expression of hCD18 (Fig. 1B)
To determine hCD18 expression levels conferred by the different LVs in LAD-I LCs, mean fluorescence intensity (MFI) values as well as mean copy numbers of each LV were determined after transduction in each population, and MFI values were normalized per copy of integrated provirus (MFI/ vector copy number (VCN)) (Fig. 1C)

Summary

Introduction

LEUKOCYTE ADHESION DEFICIENCY TYPE I (LAD-I) is an autosomal recessive primary immunodeficiency caused by deficient cell surface expression of b2 integrins. Neutrophils fail to firmly adhere to the inflamed endothelium and to extravasate from blood to infection sites. The molecular basis underlying LAD-I are mutations in the ITGB2 gene that encodes for the b2 common integrin subunit (CD18).[1,2,3]. LAD-I patients suffer from recurrent and lifethreatening infections that appear early in childhood.[4] Two different phenotypes of LAD-I have been described[5]: a severe phenotype, when levels of CD18 expression are lower than 2% of normal level, and a moderate phenotype, when levels of CD18 expression are 2–30% of the normal level. Hematopoietic stem cell transplantation (HSCT) is currently the only curative treatment for LAD-I.6

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