Lentiviral Vectors Incorporating Ubiquitous Chromatin Opening Element Driving Canine CD18 Expression

Abstract

Leukocyte adhesion deficiency type 1 (LAD1) in humans is caused due to mutations in the ITGB2 gene encoding the leukocyte CD18 subunit (b2 integrin). This results in defective leukocyte adhesion and migration leading to recurrent episodes of life-threatening bacterial infection. Canine leukocyte adhesion deficiency (CLAD) represents a disease-specific large animal model of LAD1 in which new therapeutic approaches could be tested. Our previous studies have demonstrated variable efficiency of CD18 expression under the control of several promoters. These include cellular promoters such as those of human elongation factor 1a (hEF1a): long (1169bp) and short (248bp) fragments, human phosphoglycerate kinase (hPGK), human CD11b and human CD18 genes. In addition, murine stem cell virus (MSCV) promoter has also been demonstrated to lead to very high levels of CD18 expression in CLAD CD34+ cells thereby reversing the CLAD phenotype in dogs previously treated with both foamy and lentiviral vectors. But, due to potential genotoxicity associated with the use of viral promoters, we continued our efforts in search of novel cellular promoters. One such promoter is the ubiquitous chromatin opening element (UCOE) from the human heterogeneous ribonucleoprotein A2/B1 and chromobox homolog 3 (HNRPA2B1-CBX3) loci. UCOE has been previously shown to display reproducible and stable transgene expression within the context of a self-inactivating (SIN) lentiviral vector in the absence of classical enhancer activity (Zhang et al., Blood 2007).It has also been shown to confer resistance to DNA methylation-mediated transgene silencing even upon integration into the heterochromatin regions of the host chromosome (Zhang et al., Mol Ther. 2010). Since the full-length element is about 2.6 kb, we cloned and tested different fragment lengths of the UCOE promoter in a SIN lentiviral vector (pCL20) in CLAD CD34+ cells in vitro. Efficiency of expression of CD18 obtained with the six promoter fragments of UCOE (in bp), namely U3'631, U3'1262, U3'652, U5'1357, U5'723 and U5'655 were compared to those obtained with an MSCV promoter. Functional viral titers were first determined using a human LAD EBV-transformed B-cell line that lacks endogenous human CD18. When comparable titers of each vector were used in an overnight transduction of CLAD CD34+ cells after a 24h cytokine prestimulation in vitro, the percentage of CD18+ cells 5 days after transduction were as follows: U3'631 - 8.49%, U3'1262 - 15.9%, U3'652 - 21.3% (tested at MOI 100), U5'1357 - 2.05% (tested at MOI 30), U5'723 - 2.44% (tested at MOI 20), U5'655 - 3.01% (tested at MOI 50) and MSCV - 35.3% (tested at MOI 100). The CD18 expression levels driven by some of these promoter fragments were comparable to those driven by cellular promoters mentioned previously. The UCOE is promising in that it could overcome possible gene silencing effects when used in vivo, unlike promoters such as EF1a and PGK which were largely subjected to post-transcriptional gene silencing with sub-therapeutic levels of CD18 as previously tested in the dog model. Hence, functional correction of the CD18 defect could be achieved with candidate UCOE-incorporating SIN lentiviral vector(s) when used in the treatment of CLAD dogs. DisclosuresNo relevant conflicts of interest to declare.