Development of B-lineage Predominant Lentiviral Vectors for Use in Genetic Therapies for B Cell Disorders

Abstract

Sustained, targeted, high-level transgene expression in primary B lymphocytes may be useful for gene therapy in B cell disorders. We developed several candidate B-lineage predominant self-inactivating lentiviral vectors (LV) containing alternative enhancer/promoter elements including: the immunoglobulin β (Igβ) (B29) promoter combined with the immunoglobulin µ enhancer (EµB29); and the endogenous BTK promoter with or without Eµ (EµBtkp or Btkp). LV-driven enhanced green fluorescent protein (eGFP) reporter expression was evaluated in cell lines and primary cells derived from human or murine hematopoietic stem cells (HSC). In murine primary cells, EµB29 and EµBtkp LV-mediated high-level expression in immature and mature B cells compared with all other lineages. Expression increased with B cell maturation and was maintained in peripheral subsets. Expression in T and myeloid cells was much lower in percentage and intensity. Similarly, both EµB29 and EµBtkp LV exhibited high-level activity in human primary B cells. In contrast to EµB29, Btkp and EµBtkp LV also exhibited modest activity in myeloid cells, consistent with the expression profile of endogenous Bruton's tyrosine kinase (Btk). Notably, EµB29 and EµBtkp activity was superior in all expression models to an alternative, B-lineage targeted vector containing the EµS.CD19 enhancer/promoter. In summary, EµB29 and EµBtkp LV comprise efficient delivery platforms for gene expression in B-lineage cells.

Ubiquitous high-level gene expression in hematopoietic lineages provides effective lentiviral gene therapy of murine Wiskott-Aldrich syndrome

“…Isolation, transduction, and transplantation of human CD34 ϩ cells 33 and murine lineage negative (Lin neg ) 34 cells was performed as previously described. For murine stem cell transplantation, we used a split-dose conditioning dose to deliver a total of 9 Gy irradiation over a 24-hour period unless noted otherwise.…”

Agammaglobulinemia: causative mutations and their implications for novel therapies

“…LVs are often modified to reduce the possibilities for transcriptional activation by promoters found in the long terminal repeat (LTR) region, giving rise to selfinactivating (SIN) LVs. Recently, the SIN-LV vectors have been further modified with B-lymphocyte-restricted CD19 promoters in mouse models and human HSCs [47,50,72]. Using such vectors with specific promoters and codon-optimized expression cassettes could be used for increasing transduction efficiency and specificity, thus providing safer and more potent treatments for agammaglobulinemias.…”

Epstein-Barr Virus Utilizes Ikaros in Regulating Its Latent-Lytic Switch in B Cells

“…The expression plasmids pcDNA3-R and pcDNA3-R-V5 encode wild-type and V5-tagged EBV R, respectively (28). Plasmid pCVL-EB29-MCS-T2A-GFP (number 525) (gift from David Rawlings) is a B-cell lentivirus expression vector (64). The expression vector pCDH-EF1-MSC-EF1-GFPϩPuro (abbreviated pCDH-EF1; gift from Stacy Hagemeier) was constructed by substituting the EF1 promoter for the murine stem cell virus (MSCV) promoter in pCDH-MSCV-MCS-EF1-GFPϩPuro (CD713B-1; System Biosciences).…”