64. Rescue of B Cell Development in an Animal Model of X-Linked Agammaglobulinemia (XLA) Via B Lineage-Specific Lentiviral Gene Therapy
Authors: Wenying Zhang, Stephanie Humblet-Baron, Kevin Kipp, Socheath Khim, Jordan Jarjour, Karen Sommer, Brigid Stirling, Lia Pernell, David J. Rawlings
Journal: Molecular Therapy (2006)

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X-linked agammaglobulinemia (XLA) is a human immunodeficiency caused by mutations in Bruton's tyrosine kinase (Btk); and characterized by a block in pre B-cell development leading to absence of serum immunoglobulin and recurrent bacterial infections. Using Btk and Tec double deficient (Btk/Tec|[minus]|/|[minus]|) mice as a model for XLA, we previously showed that onco-retroviral-mediated Btk gene transfer into hematopoietic stem cells (HSC) reconstituted in vivo Btk-dependent B-cell development and function (Yu et al. Blood 104(5): 1281|[ndash]|90).

In an effort to increase the safety of this approach, we developed a lentiviral system that incorporates a B lineage specific enhancer/ promoter element, E|[mu]|B29. Using SIN-lentiviral vectors expressing GFP, we observed that E|[mu]|B29 consistently promoted 3|[ndash]|5 fold higher GFP expression in human B lineage cells derived from transduced HSC in vivo and in vitro (ASGT 2002 abstract |[num]|1302). We also evaluated this vector, CSOM- E|[mu]|B29-GFP-WPRE, in lentiviral transgenic mice where it exhibited the highest GFP expression in peripheral B cells compared with all other hematopoietic lineages. Specifically, in more than 8 independent founder strains the MFI for GFP expression in B cells was 3 fold higher than that in T cells (p=0.0002).

Based upon these expression studies we generated E|[mu]|B29-huBtk SIN-lentiviral vectors with or without the insulator element derived from the chicken |[beta]|-globulin insulator (cHS4). Using both vectors to transduce Btk|[minus]|/|[minus]| DT40 B cells, followed by cloning by limiting dilution, we demonstrate Btk protein expression by intracellular staining and western blotting. All lines (including those clones containing single viral integrations) exhibited full rescue of Btk- dependent, B cell receptor (BCR)-mediated Ca2+ signaling. We next tested the capacity of these vectors to reconstitute Btk-dependent B-cell development and function in a cohort of Btk/Tec|[minus]|/|[minus]| mice. Marrow from 5-FU treated Btk/Tec |[minus]|/|[minus]| mice was harvested, cultured on fibronectin coated plates with growth factors (mIL-3,mIL-6, mSCF, mTPO and mFLT3ligand) and concentrated lentivirus (2.3|[times]|107pg/106 cells measured by p24 level). After 48h of in vitro culture, cells were transplanted into lethally or sublethally irradiated animals. Transplanted animals were serially evaluated for presence of B cells in the peripheral blood. B-cell numbers progressively increased with a significant difference as early as within 6 weeks in mice receiving transduced (16|[ndash]|18% B220+ cells) vs. control marrow (8|[ndash]|9%; mock transduced). Further, mature B cells (B220+IgMlowIgDhi) represented 14|[ndash]|20% of total B cells in treated compared to <5% in control mice. Finally, mice receiving transduced cells exhibited a rescue of total serum IgM and IgG3 levels and responses to TI-II dependent immunization. In conclusion, our data demonstrate that E|[mu]|B29-Btk SIN-lentiviral vector specifically promotes Btk expression in B lineage cells, and correction of the Btk-deficient phenotype both in vivo and in vitro.