Poly-(ADP-ribose) polymerase-2 (PARP-2) belongs to a large family of enzymes that synthesize and transfer ADPribose polymers to acceptor proteins, modifying their functional properties. PARP-2-deficient (Parp-2 À/À ) cells, similar to Parp-1 À/À cells, are sensitive to both ionizing radiation and alkylating agents. Here we show that inactivation of mouse Parp-2, but not Parp-1, produced a two-fold reduction in CD4 þ CD8 þ double-positive (DP) thymocytes associated with decreased DP cell survival. Microarray analyses revealed increased expression of the proapoptotic Bcl-2 family member Noxa in Parp-2 À/À DP thymocytes compared to littermate controls. In addition, DP thymocytes from Parp-2 À/À have a reduced expression of T-cell receptor (TCR)a and a skewed repertoire of TCRa toward the 5 0 Ja segments. Our results show that in the absence of PARP-2, the survival of DP thymocytes undergoing TCRa recombination is compromised despite normal amounts of Bcl-x L . These data suggest a novel role for PARP-2 as an important mediator of T-cell survival during thymopoiesis by preventing the activation of DNA damage-dependent apoptotic response during the multiple rounds of TCRa rearrangements preceding a positively selected TCR.

X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase catalytic subunit gp91(phox). A recent clinical trial for X-CGD using a spleen focus-forming virus (SFFV)-based γ-retroviral vector has demonstrated clear therapeutic benefits in several patients although complicated by enhancer-mediated mutagenesis and diminution of effectiveness over time due to silencing of the viral long terminal repeat (LTR). To improve safety and efficacy, we have designed a lentiviral vector that directs transgene expression primarily in myeloid cells. To this end, we created a synthetic chimeric promoter that contains binding sites for myeloid transcription factors CAAT box enhancer-binding family proteins (C/EBPs) and PU.1, which are highly expressed during granulocytic differentiation. As predicted, the chimeric promoter regulated higher reporter gene expression in myeloid than in nonmyeloid cells, and in human hematopoietic progenitors upon granulocytic differentiation. In a murine model of stem cell gene therapy for X-CGD, the chimeric vector resulted in high levels of gp91(phox) expression in committed myeloid cells and granulocytes, and restored normal NADPH-oxidase activity. These findings were recapitulated in human neutrophils derived from transduced X-CGD CD34(+) cells in vivo, and suggest that the chimeric promoter will have utility for gene therapy of myeloid lineage disorders such as CGD.

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