Nancy Pech

Rac GTPase isoform-specific regulation of NADPH oxidase and chemotaxis in murine neutrophils in vivo: Role of the C-terminal polybasic domain

The Rho family GTPase Rac acts as a molecular switch for signal transduction to regulate various cellular functions. Mice deficient in the hematopoietic-specific Rac2 isoform exhibit agonist-specific defects in neutrophil chemotaxis and superoxide production, despite expression of the highly homologous Rac1 isoform. To examine whether functional defects in rac2–/– neutrophils reflect effects of an overall decrease in total cellular Rac or an isoform-specific role for Rac2, retroviral vectors were used to express exogenous Rac1 or Rac2 at levels similar to endogenous. In rac2–/– neutrophils differentiated from transduced myeloid progenitors in vitro, increasing cellular Rac levels by expression of either exogenous Rac1 or Rac2 increased formylmethionylleucylphenylalanine- or phorbol ester-stimulated NADPH oxidase activity. Of note, placement of an epitope tag on the N terminus of Rac1 or Rac2 blunted reconstitution of responses in rac2–/– neutrophils. In rac2–/– neutrophils isolated from mice transplanted with Rac-transduced bone marrow cells, superoxide production and chemotaxis were fully reconstituted by expression of exogenous Rac2, but not Rac1. A chimeric Rac1 protein in which the Rac1 C-terminal polybasic domain, which contains six lysines or arginines, was replaced with that of the human Rac2 polybasic domain containing only three basic residues, also reconstituted superoxide production and chemotaxis, whereas expression of a Rac2 derivative in which the polybasic domain was replaced with that of Rac1 did not and resulted in disoriented cell motility. Thus, the composition of the polybasic domain is sufficient for determining Rac isoform specificity in the production of superoxide and chemotaxis in murine neutrophils in vivo.

Antibody targeting KIT as pretransplantation conditioning in immunocompetent mice

Inherited hematologic defects that lack an in vivo selective advantage following gene correction may benefit from effective yet minimally toxic cytoreduction of endogenous hematopoietic stem cells (HSCs) prior to transplantation of gene-modified HSCs. We studied the efficacy of administering a novel sequential treatment of parenteral ACK2, an antibody that blocks KIT, followed by low-dose irradiation (LD-IR) for conditioning of wild-type and X-linked chronic granulomatous disease (X-CGD) mice. In wild-type mice, combining ACK2 and LD-IR profoundly decreased endogenous competitive long-term HSC repopulating activity, and permitted efficient and durable donor-derived HSC engraftment after congenic transplantation. ACK2 alone was ineffective. The combination of ACK2 and LD-IR was also effective conditioning in X-CGD mice for engraftment of X-CGD donor HSCs transduced ex vivo with a lentiviral vector. We conclude that combining ACK2 with LD-IR is a promising approach to effectively deplete endogenous HSCs and facilitate engraftment of transplanted donor HSCs.

Donor chimerism and stem cell function in a murine congenic transplantation model after low-dose radiation conditioning: Effects of a retroviral-mediated gene transfer protocol and implications for gene therapy

OBJECTIVE We investigated low-dose radiation conditioning for the transplantation of retrovirus-transduced cells in a C57Bl6/J murine model. MATERIALS AND METHODS The effect of low-dose radiation on stem cell function was investigated using a competitive repopulation assay. Stem cell function of marrow cells that underwent a retroviral-mediated gene transfer (RMGT) protocol was examined by this assay, and donor chimerism of these cells when transplanted into 160-cGy conditioned syngeneic hosts was compared to fresh marrow. RESULTS Irradiation with 300 or 160 cGy substantially decreased stem cell function as measured by competitive repopulation. Animals conditioned with 160 cGy and transplanted with 20 x 10(6) fresh marrow cells permitted donor cell engraftment of 53.6% +/- 11.4% 6 months after transplant compared to 100% donor cell engraftment after 1100 cGy irradiation. Lymphoid and myeloid engraftment did not significantly differ from total engraftment in submyeloablated hosts. When transplanted into lethally irradiated hosts, the competitive repopulating activity of marrow treated with a single dose of 5-fluorouracil followed by ex vivo culture according to a standard RMGT protocol was equal to 5-fluorouracil-only treated marrow. However, cells treated with 5-fluorouracil or 5-fluorouracil plus ex vivo culture for RMGT repopulated less well than fresh marrow cells in 160 cGy conditioned hosts. CONCLUSIONS Low-dose irradiation decreases host stem cell function, allowing engraftment of both fresh and RMGT protocol-treated marrow, although the engraftment of 5-fluorouracil-treated cells was reduced at least two-fold, and 5-fluorouracil plus RMGT protocol-treated cells at least three-fold, compared to fresh marrow. Modification of current RMGT protocols may be important for optimizing engraftment under these conditions.

Long-Term High-Level Reconstitution of NADPH Oxidase Activity in Murine X-Linked Chronic Granulomatous Disease Using a Bicistronic Vector Expressing gp91phox and a ΔLNGFR Cell Surface Marker

A murine model of X-linked chronic granulomatous disease (X-CGD), an inherited immune deficiency with absent phagocyte NADPH oxidase activity caused by defects in the gp91(phox) gene, was used to evaluate a bicistronic retroviral vector in which expression of human gp91(phox) and a linked gene for Delta LNGFR, a truncated form of human low-affinity nerve growth factor receptor, are under the control of a spleen focus-forming virus long-terminal repeat (LTR). Four independent cohorts of 11-Gy irradiated X-CGD mice (total, 22 mice) were transplanted with or without preselection of transduced X-CGD bone marrow (BM). Transplanted mice had high-level correction of neutrophil gp91(phox) expression and reconstitution of NADPH oxidase activity. Expression lasted for at least 14 months in primary transplants, and persisted in secondary and tertiary transplants. Both gp91(phox) and Delta LNGFR were detected on circulating granulocytes, lymphocytes, lymphoid, and (for Delta LNGFR) red blood cells. Mice receiving transduced bone marrow [BM] preselected ex vivo for Delta LNGFR expression had high-level (= 80%) reconstitution with transduced cells, with an improved fraction of oxidase-corrected neutrophils posttransplant. Analysis of secondary and tertiary CFU-S showed that silencing of individual provirus integrants can occur even after preselection for Delta LNGFR prior to transplantation, and that persistent provirus expression was associated with multiple integration sites in most cases. No obvious adverse consequences of transgenic protein expression were observed.

NADPH oxidase controls neutrophilic response to sterile inflammation in mice by regulating the IL-1α/G-CSF axis.

The leukocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generates reactive oxygen species essential in microbial killing and regulation of inflammation. Inactivating mutations in this enzyme lead to chronic granulomatous disease (CGD), associated with increased susceptibility to both pyogenic infections and to inflammatory disorders. The role of the NADPH oxidase in regulating inflammation driven by nonmicrobial stimuli is poorly understood. Here, we show that NADPH oxidase deficiency enhances the early local release of interleukin-1α (IL-1α) in response to damaged cells, promoting an excessive granulocyte colony-stimulating factor (G-CSF)-regulated neutrophilic response and prolonged inflammation. In peritoneal inflammation elicited by tissue injury, X-linked Cybb-null (X-CGD) mice exhibited increased release of IL-1α and IL-1 receptor -mediated G-CSF production. In turn, higher levels of systemic G-CSF increased peripheral neutrophilia, which amplified neutrophilic peritoneal inflammation in X-CGD mice. Dampening early neutrophil recruitment by neutralization of IL-1α, G-CSF, or neutrophil depletion itself promoted resolution of otherwise prolonged inflammation in X-CGD. IL-1β played little role. Thus, we identified an excessive IL-1α/G-CSF response as a major driver of enhanced sterile inflammation in CGD in the response to damaged cells. More broadly, these results provide new insights into the regulation of sterile inflammation, and identify the NADPH oxidase in regulating the amplitude of the early neutrophilic response.

From bench to bedside: preclinical evaluation of a self-inactivating gammaretroviral vector for the gene therapy of X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by impaired antimicrobial activity in phagocytic cells. As a monogenic disease affecting the hematopoietic system, CGD is amenable to gene therapy. Indeed in a phase I/II clinical trial, we demonstrated a transient resolution of bacterial and fungal infections. However, the therapeutic benefit was compromised by the occurrence of clonal dominance and malignant transformation demanding alternative vectors with equal efficacy but safety-improved features. In this work we have developed and tested a self-inactivating (SIN) gammaretroviral vector (SINfes.gp91s) containing a codon-optimized transgene (gp91(phox)) under the transcriptional control of a myeloid promoter for the gene therapy of the X-linked form of CGD (X-CGD). Gene-corrected cells protected X-CGD mice from Aspergillus fumigatus challenge at low vector copy numbers. Moreover, the SINfes.gp91s vector generates substantial amounts of superoxide in human cells transplanted into immunodeficient mice. In vitro genotoxicity assays and longitudinal high-throughput integration site analysis in transplanted mice comprising primary and secondary animals for 11 months revealed a safe integration site profile with no signs of clonal dominance.

Granulocyte colony-stimulating factor prior to nonmyeloablative irradiation decreases murine host hematopoietic stem cell function and increases engraftment of donor marrow cells.

The use of nonmyeloablative conditioning prior to bone marrow transplantation is an important component of transplantation‐based therapies for nonmalignant blood diseases. In this study, treatment of recipient mice with granulocyte colony‐stimulating factor (G‐CSF) prior to low‐dose total body irradiation (LD‐TBI) enhanced long‐term engraftment of freshly isolated congenic marrow 1.5‐ to 2‐fold more than treatment with LD‐TBI alone. This combined regimen was also evaluated in a mouse model of X‐linked chronic granulomatous disease (X‐CGD), where neutrophils have a defective NADPH oxidase due to genetic deletion of the gp91phox subunit. Long‐term engraftment of male X‐CGD bone marrow cells cultured ex vivo for retroviral transduction of gp91phox was enhanced by ∼40% when female X‐CGD recipients were pretreated with G‐CSF prior to 300 cGy. These data confirm that sequential treatment with G‐CSF and LD‐TBI prior to transplantation increases long‐term engraftment of donor marrow, and they extend this approach to transplantation of murine donor marrow cultured ex vivo for gene transfer. Additional studies showed that the administration of G‐CSF prior to LD‐TBI did not alter early homing of donor marrow cells. However, the combined regimen significantly decreased the content of long‐term repopulating cells in recipient marrow compared with LD‐TBI alone, as assessed in competitive assays, which may contribute to the enhanced engraftment of donor marrow cells.

Retroviral vector integration in post-transplant hematopoiesis in mice conditioned with either submyeloablative or ablative irradiation

X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency with absent phagocyte NADPH-oxidase activity caused by defects in the gene-encoding gp91phox. Here, we evaluated strategies for less intensive conditioning for gene therapy of genetic blood disorders without selective advantage for gene correction, such as might be used in a human X-CGD protocol. We compared submyeloablative with ablative irradiation as conditioning in murine X-CGD, examining engraftment, oxidase activity and vector integration in mice transplanted with marrow transduced with a γ-retroviral vector for gp91phox expression. The frequency of oxidase-positive neutrophils in the donor population was unexpectedly higher in many 300 cGy-conditioned mice compared with lethally irradiated recipients, as was the fraction of vector-marked donor secondary CFU-S12. Vector integration sites in marrow, spleen and secondary CFU-S12 DNA from primary recipients were enriched for cancer-associated genes, including Evi1, and integrations in or near cancer-associated genes were more frequent in marrow and secondary CFU-S12 from 300 cGy-conditioned mice compared with fully ablated mice. These findings support the concept that vector integration can confer a selection bias, and suggest that the intensity of the conditioning regimen may further influence the effects of vector integration on clonal selection in post-transplant engraftment and hematopoiesis.

Donor chimerism and gene correction with low dose irradiation and retroviral-mediated gene transfer (Rmgt) In murine x-linked chronic granulomatous disease (X-cgd)

Abstract Our laboratory has reported the correction of neutrophil NADPH oxidase function by RMGT in murine X-CGD. Few studies, however, have employed nonmyeloablative conditioning with RMGT. We have evaluated the effect of radiation dose on donor chimerism using a congenic C57B1/6, B6.SJL mouse model. At six months post-transplant, blood cells from recipients given 20 × 10 6 fresh marrow cells and 160 or 300 cGy conditioning were 50.8 ± 14% and 82.8 ± 5.4% donor in origin, respectively. This chimerism has been stable for up to 12 months. The long-term repopulating ability (LTRA) of irradiated marrow was determined using the competitive repopulation assay. Marrow from mice given 160 or 300 cGy competed 22% or 7% as well as fresh competitor cells, respectively. We next mock-transduced marrow cells on a non-virus producing packaging cell line to examine the effects of the 5 day in vitro transduction process. Mock-transduced cells completely repopulated the marrow of lethally irradiated hosts, but recipients given 160 cGy and 20 × 10 6 mock-transduced cells showed 18.8 ± 6.1% donor chimerism at six months, compared to 50.8 ± 14% for fresh marrow. Finally, X-CGD marrow cells were transduced with MSCV-m91neo, and 20 × 10 6 transduced cells were transplanted into 160 cGy X-CGD recipients. At 4 months post-transplant, we detected 6.6 ± 5.9% corrected neutrophils by NBT assay; 1100 cGy recipients given 2 × 10 6 transduced cells had 54.2 ± 6.8% NBT positive neutrophils. These results show that low dose irradiation decreases host marrow LTRA, that ex vivo culture of donor cells for RGMT impairs subsequent engraftment, and that low numbers of gene-corrected cells can be detected following RMGT and nonmyeloablative conditioning.

NADPH oxidase activation regulates apoptotic neutrophil clearance by murine macrophages

The phagocyte reduced NAD phosphate (NADPH) oxidase generates superoxide, the precursor to reactive oxygen species (ROS) that has both antimicrobial and immunoregulatory functions. Inactivating mutations in NADPH oxidase alleles cause chronic granulomatous disease (CGD), characterized by enhanced susceptibility to life-threatening microbial infections and inflammatory disorders; hypomorphic NADPH oxidase alleles are associated with autoimmunity. Impaired apoptotic cell (AC) clearance is implicated as an important contributing factor in chronic inflammation and autoimmunity, but the role of NADPH oxidase-derived ROS in this process is incompletely understood. Here, we demonstrate that phagocytosis of AC (efferocytosis) potently activated NADPH oxidase in mouse peritoneal exudate macrophages (PEMs). ROS generation was dependent on macrophage CD11b, Toll-like receptor 2 (TLR2), TLR4, and myeloid differentiation primary response 88 (MyD88), and was also regulated by phosphatidylinositol 3-phosphate binding to the p40 phox oxidase subunit. Maturation of efferosomes containing apoptotic neutrophils was significantly delayed in CGD PEMs, including acidification and acquisition of proteolytic activity, and was associated with slower digestion of apoptotic neutrophil proteins. Treatment of wild-type macrophages with the vacuolar-type H+ ATPase inhibitor bafilomycin also delayed proteolysis within efferosomes, showing that luminal acidification was essential for efficient digestion of efferosome proteins. Finally, cross-presentation of AC-associated antigens by CGD PEMs to CD8 T cells was increased. These studies unravel a key role for the NADPH oxidase in the disposal of ACs by inflammatory macrophages. The oxidants generated promote efferosome maturation and acidification that facilitate the degradation of ingested ACs.