Little is known about the potential for engraftment of autologous hematopoietic stem cells in human adults not subjected to myeloablative conditioning regimens. Five adult patients with the p47 phox deficiency form of chronic granulomatous disease received intravenous infusions of autologous CD34 ؉ peripheral blood stem cells (PBSCs) that had been transduced ex vivo with a recombinant retrovirus encoding normal p47 phox . Although marrow conditioning was not given, functionally corrected granulocytes were detectable in peripheral blood of all five patients. Peak correction occurred 3-6 weeks after infusion and ranged from 0.004 to 0.05% of total peripheral blood granulocytes. Corrected cells were detectable for as long as 6 months after infusion in some individuals. Thus, prolonged engraftment of autologous PBSCs and continued expression of the transduced gene can occur in adults without conditioning. This trial also piloted the use of animal protein-free medium and a blood-bankcompatible closed system of gas-permeable plastic containers for culture and transduction of the PBSCs. These features enhance the safety of PBSCs directed gene therapy.

IntroductionProgressive granulomatous tissue destruction of the midface and the upper airways is most commonly caused by Wegener granulomatosis (WG) or malignancies such as natural killer (NK)/T-cell lymphomas and, to a lesser extent, cocaine abuse or infections. 1,2 The hallmark pathologic finding of destructive midline granulomatous (DMG) disease is caseating granulomas characteristically involving the nose, sinuses, palate, and upper airways, especially the subglottis. Although WG usually includes renal involvement and circulating antineutrophil antibodies (ANCAs), local nasopharyngeal WG without associated antibodies occurs. 3 Standard therapy for these diseases involves immunoablative chemotherapy to halt disease progression, in contrast to immunosupportive measures required to treat primary immune deficiencies. We describe in this report the first patient with clinical manifestations of a WG-like destructive midline granulomatosis with underlying novel compound heterozygote mutations in Rag1.The recombination activation genes, Rag1 and Rag2, perform a critical role in the somatic rearrangement and assembly of the modular genes for variable (V), diversity (D), and joining (J) gene segments of immunoglobulins and of antigen receptor genes. [4][5][6] After binding to conserved recombination signal sequences (RSSs) that flank individual V, D, or J gene segments, the Rag1/2 complex introduces a nick in the DNA, followed by formation of a coding end hairpin. Nonhomologous end joining DNA repair then takes place to generate the diverse VDJ contiguous regions that are essential for defense against many different pathogens. In addition to initiating the double-strand DNA breaks, the Rag complex stabilizes recombination intermediates to promote repair and editing of the modified genes. 6,7 Rag proteins are indispensable for lymphocyte development and differentiation, and defects in Rag manifests as T-and B-deficient severe combined immunodeficiency (T-B-SCID) or Omenn syndrome (OS) in infancy. These syndromes are characterized by early-onset profound deficiency in T and B cells for SCID and by early-onset erythroderma, hepatosplenomegaly, eosinophilia, and hyper immunoglobulin E (IgE) in OS.In stark contrast, our patient presented for evaluation of a DMG process at 14 years of age with normal numbers of CD3 ϩ T and CD19 ϩ /CD20 ϩ B cells and with normal total IgG levels. Evaluation in subsequent years showed dysregulated hyperinflammatory responses with appearance of new granulomatous lesions after environmental or viral triggers. This report further extends the clinical spectrum of Rag mutations, and our data support the view that, in addition to susceptibility to infections and autoimmunity, hyperinflammation is an important component of Rag deficiency and may be the primary clinical manifestation leading to diagnosis when lymphocyte counts are normal. Hypomorphic Rag mutations should be considered in "idiopathic" destructive granulomatous disease. MethodsThe subjects were enrolled on protocol 05-I-213 approved by...

Hematopoietic stem cells (HSCs) lose marrow reconstitution potential during ex vivo culture. HSC migration to stromal cell-derived factor (SDF)-1 (CXCL12) correlates with CXC chemokine receptor 4 (CXCR4) expression and marrow engraftment. We demonstrate that mobilized human CD34 + peripheral blood stem cells (CD34 + PBSCs) lose CXCR4 expression during prolonged culture. We transduced CD34 + PBSCs with retrovirus vector encoding human CXCR4 and achieved 18-fold more CXCR4 expression in over 87% of CD34 + cells. CXCR4-transduced cells yielded increased

IntroductionWHIM syndrome is characterized by warts, hypogammaglobulinemia, recurrent bacterial infection, and myelokathexis (severe chronic neutropenia with marrow hyperplasia and inappropriate apoptosis of mature myeloid cells in the bone marrow [BM]). [1][2][3][4][5] Many but not all cases of WHIM syndrome have been linked to autosomal dominant mutations in CXC chemokine receptor 4 (CXCR4), all of which cause truncations of the carboxy-terminus of CXCR4. Specific mutations identified in some families with myelokathexis include R334X, S339fs342X, E343X, and G335 X. 2,6,7 CXCR4 and its ligand, stromal cell-derived factor-1 (SDF-1; also known as CXCL12), play a central role in BM homing and trafficking of hematopoietic progenitor cells, mobilization of lymphocytes, and release of developing neutrophils from bone marrow. 1,[8][9][10] Mice lacking CXCR4 demonstrate defective hematopoiesis and cerebellar and cardiovascular development and die perinatally, highlighting the importance of CXCR4 in these organ systems. 11 There is 1 report of a cardiac malformation in a patient who may have had WHIM syndrome. 12 In vitro studies of WHIM variants of CXCR4 (mutated CXCR4) have provided evidence for increased agonist-dependent signaling by the mutant receptor, suggesting that the clinical manifestations may be due to hyperfunction of these receptors in vivo. In particular, we previously reported that transduction of the R334X WHIM variant into healthy human CD34 ϩ peripheral blood mobilized stem cells (PBSCs) results in enhanced chemotactic and calcium flux responses of the cells to SDF-1, and found that this effect was associated with and presumably caused by a failure of the mutant receptor to downregulate and internalize, leading to prolongation of activation. 13 In the current study we explore the mechanism of myelokathexis in WHIM syndrome using the NOD.CB17-Prkdc scid /J mouse (nonobese diabetic/severe combined immunodeficiency [NOD/SCID] mouse) xenotransplantation model engrafted with healthy human mobilized CD34 ϩ PBSCs that had been transduced with internal ribosome entry site (IRES)-containing bifunctional retrovirus vectors encoding mutated CXCR4 or wild-type (wt) CXCR4 together with a green fluorescent protein (GFP) construct or with vector-encoding GFP construct only. We will demonstrate that expression of mutated CXCR4 does not itself induce apoptosis in transduced myeloid cells differentiated in culture from the transduced PBSCs, but does result in a WHIM-type myelokathexis pattern of the transduced human cells in the in vivo xenotransplantation model (myeloid apoptosis in marrow and decreased release of cells from the marrow to the circulation). An Inside Blood analysis of this article appears at the front of this issue.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. Materials and methods G-CSF-mobilized CD34 ؉ PBSCsFollowing informed cons...

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Hematopoietic stem cell (HSC) graft cell dose impacts significantly on allogeneic transplant. Similarly, HSC gene therapy outcome is affected by loss of repopulating cells during culture required for ex vivo retrovirus transduction. Stromal cell-derived factor-1 (SDF-1) and its receptor CXCR4 play a central role in marrow trafficking of HSCs, and maneuvers that enhance CXCR4 activation might positively impact outcome in settings of limiting graft dose. CD26/dipeptidyl peptidase IV (DPP-IV) is an ectoenzyme protease that cleaves SDF-1, thus reducing CXCR4 activation. We show that injection of irradiated nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice with >or=2 micromol Diprotin A (a tripeptide specific inhibitor of CD26 protease activity) at the time of transplant of human granulocyte colony-stimulating factor (G-CSF) mobilized CD34(+) peripheral blood cells (CD34(+) PBCs) results in a >3.4-fold enhancement of engraftment of human cells. We also show that CD26 on residual stromal cells in the irradiated recipient marrow milieu, and not any CD26 activity in the human CD34(+) PBC graft itself, plays the critical role in regulating receptivity of this environment for the incoming graft. Human marrow stromal cells also express CD26, raising the possibility that Diprotin A treatment could significantly enhance engraftment of HSCs in humans in settings of limiting graft dose just as we observed in the NOD/SCID mouse human xenograft model.

In previous studies amphotropic MFGSgp91 phox (murine onco-retrovirus vector) was used in a clinical trial of X-linked chronic granulomatous disease (X-CGD) gene therapy to achieve transient correction of oxidase activity in 0.1% of neutrophils. We later showed that transduced CD34 ؉ peripheral blood stem cells (CD34 ؉ PBSCs) from this trial transplanted into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in correction of only 2.5% of human neutrophils. However, higher rates of transduction into stem cells are required. In the current study we demonstrate that the same vector (MFGS-gp91 phox ) pseudotyped with RD114 envelope in a 4-day culture/transduction regimen results in a 7-fold increase in correction of NOD/SCID mouse repopulating X-CGD CD34 ؉ PBSCs (14%-22% corrected human neutrophils; human cell engraftment 13%-67%). This increase may result from high expression of receptor for RD114 that we demonstrate on CD34 ؉ CD38 ؊ stem cells.