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Stable gene transfer to human pluripotent hematopoietic stem cells (PHSCs) is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical trials, the levels of gene transfer to this cell population have generally been low, reflecting deficiencies in both the vector systems and transduction conditions. In this study, we have used a pseudotyped murine retroviral vector to transduce human CD34(+) cells purified from bone marrow (BM) and umbilical cord blood (CB) under optimized conditions. After transduction, 71% to 97% of the hematopoietic cells were found to express a low-affinity nerve growth factor receptor (LNGFR) marker gene. Six weeks after transplantation into immunodeficient NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was detected in 6% to 57% of CD45(+) cells in eight of nine engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45% of secondary colonies derived from BM cells of engrafted NOD/SCID mice. Our data show consistent transduction of SCID-repopulating cells (SRCs) and suggest that the efficiency of gene transfer to human hematopoietic repopulating cells can be improved using existing retroviral vector systems and carefully optimized transduction conditions.

Positron emission tomography (PET) was used in the follow-up of patients with colorectal malignancies to differentiate between recurrent colorectal tumor and scar. Patients were examined with oxygen-15-labeled water and with fluorine-18-labeled deoxyglucose (FDG). FDG was injected intravenously to assess tumor metabolism. The tracer concentration was quantitatively evaluated by means of a region-of-interest technique and standardized for both injected dose and body volume. Of 29 patients, 21 had recurrent colorectal malignancy, and eight had a nonmalignant mass. All malignancies were seen on the PET cross sections. Nonmalignant lesions had a low FDG accumulation on images obtained 60 minutes after injection. While the tumor-soft tissue ratio was highest shortly after the intravenous injection of FDG, the tumor-scar ratio was highest 60 minutes after injection. It was possible to differentiate tumor from non-malignant tissue with FDG with the use of standardized concentration values and tumor-soft tissue ratios. Imaging with O-15-labeled water gave no additional information.

The potential of gene therapy as a curative treatment for monogenetic disorders has been clearly demonstrated in a series of recent Phase I/II clinical trials. Among primary immunodeficiencies, gene transfer into hematopoietic stem (HSC)/progenitor cells has resulted in the long-term correction of immune and metabolic defects in treated patients. In most cases, successes were augmented by a recognized biological selection for successfully treated cells in vivo, perhaps even to some extent at the HSC level. In contrast, similar achievements have not turned into reality for immunodeficiencies in which gene-transduced cells lack selective advantages in vivo. This is the case for chronic granulomatous disease (CGD), a primary immunodeficiency, characterized by deficient antimicrobial activity in phagocytic cells. Several attempts to correct CGD by gene transfer in combination with bone marrow conditioning have resulted in low-level long-term engraftment and transient clinical benefits despite high levels of gene marking and high numbers of reinfused cells. This review summarizes the data from clinical trials for CGD and provides some insights into treatment options that may lead to a successful application of gene therapy for CGD.

Gene-modified autologous hematopoietic stem cells (HSC) can provide ample clinical benefits to subjects suffering from X-linked chronic granulomatous disease (X-CGD), a rare inherited immunodeficiency characterized by recurrent, often life-threatening bacterial and fungal infections. Here we report on the molecular and cellular events observed in two young adults with X-CGD treated by gene therapy in 2004. After the initial resolution of bacterial and fungal infections, both subjects showed silencing of transgene expression due to methylation of the viral promoter, and myelodysplasia with monosomy 7 as a result of insertional activation of ecotropic viral integration site 1 (EVI1). One subject died from overwhelming sepsis 27 months after gene therapy, whereas a second subject underwent an allogeneic HSC transplantation. Our data show that forced overexpression of EVI1 in human cells disrupts normal centrosome duplication, linking EVI1 activation to the development of genomic instability, monosomy 7 and clonal progression toward myelodysplasia.

In order to control graft-versus-host disease after donor lymphocyte infusion, T cells can be retrovirally transduced with a suicide gene. However, the immune competence of activated T cells appears compromised, responsible for reduced alloreactivity. The present study compared different activation protocols using soluble or bead-coupled antibodies regarding. T-cell subtype expansion capacity and functionality. T cells were purified on a laboratory and clinical scale using both CD3 and CD4/CD8 antibodies for selection, leading to a mean purity of 96%. Transductions were performed with a GMP-grade CD34/HSV-TK vector. Activation with soluble CD3/CD28-antibodies +1000 U/ml IL-2 induced a 50-fold expansion of T cells over 14 days, whereas T cells activated with bead-coupled antibodies only expanded 2-4-fold restricted to the first week. Apart from using soluble antibodies, proliferation was highly IL-2 dependent. Expansion of CMV-specific T cells coincided with the expansion of whole CD3(+) cells. Soluble antibodies and higher IL-2 concentrations preferentially stimulated CD8(+) T cells, while bead-coupled antibodies +20 U/ml IL-2 preserved the CD4/CD8 ratio. Irrespective of the activation protocol, there was a shift from a naive to memory phenotype. When activated with soluble antibodies, mainly CD8(+) T cells were transduced. Furthermore, Th1/Th2 cytokine secretion was reduced. In contrast, CD4(+)/CD8(+) T cells activated with bead-coupled antibodies were rather homogenously transduced and cytokine secretion did not appear to be affected. (C) 2007 Elsevier B.V. All rights reserved. In order to control graft-versus-host disease after donor lymphocyte infusion, T cells can be retrovirally transduced with a suicide gene. However, the immune competence of activated T cells appears compromised, responsible for reduced alloreactivity. The present study compared different activation protocols using soluble or bead-coupled antibodies regarding. T-cell subtype expansion capacity and functionality. T cells were purified on a laboratory and clinical scale using both CD3 and CD4/CD8 antibodies for selection, leading to a mean purity of 96%. Transductions were performed with a GMP-grade CD34/HSV-TK vector. Activation with soluble CD3/CD28-antibodies +1000 U/ml IL-2 induced a 50-fold expansion of T cells over 14 days, whereas T cells activated with bead-coupled antibodies only expanded 2-4-fold restricted to the first week. Apart from using soluble antibodies, proliferation was highly IL-2 dependent. Expansion of CMV-specific T cells coincided with the expansion of whole CD3(+) cells. Soluble antibodies and higher IL-2 concentrations preferentially stimulated CD8(+) T cells, while bead-coupled antibodies +20 U/ml IL-2 preserved the CD4/CD8 ratio. Irrespective of the activation protocol, there was a shift from a naive to memory phenotype. When activated with soluble antibodies, mainly CD8(+) T cells were transduced. Furthermore, Th1/Th2 cytokine secretion was reduced. In contrast, CD4(+)/CD8(+) T cells activated with bead-coupled antibodies were rather homogenously transduced and cytokine secretion did not appear to be affected. (C) 2007 Elsevier B.V. All rights reserved.

Regulated transgene expression may improve the safety and efficacy of hematopoietic stem cell (HSC) gene therapy. Clinical trials for X-linked chronic granulomatous disease (X-CGD) employing gammaretroviral vectors were limited by insertional oncogenesis or lack of persistent engraftment. Our novel strategy, based on regulated lentiviral vectors (LV), targets gp91(phox) expression to the differentiated myeloid compartment while sparing HSC, to reduce the risk of genotoxicity and potential perturbation of reactive oxygen species levels. Targeting was obtained by a myeloid-specific promoter (MSP) and posttranscriptional, microRNA-mediated regulation. We optimized both components in human bone marrow (BM) HSC and their differentiated progeny in vitro and in a xenotransplantation model, and generated therapeutic gp91(phox) expressing LVs for CGD gene therapy. All vectors restored gp91(phox) expression and function in human X-CGD myeloid cell lines, primary monocytes, and differentiated myeloid cells. While unregulated LVs ectopically expressed gp91(phox) in CD34(+) cells, transcriptionally and posttranscriptionally regulated LVs substantially reduced this off-target expression. X-CGD mice transplanted with transduced HSC restored gp91(phox) expression, and MSP-driven vectors maintained regulation during BM development. Combining transcriptional (SP146.gp91-driven) and posttranscriptional (miR-126-restricted) targeting, we achieved high levels of myeloid-specific transgene expression, entirely sparing the CD34(+) HSC compartment. This dual-targeted LV construct represents a promising candidate for further clinical development.