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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. [ABSTRACT FROM AUTHOR]

An abstract of the article "Correction of Chronic Granulomatous Disease by Gene Therapy," by Manuel Grez, Marion G. Ott, Stefan Stein, Ulrich Siler, Ulrike Koehl, Hana Kunkel, Andrea Schilz, Klaus Kuehlcke, Dieter Hoelzer and Reinhard Seger is presented.

An abstract of the article "Chronic Granulomatous Disease Gene Therapy Functionally Corrects the Phenotype of Polymorphonuclear Leukocytes (PMN)," by Ulrich Siler, Marion G. Ott, Stefan Stein, Elke Karaus, Maja Rutishauser, Corinne Wenk, Dieter Hoelzer, Manuel Grez and Reinhard Seger is presented.

Vector-associated side effects in clinical gene therapy have provided insights into the molecular mechanisms of hematopoietic regulation in vivo. Surprisingly, many retrovirus insertion sites (RIS) present in engrafted cells have been found to cluster nonrandomly in close association with specific genes. Our data demonstrate that these genes directly influence the in vivo fate of hematopoietic cell clones. Analysis of insertions thus far has been limited to individual clinical studies. Here, we studied >7,000 insertions retrieved from various studies. More than 40% of all insertions found in engrafted gene-modified cells were clustered in the same genomic areas covering only 0.36% of the genome. Gene classification analyses displayed significant overrepresentation of genes associated with hematopoietic functions and relevance for cell growth and survival in vivo. The similarity of insertion distributions indicates that vector insertions in repopulating cells cluster in predictable patterns. Thus, insertion analyses of preclinical in vitro and murine in vivo studies as well as vector insertion repertoires in clinical trials yielded concerted results and mark a small number of interesting genomic loci and genes that warrants further investigation of the biological consequences of vector insertions. [ABSTRACT FROM AUTHOR]

The potential of gene therapy to cure inherited immunodeficiencies of the lymphoid compartment has been demonstrated in clinical trials of ADA-SCID and SCID-X1. To circumvent the missing in vivo selection advantage of the corrected cells in myeloid diseases the two patients in the recent successful chronic granulomatous disease (CGD) gene therapy trial received nonmyeloablative conditioning before transplantation of the corrected cells. 3 months after transplantation 20% and 10% of the granulocytes in the peripheral blood expressed gp91phox in patient 1 and patient 2, respectively. 5 to 9 months after therapy a 3 to 4 fold expansion of marked granulocytes in the peripheral blood appeared in both patients. The proportion of gene-corrected granulocytes in both patients was then stable until 524 days post transplantation in patient 1 and 343 days post transplantation in patient 2 which were the last time points analyzed. To characterize the clonality of the hematopoietic repopulation, the long term activity of individual clones and the distribution of the SFFV vector integration sites we analyzed peripheral blood leucocytes and sorted cells derived from different time points post transplantation with linear amplification mediated PCR (LAM-PCR) and accomplished high throughput sequencing and mapping of the insertion sites. For patient 1 we could identify 435 exactly mappable insertion sites. For patient 2 330 unique integrations could be assigned to the human genome. We detected 3 common integrations sites (CIS) in or near the loci of the zinc finger transcription factor homologous MDS1/EVI1 and PRDM16 in both patients and SETBP1 as a third CIS gene in patient 1. RNA analysis demonstrated an activating influence of the retroviral vector on the CIS genes in both patients. LAM-PCR analysis of CFU-GM and BFU-E derived colonies from both patients revealed that 1 to 4 integrations are present per cell and confirmed the increasing dominance of one MDS1/EVI1 clone in patient 1 seen in the peripheral blood LAM-PCR pattern. Our data show that the upregulation of specific human genes as a side effect of retroviral integration may lead to an unprecedented in vivo expansion of the concerned cell clones and have therefore great impact on future gene therapy strategies.Molecular Therapy (2006) 13, S279–S279; doi: 10.1016/j.ymthe.2006.08.803 [ABSTRACT FROM AUTHOR]

An abstract of the article "Stable Polyclonal Hematopoietic Repopulation after Successful Clinical Gene Therapy of Chronic Granulomatous Disease (CGD)," by Manfred Schmidt and colleagues is presented.