Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1

Abstract

Gene transfer into hematopoietic stem cells has been used successfully for correcting lymphoid but not myeloid immunodeficiencies. Here we report on two adults who received gene therapy after nonmyeloablative bone marrow conditioning for the treatment of X-linked chronic granulomatous disease (X-CGD), a primary immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes resulting from mutations in gp91(phox). We detected substantial gene transfer in both individuals' neutrophils that lead to a large number of functionally corrected phagocytes and notable clinical improvement. Large-scale retroviral integration site-distribution analysis showed activating insertions in MDS1-EVI1, PRDM16 or SETBP1 that had influenced regulation of long-term hematopoiesis by expanding gene-corrected myelopoiesis three- to four-fold in both individuals. Although insertional influences have probably reinforced the therapeutic efficacy in this trial, our results suggest that gene therapy in combination with bone marrow conditioning can be successfully used to treat inherited diseases affecting the myeloid compartment such as CGD.

Significant increase of self-renewal in hematopoietic cells after forced expression of EVI1

“…A constellation of intrinsic and extrinsic cellular mechanisms regulates the balance of self-renewal and differentiation in all stem cells and the genetic and molecular mechanisms responsible for the choice of a HSC between these two directions remain to be elucidated. It has recently been reported that retroviral integration in the MDS1/ EVI1 locus leads to the emergence of a dominant hematopoietic clone with enhanced expansion and self-renewal potential [3][4][5]. The MDS1/EVI1 locus encodes 2 major proteins, MDS1/EVI1 and EVI1, but it is not yet known whether one of them is responsible for the emergence of the dominant hematopoietic clone.…”

“…In both studies the investigators observed a significant frequency of insertion into the MDS1/EVI1 region and the retrovirus was detected primarily in long-term myeloid cells, suggesting that the selfrenewal and engraftment potential of CD34 + progenitor cells are enhanced by insertion mutagenesis at this specific locus across species. As with the mice, these investigators noted no evidence of in vivo malignant clones in the MDS1/EVI1 populations and the hematopoietic system of all animals and patients appeared normal without evidence of leukemia [4,5].…”

“…This effect of retroviral integration in the MDS1/EVI1 locus leading to the emergence of a dominant hematopoietic stem cell clone is not limited to the mouse. Two other groups analyzed hundreds of integration sites either in non-human primates seven years after transplantation of autologous CD34 + cells transduced with a retroviral vectors [4] or in two patients following a gene-therapy treatment a year earlier that had required infection of autologous bone marrow with a retrovirus [5]. In both studies the investigators observed a significant frequency of insertion into the MDS1/EVI1 region and the retrovirus was detected primarily in long-term myeloid cells, suggesting that the selfrenewal and engraftment potential of CD34 + progenitor cells are enhanced by insertion mutagenesis at this specific locus across species.…”

Physiological regulation of transgene expression by a lentiviral vector containing the A2UCOE linked to a myeloid promoter

“…One particular notorious example of this is the CpG methylation at the promoter region of the spleen focus-forming virus (SFFV) long terminal repeat that occurred in our gene therapy trial for X-CGD, leading to the progressive silencing of gp91 phox expression and reversal of the therapeutic effect. 7,8 Functional elements such as insulators, replicators, scaffold attachment regions or CpG islands have been reported to counteract silencing of gene expression by shielding vector sequences such as positional effects and silencing epigenetic modifications (reviewed in Emery 13 ). Although insulators, such as the chicken hypersensitive site 4 element, have been shown to reduce silencing and to protect vectors from chromosomal position effects, their effects are variable.…”

Automated analysis of viral integration sites in gene therapy research using the SeqMap web resource

“…2,3 These studies, and their resulting publications, have described the abundance and location of vector integration sites in normal and malignant cells using experimental methods such as ligation-mediated PCR (LM-PCR) or linear amplification-mediated PCR (LAM-PCR) in both human and model organisms. 2,4,5 Ligation-mediated PCR and linear amplificationmediated PCR seek to identify retroviral vector integration sites by digesting genomic DNA with an appropriate restriction enzyme and by using PCR primers within the long terminal repeats and an adapter fragment to amplify the long terminal repeat-genomic DNA junctions. 6,7 Investigators may sequence the PCR product directly or through Topo-cloning methods.…”