Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous disease

Insertional mutagenesis represents a serious adverse effect of gene therapy with integrating vectors. However, although uncontrolled activation of growth-promoting genes in stem cells can predictably lead to oncological processes, this is far less likely if vector transcriptional activity can be restricted to fully differentiated cells. Diseases requiring phenotypic correction only in mature cells offer such an opportunity, provided that lineage/stage-restricted systems can be properly tailored. In this study, we followed this reasoning to design lentiviral vectors for the gene therapy of chronic granulomatous disease (CGD), an immune deficiency due a loss of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in phagocytes, most often secondary to mutations in gp91(phox). Using self-inactivating HIV1-derived vectors as background, we first expressed enhanced green fluorescent protein (eGFP) from a minimal gp91(phox) promoter, adding various natural or synthetic transcriptional regulatory elements to foster both specificity and potency. The resulting vectors were assessed either by transplantation or by lentiviral transgenesis, searching for combinations conferring strong and specific expression into mature phagocytic cells. The most promising vector was modified to express gp91(phox) and used to treat CGD mice. High-level restoration of NADPH activity was documented in granulocytes from the treated animals. We propose that this lineage-specific lentiviral vector is a suitable candidate for the gene therapy of CGD.

Lentiviral vector transduction of NOD/SCID repopulating cells results in multiple vector integrations per transduced cell: risk of insertional mutagenesis

Didier Trono

Efficient vector transduction of hematopoietic stem cells is a requirement for successful gene therapy of hematologic disorders. We asked whether human umbilical cord blood CD34(+)CD38(lo) nonobese diabetic/severe combined immunodeficiency (NOD/SCID) repopulating cells (SRCs) could be efficiently transduced using lentiviral vectors, with a particular focus on the average number of vector copies integrating into these primitive progenitor cells. Mouse bone marrow was analyzed by fluorescence-activated cell-sorter scanner and by semiquantitative polymerase chain reaction (PCR) to determine the transduction efficiency into SRCs. Lentiviral vector transduction resulted in an average of 22% (range, 3%-90%) of the human cells expressing green fluorescent protein (GFP), however, multiple vector copies were present in human hematopoietic cells, with an average of 5.6 +/- 3.3 (n = 12) copies per transduced cell. To confirm the ability of lentiviral vectors to integrate multiple vector copies into SRCs, linear amplification mediated (LAM)-PCR was used to analyze the integration site profile of a selected mouse showing low-level engraftment and virtually all human cells expressing GFP. Individually picked granulocyte macrophage colony-forming unit colonies derived from the bone marrow of this mouse were analyzed and shown to have the same 5 vector integrants within each colony. Interestingly, one integration site of the 5 that were sequenced in this mouse was located in a known tumor-suppressor gene, BRCA1. Therefore, these findings demonstrate the ability of lentiviral vectors to transduce multiple copies into a subset of NOD/SCID repopulating cells. While this is efficient in terms of transduction and transgene expression, it may increase the risk of insertional mutagenesis.

2003

Dual-regulated Lentiviral Vector for Gene Therapy of X-linked Chronic Granulomatosis

Didier Trono

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 post-transcriptionally 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.

Elsevier2014

Efficient transduction of primary human B lymphocytes and nondividing myeloma B cells with HIV-1-derived lentiviral vectors

Didier Trono

We studied the transduction of primary human B lymphocytes and myeloma cells with lentiviral vectors. In peripheral blood B cells that had been activated with helper T cells (murine thymoma EL-4 B5) and cytokines, multiply attenuated HIV-1-derived vectors pseudotyped with vesicular stomatitis virus (VSV) G-envelope protein achieved the expression of green fluorescence protein (GFP) in 27% +/- 12% (mean +/- 1 SD; median, 27%) of B cells in different experiments. When compared in parallel cultures, the transducibility of B cells from different donors exhibited little variation. The human cytomegalovirus (CMV) promoter gave 4- to 6-fold higher GFP expression than did the human elongation factor-1alpha promoter. A murine retroviral vector pseudotyped with VSV G protein proved inefficient even in mitotically active primary B cells. B cells freshly stimulated with Epstein-Barr virus were also transducible by HIV vectors (24% +/- 9%), but B cells activated with CD40 ligand and cytokines resisted transduction. Thus, different culture systems gave different results. Freshly isolated, nondividing myeloma cells were efficiently transduced by HIV vectors; for 6 myelomas the range was 14% to 77% (median, 28%) GFP(+) cells. HIV vectors with a mutant integrase led to no significant GFP signal in primary B or myeloma cells, suggesting that vector integration was required for high transduction. In conclusion, HIV vectors are promising tools for studies of gene functions in primary human B cells and myeloma cells for the purposes of research and the development of gene therapies.

2003