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

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.

Lentiviral gene transfer into primary and secondary NOD/SCID repopulating cells

Didier Trono, Romain Zufferey

The ability of lentiviral vectors to transfer genes into human hematopoietic stem cells was studied, using a human immunodeficiency virus 1 (HIV-1)-derived vector expressing the green fluorescence protein (GFP) downstream of the phosphoglycerate kinase (PGK) promoter and pseudotyped with the G protein of vesicular stomatitis virus (VSV). High-efficiency transduction of human cord blood CD34(+) cells was achieved after overnight incubation with vector particles. Sixteen to 28 percent of individual colony-forming units granulocyte-macrophage (CFU-GM) colonies derived from cord blood CD34(+) cells were positive by polymerase chain reaction (PCR) for the GFP gene. The transduction efficiency of SCID-repopulating cells (SRC) within the cord blood CD34(+) population was assessed by serial transplantation into nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. When 400,000 cord blood CD34(+) cells were transplanted into primary recipients, all primary and secondary recipients contained and expressed the transgene. Over 50% of CFU-GM colonies derived from the bone marrow of these primary and secondary recipients contained the vector on average as determined by PCR. Transplantation of transduced cells in limiting dilution generated GFP(+) lymphoid and myeloid progeny cells that may have arisen from a single SRC. Inverse PCR analysis was used to amplify vector-chromosomal junctional fragments in colonies derived from SRC and confirmed that the vector was integrated. These results show that lentiviral vectors can efficiently transduce very primitive human hematopoietic progenitor and stem cells. (Blood. 2000;96:3725-3733)

American Society of Hematology2000

Prospective identification of hematopoietic and neural stem/progenitor cells using transgenic and lentiviral based approaches

Didier Surdez

Adult stem cells represent only a minor proportion of a given tissue and are difficult to amplify in vitro without affecting their biological properties. To circumvent this problem, we used a transgenic/lentiviral based approach to isolate and characterize these cells by driving a fluorescent reporter protein under the control of Spi2a, H2Kb or MELK promoter/enhancer elements, which were previously shown to be active in various stem cells. The first chapter focuses on Spi2a, a serine protease inhibitor (Serpin), which is known to be preferentially expressed in hematopoietic stem cells (HSCs). Taking advantage of the previously characterized minimal Spi2a promoter, we were able to determine its activity profile in the hematopoietic system. For this purpose, we infected HSCs with a lentivirus driving d2eGFP expression under the control of the minimal Spi2a promoter. These cells were then injected into lethally irradiated recipient mice that were analyzed at different time points (≥2 months) after transplantation. Despite variable d2eGFP expression in the hematopoietic compartment, we found a population of bright d2eGFP-expressing bone marrow cells (Spi2a0.1%) that were mostly restricted to HSCs and early myeloid progenitors. Spi2a0.1% cells displayed multilineage colony forming potential in vitro and exhibited long-term multilineage repopulating capacity in vivo, when a restricted numbers of these cells (≥ 300 cells) were transplanted into secondary recipient mice. Furthermore, we generated transgenic mice using a similar construct as used for the lentiviral approach. These mice indicated that the Spi2a promoter may be activated in non-hematopoietic adult stem cells, such as in the brain, the testis or the pancreas. In the second chapter, we describe a transgenic mouse line that expresses a green fluorescent protein variant (zFP) under the control of H2Kb promoter/enhancer element. Despite the broad zFP expression, transgenic HSCs expressed exceptionally high levels of zFP, allowing prospective isolation of a population highly enriched in HSCs by sorting the 0.2% of the brightest green cells from the enriched bone marrow of H2Kb-zFP mice. Up to 90% of zFPbright cells were also c-kithigh, Sca-1high, Linneg, Flk-2neg, which is a bona fide phenotype for long-term HSCs. Double-sorted zFPbright HSCs were capable of long-term multilineage reconstitution at a limiting dilution dose of approximately 12 cells, which is comparable to that of highly purified HSCs obtained by conventional multicolor flow cytometry. Thus, the H2Kb-zFP transgenic mice provide a straightforward and easy setup for the simple and highly efficient enrichment for genetically labeled HSCs. The last chapter is dedicated to MELK. This kinase of the snf1/AMPK family was recently identified to regulate the proliferation of multipotent neural progenitor cells and was also found to be expressed in hematopoietic stem cells and in spermatogonia. Using a transgenic mouse approach by expressing eGFP under a 3.5kb MELK promoter/enhancer element, we demonstrated that eGFP-expressing cells were localized in the subventricular zone (SVZ) of the lateral ventricle and in the subgranular zone of the dentate gyrus, the two major regions of adult neurogenesis. Moreover, the eGFP expression level in SVZ-derived cells positively correlated with their ability to form neurospheres. We also observed strong and restricted expression of eGFP in spermatogonia cells, indicating that MELK may play an important role in early spermatogenesis. Additionally, activation of the MELK promoter in hematopoietic stem/progenitor cells suggested a widespread implication of MELK in various adult stem cells. To conclude, we have generated three different reporter strains and one lentiviral based approach that allow for in vivo identification of stem/progenitor cells in different compartment such as the brain, the bone marrow or the testis. With the raising interest of stem-cell-based therapies in the neurodegenerative or the cancer field, these mice should provide novel and alternative tools to study these phenomena in vivo.

EPFL2008

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

Prospective identification of hematopoietic and neural stem/progenitor cells using transgenic and lentiviral based approaches

Didier Surdez

EPFL2008