Genetic engineering of hematopoiesis for targeted IFN-α delivery inhibits breast cancer progression

The immunosuppressive tumor microenvironment represents a major hurdle to cancer therapy. We developed a gene transfer strategy into hematopoietic stem cells (HSCs) to target transgene expression to tumor-infiltrating monocytes/macrophages. Using a combination of transcriptional and microRNA-mediated control, we achieved selective expression of an interferon-α (IFN-α) transgene in differentiated monocytes of human hematochimeric mice. We show that IFN-α transgene expression does not impair engraftment and long-term multilineage repopulation of NSG (NOD/LtSz-scidIL2Rγ(null)) mice by transplanted human HSCs. By providing a source of human cytokines in the mice, we improved the functional reconstitution of human myeloid, natural killer, and T cell lineages, and achieved enhanced immune-mediated clearance of transplanted human breast tumors when hematopoiesis was engineered for tumor-targeted IFN-α expression. By applying our strategy to mouse breast cancer models, we achieved inhibition of tumor progression and experimental metastases in an autologous setting, likely through enhanced generation of effector T cells and their recruitment to the neoplastic tissues. By forcing IFN-α expression in tumor-infiltrating macrophages, we blunted their innate protumoral activity and reprogrammed the tumor microenvironment toward more effective dendritic cell activation and immune effector cell cytotoxicity. Overall, our studies validate the feasibility, safety, and therapeutic potential of a new cancer gene therapy strategy, and open the way to test this approach as adjuvant therapy in advanced breast cancer patients.

Role of Interferon-Gamma in Interleukin 12-Induced Pathology in Mice

Michel Aguet

Interleukin 12 (IL-12) activates natural killer (NK) and T cells with the secondary synthesis and release of interferon-gamma (IFN-gamma) and other cytokines. IL-12-induced organ alterations are reported for mice and the pathogenetic role of IFN-gamma is investigated by the use of mice deficient in the IFN-gamma receptor (IFN-gamma R(-/-)). IL-12 caused a rapid infiltration of liver and splenic red pulp with activated macrophages; this and increased NK cells resulted in a fivefold increase of splenic weight in wild-type mice. Splenomegaly was associated with myelosuppression and decreasing peripheral leukocyte counts. IL-12-induced changes in wild-type mice were associated with markedly increased IFN-gamma serum levels and up-regulation of major histocompatibility complex (MHC) class I and II expression in various epithelia. IL-12 induced a qualitatively similar macrophage infiltration in IFN-gamma R(-/-) mice, less marked splenomegaly (to 2 x normal), and no MHC upregulation. Strikingly increased vascular endothelial intercellular adhesion molecule-1 expression was apparent in both IFN-gamma R(-/-) and IFN-gamma R(+/+) mice. Restricted to mutant mice was a severe, invariably lethal, interstitial, and perivascular pulmonary macrophage infiltration with diffuse pulmonary edema. Extensive quantitative reverse transcriptase polymerase chain reaction analysis revealed an increase of only IL-6 and IL-10 pulmonary gene transcripts in IFN-gamma R(-/-) mice compared with wild-type mice. IL-12-induced myelosuppression is due to IFN-gamma-release from NK cells and T cells, and is associated with macrophage activation and distinct MHC class I and II antigen upregulation. The pulmonary pathology in IFN-gamma R(-/-) mice, however, reveals a toxic potential for IL-12 and suggests that endogenous IFN-gamma plays a protective role in preventing fatal pulmonary disease in these mice.

1995

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

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

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

Didier Surdez

EPFL2008