Research

Our lab focuses on the following three research lines:
   1. Development of Foamy Virus (FV) Vectors.
   2. The role of b-catenin in the establishment of human leukemias.
   3. Characterization of the Neuroblastoma Stem Cells.

The lab has developed expertise in retroviral vector production but have a specific interest on FV vectors since they are non pathogenic and give long term expression in hematopoietic stem cells. In addition, since

1. Foamy Virus Vectors.
FVs are non-pathogenic retroviruses that use primates as their natural reservoir. Human transmission has been documented in zoo keepers and African hunters who have been bitten by primates; in these individuals, long-term observations have documented that the infection has resulted in sero-conversion (devel opment of antibodies) and no pathology, indicating the enhanced safety profile of FVs. We and others have shown that vectors derived from these viruses can transduce murine, canine and human hematopoietic stem cells (HSC); in addition, in the canine model of leukocyte adhesion deficiency (lack of CD18), gene transfer of CD18 in HSC has resulted in the genetic correction of the disorder. In essence, FV vectors offer a safe alternative to the lenti-vectors that are currently widely used in the gene therapy field. We are developing FV vectors for the following aims:
(i) genetic correction of beta-thalassemia and chronic granulomatous disease,
(ii) coordinated transgene expression using endogenous bidirectional promoters and
(iii) short-hairpin RNA expression for targeted gene silencing

2. b-catenin role in human leukemia.
It has been documented that overexpression of b-catenin (a signal transducer for the WNT pathway) is an adverse prognostic marker in acute myeloid leukemia, indicating the central role that the aberrant regulation of WNT may have in AML. To delineate its role, we down-regulated its levels in AML lines and tested various aspects of cell behaviour after transplantation in NOD/SCID animals. Our results showed that there was an effect in the rate of cell proliferation and, most importantly, that the cell could not engraft and establish leukemias in the host animals. The latter was mediated through the downregualtion of CD44, an adhesion molecule in the BM microenvironment and a transcriptional target of b-catenin.
We are currently expanding our research in the aberrant wnt signaling in hematopoietic neoplasms and are exploring its role in myelodysplastic syndromes (MDS). MDS is a preleukemic condition characterized by cytopenias in the peripheral blood. The aetiology is unknown but a large body evidence shows that the microenvironment may play a significant role in the development of the phenotype. There is also evidence that osteoblasts (OB) and HSC are in close proximity in the bone marrow and OB are of paramount importance in the formation of the HSC niche and the regulation of the HSC phenotype. A common link for both OB and HSC is the wnt signaling path that provides growth signals for both cell types while stroma-cell secreted wnt ligands are though to act in a paracrine fashion to support HSC regulation. In addition, an MDS phenotype was recently described in APC-/- animal which is also a transcriptional target of b-catenin. To directly test the hypothesis of aberrant wnt signaling in the pathogenesis of MDS, we are growing stromal cells from MDS patients and are quantitating b-catenin expression by RT-PCR; we also plan to analyze the wnt path by SuperArrays to identify differentially expressed targets that could provide clues to the pathogenesis of this disorder.

3. Neuroblastoma Stem Cells.
Solid tumor formation is also characterized by the presence of stem cells that are responsible for self renewal. We sought to define the profile of the Neuroblastoma Stem Cells based on the expression of CD44, a stem cell marker. Using a xenograft model in NOD/SCID animals, we found that in contrast to what is happening in other tumors, neuroblastoma stem cells are CD44 negative. Using array gene expression analysis, we showed that CD44 defines molecularly discrete cell types with the CD44 negative cells expressing proteins associated with uncontrolled cell cycle progression, immune evasion and a reduced capacity to undergo apoptosis, all properties of a tumor initiating cell.