Research Projects

Development of cellular innovative strategies for the treatment of post transplant complications (infection and graft versus host disease) in leukemic patients

Hematopoietic stem cell transplantation is currently used to treat childhood leukemia when chemotherapy yields a poor outcome. Allogeneic HSCT involves complete replacement of the blood and immune systems of the patient with those of the donor. Severe acute graft-versus-host disease (GVHD) after transplantation is associated with a high mortality rate and is a major threat to a successful outcome. There is no effective therapy for severe steroid-refractory acute GVHD. Moreover, HSCT is associated with marked immunosuppression, which continues for several months after transplantation until donor T cells can be generated de novo from donor HSCs. The immunosuppression associated with HSCT results from several factors. Chemotherapy and radiotherapy, used before transplantation to bring malignant disease under control, can suppress immune function, as do similar drugs that are used in the transplant-conditioning regimen to allow graft acceptance. Finally, immunosuppressive drugs are administered to suppress GVHD. The net result of these influences is a marked cellular immunodeficiency in the first few months after transplantation, which leaves the host susceptible to a wide range of infections. Cytomegalovirus (CMV), adenoviruses (HAdV) are the commonest viral pathogens causing early as well as late onset infection after transplant, especially in leukemia patients receiving mismatched related or unrelated donor stem cell grafts. Two will be the targets of our research : 1. developing of novel supportive therapy of CMV and AdV-infectious complications of transplanted pediatric leukemia patients, capable of overcoming the limitations and side effects of conventional therapy leukemia patients. 2. improving the immunomodulatory function of mesenchymal cells for their potential use in GVHD treatment
 

Exploiting miRNA as a target for immuno-intervention

The complete scope of miRNA involvement in immunity will probably take some time to emerge, but it is already clear that these tiny players can have a big impact on this complicated and life-sustaining system. Because miRNAs appear to provide quantitative regulation of genes, rather than on-off decisions, they can be seen as fine tuning a cell’s responses to external influences. The ability of environmental factors, such as inflammatory ligands or immunosuppressive molecules, to alter miRNA expression is just beginning to be explored. Inadequate expression of cytokines have been suggested to complement genetic aberrations in leukomogenesis, thus, supporting survival and proliferation of leukemic cells. In addition, secreted cytokines have critical effects on the immune system by impairing efficient immune surveillance of immunocompetent cells. It has been demonstrated that bone marrow Th2 cytokine expression could be used as predictor for relapse in childhood ALL. In particular, IL-10 expression is considered a significant adverse prognostic indicator in childhood BCP-ALL. Moreover, an increased expression of IL-4 and vascular endothelial growth factor (VEGF) mRNA was observed in patients with late relapses. To better understand the leukemia-derived signals guiding polarisation of innate and adaptive immunity in leukemia bearers and to identify molecular mechanisms that might be amenable to therapeutic intervention, we will analyze the potential involvement of miRNAs in immunoregulatory functions of monocytes and lymphocytes exposed to leukaemia-like microenviroment..

References

Biagi E, Bambacioni F, Gaipa G, Casati C, Golay J, Biondi A, Introna M. Efficient lentiviral transduction of primary human acute myelogenous and lymphoblastic leukemia cells. Haematologica. 2001; 86:13-6.

Biagi E, Rousseau RF, Yvon E, Vigouroux S, Dotti G, Brenner MK. Cancer vaccines: dream, reality, or nightmare? Clin Exp Med. 2002; 2:109-18.

Longoni D., D'Amico G., Gaipa G., Vulcano M., Niemeyer C.M., Allavena P.and Biondi A.Committment of pathological monocytic cells from JMML patients to spontaneously differentiate into dendritic cells. The Hematol. Journal. 2002;3:302-306.

Biagi E, Yvon E, Dotti G, Amrolia PJ, Takahashi S, Popat U, Marini F, Andreeff M, Brenner MK, Rousseau RF. Bystander transfer of functional human CD40 ligand from gene-modified fibroblasts to B-chronic lymphocytic leukemia cells. Human Gene Therapy. 2003;14:545-559.

Bonamino M, Serafini M, D'Amico G, Gaipa G, Todisco E, Bernasconi S, Golay J, Biondi A, Introna M. Functional transfer of CD40L gene in human B-cell precursor ALL blasts by second-generation SIN lentivectors. Gene Ther. 2004;11:85-93.

Vigouroux S, Yvon E, Biagi E, Brenner MK. Antigen-induced regulatory T cells. Blood. 2004;104:26-33. D’Amico G., Vulcano M., Bugarin C., Bianchi G., Pirovano G., Bonamino M., Marin V., Allavena P, Biagi E. and Biondi A. CD40 activation of BCP-ALL cells generates IL-10 producing, IL-12 defective APCs that induce allogeneic T-cell anergy. Blood. 2004;104: 744-751.

V. Marin, E. Dander, E. Biagi, M. Introna, G. Fazio, A. Biondi and G. D’Amico. Characterization of in vitro migratory properties of anti-CD19 chimeric receptor-redirected CIK cells for their potential use in B-ALL immunotherapy. Exp Hematol. 2006. 34:1219-1229.

D’Amico G., Bonamino M., Dander E., Marin V., Basso G., Balduzzi A., Biagi E., Biondi A. T cells stimulated by CD40L positive leukemic blasts-pulsed dendritic cells meet optimal functional requirements for adoptive T-cell therapy. Leukemia. 2006;20:2015-24.

Marin V., Kakuda H, Dander E, Imai C, Campana D, Biondi A and D’Amico G. Enhancement Of The Anti-leukemic Activity of Cytokine Induced Killer cells With An Anti-CD19 Chimeric Receptor Delivering a 4-1BB Activating Signal. Exp Hematol. 2007. 35:1388-1397.

Biagi E, Marin V, Giordano Attianese GM, Dander E, D'Amico G, Biondi A. Chimeric T-cell receptors: new challenges for targeted immunotherapy in hematologic malignancies. Haematologica. 2007. 9:381-388.

Salvadè A, Belotti D, Donzelli E, D'Amico G, Gaipa G, Renoldi G, Carini F, Baldoni M, Pogliani E, Tredici G, Biondi A, Biagi E.GMP-grade preparation of biomimetic scaffolds with osteo-differentiated autologous mesenchymal stromal cells for the treatment of alveolar bone resorption in periodontal disease. Cytotherapy. 2007. 9:427-438.

Dander E., Lipira G., Biagi E., Perseghin P., Introna M., Marin V., Manca F., Biondi A., and D’Amico G. Adoptive T-cell therapy with CMV-specific lymphocytes by use of GMP-grade peptides: extensive characterization of functional immune features for improved clinical use. Exp Hematol. In press.
 

Chimeric T cells for the treatment of pediatric cancers

Leukemias are the most common cancers affecting children while malignant lymphomas, including non-Hodgkin lymphomas (NHL), comes in third position after brain tumors. A significant number of children with leukemia/lymphomas still fail current therapies. The aim of the CHILDHOPE project is to develop a safe and efficient adoptive immunotherapy for children with advanced or refractory malignancies. CHILDHOPE particularly focuses on three pediatric tumors: acute B-lineage lymphoblastic leukemia, non-hodgkin B-lineage lymphoma and acute myeloid leukemia. The CHILDHOPE project is a new approach in pediatric cancer treatment since it brings from bench to bedside (and back) an innovative technology as yet never applied in children with advanced or refractory hematopoietic malignancies. The CHILDHOPE translational research project will focus on: 1. Improving and testing the efficacy and the safety of anti-leukemia/lymphoma chimeric T cells in relevant preclinical models in vitro and in vivo in mice. 2. Scaling-up this technology to numbers suitable for a clinical application in children with hematopoietic malignancies. Based on biological material obtained from our preclinical models and from children treated with these genetically engineered T cells, dissecting the interface between the host and tumor and immune cells and use this knowledge to understand the mechanisms of anti-tumor action, validate novel targets and diagnostic tools specific to children affected with leukemia or lymphomas. The CHILDHOPE project is built on the excellence of a network of EU-based partners with a broad experience in the field of pediatric hematology and oncology, immunology and cell & gene therapies and integrates the international confederation of parents of children with cancer and an SME specialized in the project management.
 

Molecular dissection of the etiopathogenesis of childhood Acute Lymphoblastic Leukemia (ALL)

1) The pre-leukemic state of the TEL-AML1 positive cell. TEL-AML1 fusion is the most frequent genetic abnormality in paediatric cancer, and it is usually an early or initiating and pre-natal event in childhood ALL. Transformation results in the generation of a persistent pre-leukemic clone, which converts to frank ALL post-natally (at 1-15 years) following the acquisition of secondary genetic alterations. The mechanism by which the transcriptional dysregulation imposed by TEL-AML1 impacts on the pre-leukemic phenotype and disease natural history is unknown. We showed that the TEL-AML1 protein inhibits the TGFb response pathway, facilitating the selective expansion of otherwise more slowly expanding TEL-AML1 expressing progenitors. We aim to further explore the pre-leukemic phase by analyzing the migration/adhesion properties of the TEL-AML1 positive cells, and their interactions with the bone marrow niche. 2) Functional characterization of PAX5 genomic lesions. PAX5 is a transcription factor essential for B-cell development. Recently, it has been found as frequent target of aberrancies in childhood ALL (30% of B-cell ALL cases), showing monoallelic loss, point mutations or chromosomal translocations. The role of these aberrancies is still poorly understood. We reported the first evidence of the PAX5/TEL fusion gene in an ALL patient with the t(9;12) translocation, and we further investigated the molecular and functional roles of PAX5/TEL protein in vitro, in murine wild type preBI cells. In vitro, PAX5/TEL protein has a dominant negative effect on target genes, it interferes with the process of B-cell differentiation and migration; and it induces resistance to apoptosis, key events in the process of B-cell transformation. We aim to further investigate the effect of the PAX5-TEL chimeric protein on the differentiation program of preBI cells, to evaluate the migration/adhesion properties of the PAX5 aberrant cells, and to study the in vivo role of the PAX5-TEL fusion in mice models. We are also characterizing additional PAX5 lesions in childhood ALL patients. 3) Identification and characterization of the “leukemia-initiating stem cell” in Infant ALL with t(4;11). The reciprocal t(4;11), giving rise to the expression of MLL/AF4 fusion protein, is the most frequent aberration involving the MLL gene, and it is mainly associated to infant ALL (less than 12 months old at diagnosis). These patients have an overall a very poor prognosis, and their cells have a peculiar gene signature and phenotype that confer distinct properties to the disease (different form other form of common leukemia), which may be driven and sustained by a unique clone of stem cells. Aim of this task is the identification of the subset of cells responsible for initiating, expanding and maintaining the tumor, the “cancer stem cell” of Infant ALL/t(4;11). Identifying the nature of this cell not only may be key to unravelling the origin and the dismal prognosis of the disease, but also it is crucial to develop new therapies able to target these cells in patients. We started a combined multiparameter immunophenotype and FISH analyses on different subpopulations of PB/BM-derived cells from infant ALL/t(4;11) patients at diagnosis. We will set up an in vitro assay to confirm the clonogenic potential and self-replating ability of these cell. The NOD/SCID repopulating assay, by injecting purified sub-populations, will assess the ability of the candidate leukemia stem cell to transfer the leukemia in vivo.

References

Wiemels, J. L. et al. Prenatal origin of acute lymphoblastic leukaemia in children. Lancet 354, 1499-1503 (1999).

Greaves, M. F. et al. Leukemia in twins: lessons in natural history. Blood 102, 2321-2333 (2003).

Tsuzuki, S., et al. Modelling first-hit functions of the t(12;21) TEL-AML1 translocation in mice. Proc. Natl. Acad. Sci., U S A 101, 8443-8448 (2004).

Morrow, M., et al. TEL-AML1 promotes development of specific hematopoietic lineages consistent with preleukemic activity. Blood 103, 3890-3896 (2004).

Mishra, L., et al. Transforming growth factor-b signaling in stem cells and cancer. Science 310, 68-71 (2005).

Cobaleda C SA, et al. Pax5: the guardian of B cell identity and function. Nature Immunology. 2007;8:463-470.

Mullighan CG, et al. Genome-wide analysis of genetic alterations in acute lymphoblastic leukemia. Nature. 2007;446:758-764.

Cazzaniga G, et al. The Paired Box Domain Gene PAX5 Is Fused to ETV6/TEL in an Acute Lymphoblastic Leukemia Case. Cancer Research. 2001;61:4666-4670.

Fazio C, et al. PAX5/TEL acts as a transcriptional repressor causing down modulation of CD19, enhances migration to CXCL12 and confers survival advantage in preBI cells. Cancer Research, in press.

Strehl S, et al. PAX5/ETV6 fusion defines cytogenetic entity dic(9;12)(p13;p13). Leukemia. 2003;17:1121–1123.

Nutt SL, et al. Commitment to the B-lymphoid lineage depends in the transcription factor Pax5. Nature. 1999;401:556-562.

Biondi A, et al. Biological and therapeutic aspects of infant leukaemia. Blood. 2000 Jul;96(1):24-33.

Armstrong SA, et al. MLL translocations specify a distinct gene expression profile that distinguishes a unique leukemia. Nat Genet. 2002 Jan;30(1):41-7.

Hotfilder M, et al. Leukemic stem cells in childhood high-risk ALL/t(9;22) and t(4;11) are present in primitive lymphoid-restricted CD34+CD19- cells. Cancer Res. 2005 Feb 15;65(4):1442-1449.

Castor A, et al. Distinct patterns of hematopoietic stem cell involvement in acute lymphoblastic leukemia. Nat Med. 2005 Jun;11(6):630-7.

Somervaille TC, Cleary ML. Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell. 2006 Oct;10(4):257-68.
 

Therapeutic potential of adult pluripotent stem cells

1) Multipotent Adult Progenitor Cells (MAPC)
In the last seven years the group directed by C.Verfaillie published a series of papers in the most prestigious scientific journals reporting the discovery of a rare population of stem cells, named multipotent adult progenitor cells or MAPC, isolated from human as well as mouse bone marrow. MAPC have characteristics unlike most adult stem cells in that they grow without aging (senescing) and can differentiate not only to cells present in bone marrow, like blood and bone, but also muscle, blood vessels, liver and brain-like cells in vitro. Moreover, when MAPC cells are injected in the blastocyst (cell created from a mouse fertilized egg that when implanted in the uterus can give rise to a whole mouse), MAPC participate in the formation of most if not all organs from the mouse born from such a mixed blastocyst, Therefore, MAPC resemble embryonic stem cells, even though they are generated from bone marrow from a born human or mouse. In contrast to embryonic stem cells, MAPC can be selected from bone marrow from the patient him or herself and used in local or systemic therapies, without rejection. Because of these characteristics, MAPC hold great promise for the treatment of degenerative or inherited diseases such as heart disease, Parkinson’s disease, diabetes, heart disease, hemophilia, and metabolic disorders, similarly to embryonic stem cells.

2) Derivation and culture of MAPC from pre-term cord blood
To date, the most common source of MAPC has been the bone marrow, but aspirating bone marrow from the iliac crest of a donor is an invasive procedure. Therefore, the search for alternative sources of MAPC is of significant value. It is well accepted that umbilical cord blood is a good source for hematopoietic (blood) stem cells, and transplantation of cord blood has been part of clinical practice for more than 10 years. However, it is not clear whether MAPC can be isolated from cord blood. In preliminary studies we have shown that a population of cells morphologically similar to bone marrow derived MAPC can be found in mixed culture of umbilical cord blood derived cells, suggesting that isolation of such potent MAPC may be possible. If this can be solidified, we believe that cord blood would be an excellent source of cells to generate, for several reasons. Cord blood is already stored in many cord blood banks and immunologically matched cords may be readily identified for many patients. There is very good evidence that blood stem cells in cord blood are more potent and age less fast than blood stem cells from bone marrow. Hence we believe that a similar phenomenon may be true for cord blood MAPC. Finally, cord blood MAPC would potentially be safer as the infant has not been exposed to the many toxins that adult donors may have been exposed to, and the stem cells may therefore less likely be mutated or altered by environmental factors.

3) Stem cell-gene therapy for Hurler Syndrome
The main interest of our group is focussed on the development of new stem cells therapy approaches to treat mucopolysaccharidosis type I (Hurler syndrome). Affecting one in 100,000 people, Hurler syndrome is a rare genetic metabolic disorder where the enzyme (alpha-L-iduronidase), which normally breaks down the mucopolysaccharides dermatan and heparan sulphate, is missing. These mucopolysaccharides build up in all tissues in the body causing progressive deterioration, abnormal function of multiple organs, and in severe cases, early death. Bone marrow transplantation (BMT) is the only treatment available that stabilizes the progression of this disease. BMT provides healthy cells that can produce the missing enzyme or protein leading to the normalization of cell processes. Unfortunately, the high incidence of rejection, with an estimated frequency of 1 in 4 patients, limits the success of this treatment. Hence, the need persists to evaluate novel sources of stem cells. The major goal of our group is to develop alternative stem cells-gene therapies to treat this disease. We aim to determine if MAPCs may facilitate hematopoietic repopulation and tissue repair in a MPS-I mouse model. Subsequently we want to explore if MAPCs isolated from MPS-I mice can be genetically modified to express IDUA gene and, once transplanted into MPS-I mice, the cells can offer a significant beneficial effect on the phenotypic abnormalities of MPS-I. These studies may be translated to clinical stem cell-gene therapy of patients with Hurler disease and will contribute to improve current cell therapy approaches and advance in patients’ care.

References

Verfaillie C.M. and Serafini M. “Multipotent progenitor cell populations obtained from bone marrow”, chapter in Cell Therapy, edited by D.Garcia-Olmo, J.M.Garcia-Verdugo, J.Alemany, M.A.Gonzales, J.A.Gutierrez-Fuentes, in press.

Serafini M. and Verfaillie C.M. Multipotent progenitor cells: a new era in stem cells-mediated gene therapy? Progress in gene therapy, in press.

Serafini M., Dylla S.J., Oki M., Heremans Y., Jiang Y., Tolar J., Jiang Y., Buckley S.M., Pelacho B., Burns T.C., Frommer S., Rossi D.J., Bryder D., Panoskaltsis-Mortari A., O’Shaughnessy M.J., Nelson-Holte M., Fine G.C., Weissman I.L., Blazar B.R. and Verfaillie C.M. Hematopoietic reconstitution by multipotent adult progenitor cells: precursors to long-term hematopoietic stem cells. Journal of Experimental Medicine, 2007 Jan 22; 204(1):129-39. Comment on Science, 2007 Feb, 315: 760-761.

Serafini M. and Verfaillie C.M. Adult stem cells pluripotency: state of the art. Seminars in Reproductive Medicine, 2006 Nov; 24(5): 379-388.

Serafini M. and Biondi A. Adult Multipotent Stem Cells: fact or fiction? Journal of medicine and the person, 2005 March ; 3(1).

Serafini M., Naldini L., Introna M. Molecular evidence of integration in a small fraction of proliferating human B lymphocytes transduced by VSV-pseudotyped HIV-1 derived lentivector. Virology, 2004 Aug 1; 325(2): 413-24.

Serafini M., Bonamino M., Bernasconi S., Golay J., Introna M. EF1- promoter in a lentiviral backbone improves the CD20 suicide gene expression in primary T lymphocytes allowing efficient Rituximab-mediated lysis. Haematologica, 2004 Jan; 89(1): 86-95.

Serafini M., Manganini M., Borleri G., Bonamino M., Imberti L., A. Biondi, J. Golay, A. Rambaldi, M. Introna. Characterization of CD20 transduced T lymphocytes as an alternative suicide gene therapy approach for the treatment of the Graft Versus Host Disease. Human Gene Therapy, 2004 Jan; 15: 63-76.

Bonamino M., Serafini M., D’Amico G., Gaipa G., Todisco E., Bernasconi S., Golay J., Biondi A., Introna M. Functional transfer of CD40L gene in human B-cell precursor ALL blasts by second generation SIN lentivectors. Gene Therapy, 2004 Jan; 11(1): 85-93.

Erba E., Serafini M., Gaipa G., Tognon G., Marchini S., Celli N., Rotilio D., Broggini M., Jimeno J., Faircloth G., Biondi A., D’Incalci M. Effect of aplidine in acute lymphoblastic leukaemia cells. British Journal of Cancer, 2003 Aug; 89(4): 763-73.

Manganini M., Serafini M., Bambacioni F., Casati C., Erba E., Follenzi A., Naldini L., Bernasconi S., Gaipa G., Rambaldi A., Biondi A., Golay J., Introna M. A Hiv-1 pol gene derived sequence increases the efficiency of transduction of human non dividing monocytes and T lymphocytes by lentiviral vectors. Human Gene Therapy, 2002 Oct; 13(15): 1793-1807.

Bambacioni F., Casati C., Serafini M., Manganini M., Golay J., Introna M. Lentiviral vectors show dramatically increased efficiency of transduction of human leukemic cell lines. Haematologica, 2001 Oct; 86(10): 1095-6.

• Curriculum Vitae
• Selected Publications