Human lymphohaematopoiesis and its use for setting up innovative clinical protocols

1. Marina Cavazzana-Calvo The treatment of life-threatening immunodeficiencies relies on allogeneic haematopoietic stem cell transplantation (HSCT). The generation of new T-lymphocytes deriving from transplanted HSC requires several months, during which a profound immunodeficiency is present and accounts for 40% of the mortality observed in patients receiving an allograft from an HLA-incompatible donor. In this context, our group has focused on understanding the early steps of lympho/myeloipoiesis in normal and pathological settings, on the development of new cell and gene therapies and on the discovery of new sources of haematopoietic stem cells. a) Development of gene therapy Following extensive preclinical work, a clinical trial was performed between 1999 and 2002 in 10 patients suffering from SCID (Severe Combined Immune Deficiency)-X1 disease caused by c gene mutation. It was based on the ex vivo transfer of the c gene into CD34(+) bone marrow cells by using a first-generation, LTR-competent, non-replicative Moloney retroviral vector. This led to sustained correction of the T cell immunodeficiency and clinical benefit in 9 patients with a follow-up of 6 to 9 years. The trial was suspended following occurrence of a leukaemic disease in 4 subjects (reversible in 3) as a consequence of LTR-driven, aberrant expression of an oncogene close to the location at which the retrovirus had integrated into the genome. A new SCID-X1 clinical trial (based on the use of a self-inactivated retroviral vector) will be implemented. Predictive toxicity assays for oncogenesis are being generated by using p19 arf-deficient, cancer-prone mice crossed with the relevant immunodeficient models (c-,Rag-1- Artemis-deficient) for gene therapy with the vectors to be tested before their clinical application. Long-term assessment of both clinical and experimental models of SCID gene therapy provides an interesting setting for analyzing the dynamics of T cell populations, based on a combined high-throughput analysis of the retroviral integration sites (clonal signature) with the TCR repertoire and T cell subset phenotype. b) Characterization of human lymphoid progenitors and their usage in HSCT Our laboratory has characterized a population of human lymphocyte progenitors that can differentiate into T, B and NK cells and that is detectable in the cord blood, bone marrow, adult blood and thymus. One goal is now to understand the mechanisms used to migrate from the bone marrow to the thymus by further characterization based of chemokine and cytokine receptor gene expression patterns. Preliminary data indicate that lymphocyte progenitors can be both extensively amplified (x 80) and specified toward the T cell lineage in vitro on stromal cell lines expressing the delta 1 Notch ligand. Our aim is to validate this observation, optimize expansion conditions both in vitro and in vivo in xenogeneic murine models and study the population's migration capacity and ability to differentiate into mature functional T cells. This would constitute a prerequisite for potential clinical application, by speeding up T cell reconstitution in allogeneic HSCT or gene therapy. c) Description of a newly characterized SCID: reticular dysgenesis. We have recently identified the gene mutation responsible for a very rare form of SCID: reticular dysgenesis (RD). Despite its rareness, this SCID form is particularly interesting because it is the only form of SCID with profound lymphopenia (i.e. T- NK-) associated with (i) an early maturation arrest in the myeloid lineage at the myeloblast-promyelocyte stage and (ii) bilateral sensorineural deafness. Our results suggest a novel mechanism regulating energy metabolism and/or controlling apoptosis and which is involved in one of the most severe human immunodeficiency syndromes. Our goal focuses on understanding the mechanism of action of AK2 in haemato/lymphopoiesis based on the silencing of AK2 expression by SiRNA and the generation of conditional KO mouse. d) Characterization of stem cells present in the amniotic fluid We have recently described the presence of haematopoietic cells in murine and human amniotic fluid. These cells express genes typically associated with a haematopoietic potential and are able to generate in vitro all the blood lineages. The great question raised by this observation concerns the intra or extra-embryonic origin of these cells and experiments are ongoing to try to solve it. Salima Hacein-Bey-Abina Chantal Lagresle-Peyrou Isabelle André-SchmutzEmmanuelle Six Delphine Bonhomme Corinne de Chappedelaine Julie RivièreJulien Rouiller 5 best publications : Hacein-Bey-Abina S, Garrigue A, Wang GP, Soulier J, Lim A, Morillon E, Clappier E, Caccavelli L, Delabesse E, Beldjord K, Asnafi V, MacIntyre E, dal Cortivo L, Radford I, Brousse N, Sigaux F, Moshous D, Hauer J, Borkhardt A, Belohradsky BH, Wintergerst U, Velez MC, Leiva L, Sorensen R, Wulffraat N, Blanche S, Bushman FD, Fischer A, Cavazzana-Calvo M. (2008) Insertional Oncogenesis in Four Patients after Retrovirus-Mediated Gene Therapy of SCID-X1. J Clin Invest 118:3132-42. Cavazzana-Calvo M, Fischer A. (2007) Gene therapy for severe combined immunodeficiency: are we there yet ? J Clin Invest 117:1456-65 Six EM, Bonhomme D, Monteiro M, Beldjord K, Jurkowska M, Cordier-Garcia C, Garrigue A, dal Cortivo L, Rocha B, Fischer A, Cavazzana-Calvo M, André -Schmutz I. (2007) A human postnatal lymphoid progenitor capable of circulating and seeding the thymus. J Exp Med 204:3085-93 Lagresle-Peyrou C, Six EM, Picard C, Rieux-Laucat F, Michel V, DITAID A, Demerens-de Chappedelaine C, Morillon E, Valensi F, Simon-Stoos KL, Mullikin JC, Noroski LM, Besse C, Wulffraat NM, Ferster A, Abecasis MM, Calvo F, Petit C, Candotti F, Abel L, Fischer A, Cavazzana-Calvo M. (2009) Human adenylate kinase 2 deficiency causes a profound haematopoietic defect. Nat Genet 41:106-11 Ditadi A, De Coppi P, Picone O, Gautreau L, Smati R, Six E, Bonhomme D, Ezine S, Frydman R, Cavazzana-Calvo M, André-Schmutz I. (2009) Human and murine amniotic fluid c-Kit+Lin- cells display hematopoietic activity. Blood in press Human lymphohaematopoiesis and its use for setting up innovative clinical protocols