Congenital Amegakaryocytic Thrombocytopenia (CAMT) is an inherited stem cell disorder caused by loss of function mutations in MPL,the receptor for thrombopoietin (TPO). Affected individuals are born thrombocytopenic and over months to years develop lethal pancytopenia unless they are successfully treated by allogeneic stem cell transplantation. Unfortunately, a significant number of CAMT patients do not have a suitable HLA matched donor. Hematopoietic stem cell (HSC) gene therapy offers the promise of treating all patients with CAMT by genetically modifying autologous stem cells through retroviral mediated transfer of a normal copy of the MPL gene. In this proposal we outline pre-clinical work for developing Prototype Foamy Virus (PFV) vectors and HSC gene transfer protocols to treat CAMT. Vector expression of MPL in CAMT patients needs to produce normal expression patterns to ensure stem cell survival and megakaryocyte development, without inducing dyshematopoiesis.
Aim 1 proposes the development of vectors with tissue specific promoters. We will generate and test vectors with promoters that limit gene expression to primitive hematopoietic cells and those committed to the megakaryocyte lineage.
In Aim 2 we will evaluate if Mpl gene addition can reverse the hematopoietic defects in a mouse model of CAMT. Using standard transgenic techniques an Mpl transgene under direction of the Mpl gene promoter has been added to the germ line cells of CAMT mice and we now have a stable breeding colony of gene corrected mice. In addition we will use PFV vectors, with the tissue specific promoters developed in Aim I, to transfer the Mpl gene into the HSCs of CAMT mice. Gene corrected mice will be evaluated for peripheral blood counts, megakaryocyte lineage development, and the restoration of the HSC compartment. We will check for vector safety by looking for morphological evidence of dyshematopoiesis and molecular evidence of clonal hematopoiesis in PFV vector corrected mice kept alive for at least 1 year.
Aim 3 of the grant proposes using PFV vectors with the human MPL transgene and the promoters evaluated in Aims 1 and 2 to transduce human CD34+ cells from patients with CAMT. Transduced cells will be evaluated for both megakaryocyte development and clonogenic progenitor growth. We project that, as a result of the studies completed in this research plan, a clinical trial for the treatment of CAMT with PFV vectors will be proposed at the completion of this grant.
