This supplement proposal to our Gene Therapy Program Grant has been prompted by the unanticipated success, ahead of schedule, of the correction of sickle transgenic mice and severe transgenic B- thalassemic mice with our lentivirus vector. The present proposal aims at accelerating, as much as it is possible and safe, the advent of clinical trials. In the absence of unanticipated set backs, we estimate that within 2 years we should be in a position of contemplating clinical trials. Hence, this proposal includes expansion and retargeting of previous Aims but also completely new experimental Aims. The specific reasons for a supplemental proposal are the following: 1) The effort, initially centered on sickle cell anemia, has been significantly expanded recently, to the gene therapy of B-thalassemia, with aspects not included in the original proposal; 2) We have significantly expanded our strategy of anti-sickling in order to improve even further the antisickling properties of our vector, including improvement of the thalassemia-targeted vector by adding an increased capacity of delivering oxygen. Recently data from our laboratory suggest the need to pursue a novel anti-sickling globin construct, involving simultaneously vertical, lateral and inter-double strand contact site mutations. These new objectives have as background our grossly underestimation of the animal facility cost of our large transgenic mice colony. 3) As a completely new aspect of the project is the use of a Primate model. Since they are significant differences in the behavior of murine and human hematopoietic stem cells, it is, therefore, imperative to test gene therapy strategies in an animal model system which closely resembles man before considering clinical trials. The objectives include the assessment of: a) Potential toxicity of genetically modify marrow repopulating cells, b)The efficacy and duration of transgene expression, c)The various sources and doses of hematopoietic stem cells required for clinically achievable levels of genetic modification will be evaluated, d) The conditioning regimen required for engraftment of the genetically modified stem cell graft. 4) Another area of novel expansion is the extension to testing of B-globin therapeutic vectors and gene transfer strategies to human thalassemia-patient hematopoietic cells assessed both in vitro and in in vivo immunodeficient mouse models. Also, we plan to extend to the thalassemia model studies to developing effective procedures for transplantation under non-myeloablative conditions including assessment of selective expansion of genetically modified stem cells. In the expansion to primate models, the Vancouver site will optimize and carry out clinical scale transductions of non-human primate hematopoietic stem cells. Additionally, we will exploit the recently developed nude-NOD/SCID mouse model to enable long term (over 1 year follow-up) comparative studies of the recovery and repopulating function of transduced non-human primate hematopoietic stem cells.
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