Optimal delivery and expression of potentially therapeutic genes into human cells is an evolving technology whose ultimate goal is to cure or ameliorate human inherited and acquired diseases. We have previously described oncoretroviral products containing therapeutic genes that can be produced in large amounts, contain no replication-competent retrovirus and that can be used successfully in human clinical trials to deliver genes to human hematopoietic stem cells (HSC) and their progeny. More recently, lentiviral vectors have been shown to be more efficient than oncoretroviruses in delivering certain therapeutic genes such as the human b globin gene to murine and human HSC. The specific goals of this grant are to: (1) develop novel oncoretroviral vectors that can be used to transfer and express human genes more efficiently in human HSC than those currently available by utilizing newer retroviral envelopes, such as RD114, an envelope that allows efficient transduction of human HSC; and (2) develop lentiviral packaging systems that are more amenable for human use than those described to date. Most recently, we have constructed and characterized a stable RD114 pseudotyped oncoretroviral packaging line. We have shown that retroviral RD114 supernatants derived from this stable line can be concentrated to high titer and are capable of high-level transduction of human CD34+ cells. Currently, in lentiviral gene transfer, """"""""transient"""""""" systems are used, involving adding several plasmids to human cells in culture for limited times because of the toxicity of the VSV-G envelope. We intend to develop """"""""stable"""""""" lentiviral vector systems comparable to our best oncoretroviral systems in which the required genes are stably integrated into the genome of the cell lines utilized; supernatants from these stable lines are more easily tested for safety and can generate retroviral supernatants more efficiently and reproducibly in larger amounts than transient supernatants for use in human clinical trials. One potentially therapeutic construct we will use in both oncoretroviral and lentiviral systems is a small interfering RNA (siRNA) directed against the human sickle beta-globin gene mRNA to decrease beta-s globin protein production. In addition, we will deliver a normal human beta-globin gene in these systems. The ultimate goal of these experiments is to provide safe and novel systems to deliver and express therapeutic genes in human HSC and to cure or ameliorate hematologic diseases such as sickle cell disease and beta-thalassemia. The gene delivery technology should also be applicable for use in treating other human diseases as well.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-GDD (01))
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Evans, Gregory
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Columbia University (N.Y.)
Schools of Medicine
New York
United States
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Bell Jr, Anthony J; Fegen, David; Ward, Maureen et al. (2010) RD114 envelope proteins provide an effective and versatile approach to pseudotype lentiviral vectors. Exp Biol Med (Maywood) 235:1269-76