Gene Delivery into Human Hematopoietic Cells

描述（由申请人提供）：将潜在治疗性基因最佳递送和表达到人类细胞中是一项不断发展的技术，其最终目标是治愈或改善人类遗传和获得性疾病。我们之前已经描述了含有治疗基因的肿瘤逆转录病毒产品，这些产品可以大量生产，不含复制能力强的逆转录病毒，并且可以成功地用于人类临床试验，将基因传递给人类造血干细胞（HSC）及其后代。最近，慢病毒载体已被证明在将某些治疗性基因（如人b球蛋白基因）传递到鼠和人HSC中比逆转录病毒更有效。该基金的具体目标是：(1)开发新的逆转录病毒载体，与目前使用更新的逆转录病毒包膜（如RD114）相比，这种载体可以更有效地在人类HSC中转移和表达人类基因；(2)开发慢病毒包装系统，比迄今为止所描述的更适合人类使用。最近，我们构建并鉴定了一个稳定的RD114假型肿瘤逆转录病毒包装系。我们已经证明，来自这一稳定系的逆转录病毒RD114上清液可以浓缩到高滴度，并且能够高水平地转导人CD34+细胞。目前，在慢病毒基因转移中，使用了“瞬时”系统，由于VSV-G包膜的毒性，需要在有限的时间内将几个质粒添加到培养的人类细胞中。我们打算开发“稳定的”慢病毒载体系统，与我们最好的肿瘤逆转录病毒系统相媲美，在这种系统中，所需的基因被稳定地整合到所用细胞系的基因组中；来自这些稳定系的上清液更容易进行安全性测试，并且比用于人体临床试验的瞬时上清液更有效和可重复地产生大量逆转录病毒上清液。我们将在肿瘤逆转录病毒和慢病毒系统中使用的一种潜在的治疗结构是针对人类镰状β -珠蛋白基因mRNA的小干扰RNA (siRNA)，以减少β -s珠蛋白的产生。此外，我们将在这些系统中植入正常的人类-珠蛋白基因。这些实验的最终目标是提供安全和新颖的系统来传递和表达人类HSC中的治疗性基因，并治愈或改善血液学疾病，如镰状细胞病和-地中海贫血。基因传递技术也应该适用于治疗其他人类疾病。

DESCRIPTION (provided by applicant): 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.