Gene Delivery into Human Hematopoietic Cells

描述(由申请人提供)：将潜在的治疗性基因最佳地输送和表达到人类细胞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性和获得性疾病。我们之前曾描述过含有治疗性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，将基因输送到人类造血干细胞(HSC)及其后代。最近，慢病毒载体被证明比肿瘤逆转录病毒更有效地将某些治疗性基因，如人b珠蛋白基因传递给小鼠和人的HSC。这笔赠款的具体目标是：(1)开发新的肿瘤逆转录病毒载体，可用于在人类HSC中更有效地转移和表达人类基因，而不是通过利用较新的逆转录病毒包膜，如RD114，这是一种允许有效转导人类HSC的包膜；以及(2)开发比迄今所描述的更适合人类使用的慢病毒包装系统。最近，我们已经构建并鉴定了一株稳定的RD114假型肿瘤逆转录病毒包装株。我们已经证明，从该稳定株衍生的逆转录病毒RD114上清液可以浓缩到高滴度，并能够高水平转导人CD34+细胞。目前，在慢病毒基因转移中，由于VSV-G包膜的毒性，需要在培养的人类细胞中有限次地加入几个质粒，这是一种“瞬时”系统。我们打算开发可与我们最好的肿瘤逆转录病毒系统相媲美的“稳定的”慢病毒载体系统，在该系统中，所需的基因稳定地整合到所使用的细胞系的基因组中；来自这些稳定株的上清液更容易进行安全性测试，并且可以比瞬时上清液更高效、更可重复性地产生逆转录病毒上清液，用于人体临床试验。我们将在肿瘤逆转录病毒和慢病毒系统中使用的一个潜在的治疗结构是针对人类镰状β-珠蛋白基因的小干扰RNA(SiRNA)，以减少β-S珠蛋白的产生。此外，我们将在这些系统中传递正常的人类β-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，在人类HSC中传递和表达治疗性基因，并治愈或改善诸如镰状细胞病和β-地中海贫血等血液病。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.