Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, MS, USA.
Exp Biol Med (Maywood). 2010 Oct;235(10):1269-76. doi: 10.1258/ebm.2010.010053. Epub 2010 Sep 27.
Lentiviral vectors derived from the HIV-1 genome offer great promise for gene therapy due to their ability to transduce non-dividing cells and sustain long-term expression of transgenes. The majority of current lentiviral vectors are pseudotyped with the vesicular stomatitis viral envelope (VSV-G). VSV-G equips lentiviral vectors with a broad host cell tropism and increased stability. Increased particle stability enables viral supernatants to be concentrated by high-speed centrifugation to enhance their infectivity. Despite its efficacy, VSV-G is cytotoxic - a feature that prohibits the development of stable cell lines that constitutively express this envelope. Therefore, non-toxic envelope proteins are being investigated. RD114 is an attractive alternative because it also provides increased particle stability and its receptor is widely expressed on hematopoietic stem cells (HSCs). In this study, the packaging efficiency of three envelope proteins, RD114, RDpro and VSV-G, were evaluated with two lentiviral vectors (TRIP GFP and HPV-402). RDpro is an RD114-HIV chimera designed to pseudotype lentiviral vectors more efficiently. In transient systems, VSV-G generated titers of 10(8) and 10(7) viral particles/mL for TRIP GFP and HPV-402. RDpro possessed titers of 10(7) and 10(6), while RD114 titers were one log lower for each vector. Despite having relatively lower titers, RD114 proteins are less toxic; this was demonstrated in the extension of transient transfection reactions from 48 to 96 h. VSV-G transfections are generally limited to 48 h. In regard to gene therapy applications, we show that RDpro supernatants efficiently transduce peripheral blood HSCs. The versatility of RD114 envelopes was again demonstrated by using a 'mixed' expression system; composed of stably expressed RD114 envelope proteins to pseudotype lentiviral vectors generated in trans (titer range 10(3)-10(5)). Our data show that RD114 envelope proteins are effective and versatile constructs that could prove to be essential components of therapeutic lentiviral gene transfer systems.
慢病毒载体来源于 HIV-1 基因组，由于其能够转导非分裂细胞并维持转基因的长期表达，因此在基因治疗方面具有很大的应用前景。目前大多数慢病毒载体都被水疱性口炎病毒包膜（VSV-G）假型化。VSV-G 赋予慢病毒载体广泛的宿主细胞嗜性和增强的稳定性。增加的颗粒稳定性使病毒上清液能够通过高速离心浓缩，从而提高其感染力。尽管 VSV-G 具有功效，但它具有细胞毒性 - 这一特征阻止了稳定表达这种包膜的细胞系的发展。因此，正在研究非毒性包膜蛋白。RD114 是一种有吸引力的替代物，因为它还提供了更高的颗粒稳定性，并且其受体广泛表达于造血干细胞（HSCs）上。在这项研究中，使用两种慢病毒载体（TRIP GFP 和 HPV-402）评估了三种包膜蛋白 RD114、RDpro 和 VSV-G 的包装效率。RDpro 是一种 RD114-HIV 嵌合体，旨在更有效地假型化慢病毒载体。在瞬时系统中，VSV-G 为 TRIP GFP 和 HPV-402 产生了 10(8)和 10(7)病毒颗粒/mL 的滴度。RDpro 具有 10(7)和 10(6)的滴度，而 RD114 的滴度则低一个对数。尽管 RD114 蛋白的滴度相对较低，但它们的毒性较小；这在将瞬时转染反应从 48 小时延长至 96 小时时得到了证明。VSV-G 转染通常限于 48 小时。关于基因治疗应用，我们表明 RDpro 上清液有效地转导外周血 HSCs。RD114 包膜的多功能性再次通过使用“混合”表达系统得到证明；由稳定表达的 RD114 包膜蛋白组成，用于转染（滴度范围 10(3)-10(5))生成的慢病毒载体假型化。我们的数据表明，RD114 包膜蛋白是有效的多功能构建体，它们可能成为治疗性慢病毒基因转移系统的重要组成部分。
