Turan Soeren, Qiao Junhua, Madden Sally, Benham Craig, Kotz Marina, Schambach Axel, Bode Juergen
Institute of Experimental Hematology, Hannover Medical School, D-30625 Hannover, Germany; Department of Genetics, Stanford University School of Medicine, Stanford, CA, USA.
WuXi AppTec Co. Ltd., 288 Fute Zhong Road, Shanghai 200131, China.
Gene. 2014 Aug 10;546(2):135-44. doi: 10.1016/j.gene.2014.06.002. Epub 2014 Jun 4.
Where possible, developments enabling the establishment of cell lines with predictable, long-term stable expression capacity are based on single-copy integrations at safe genomic loci with predictable properties. Robust performance could be assigned to lentiviral transduction systems anchoring single LV-units at sites with adequate transcription potential. In the case of gene therapeutic vectors it is essential that the expression interval can be safely terminated following individual requirements, which has mostly been achieved by lox-mediated excision ("floxing"). To extend the spectrum of possible applications we replaced the common, phage-derived Cre/loxP-setup by modules derived from the yeast "Flp/FRT" site-specific recombination system. This change enables a variety of additional options, for instance by "multiplexing" strategies, which rely on a variety of heterospecific FRT-site variants (F'). If we provide lentiviral LTRs with a "twin-site", here an FF3 fusion, the presence of Flp-recombinase will effectively excise the expression cassette, leaving behind a single neutral, genomically anchored FF3 unit. This tag serves to identify the integration locus and to apply sequence- and structural (SIDD-) analyses to predict its functions. Candidate loci are then used to accommodate, at the given site, other genes of interest by "Recombinase-Mediated Twin Site Targeting" (RMTT), a contemporary extension of existing cassette exchange (RMCE-) routines. Supported by the fact that FF3 twins remain accessible within the host genome, RMTT provides access to certified cell lines as it complies with recently defined stringent genomic safe harbor criteria. Our discussion- and outlook-sections will cover lentiviral targeting strategies and current possibilities to enable their fine-tuning.
在可能的情况下，能够建立具有可预测的长期稳定表达能力的细胞系的技术进展，是基于在具有可预测特性的安全基因组位点进行单拷贝整合。强大的性能可归因于慢病毒转导系统，该系统将单个慢病毒载体单元锚定在具有足够转录潜力的位点。对于基因治疗载体而言，至关重要的是表达间隔能够根据个体需求安全终止，这主要通过lox介导的切除（“floxing”）来实现。为了扩展可能的应用范围，我们用源自酵母“Flp/FRT”位点特异性重组系统的模块取代了常见的噬菌体衍生的Cre/loxP系统。这种改变带来了多种额外的选择，例如通过“多重化”策略，该策略依赖于多种异源特异性FRT位点变体（F'）。如果我们为慢病毒长末端重复序列（LTR）提供一个“双位点”，这里是FF3融合，Flp重组酶的存在将有效地切除表达盒，留下单个中性的、基因组锚定的FF3单元。这个标签用于识别整合位点，并应用序列和结构（SIDD）分析来预测其功能。然后，通过“重组酶介导的双位点靶向”（RMTT），即现有盒式交换（RMCE）程序的当代扩展，利用候选位点在给定位置容纳其他感兴趣的基因。由于FF3双位点在宿主基因组中仍然可及，RMTT符合最近定义的严格基因组安全港标准，从而能够获得经过认证的细胞系。我们的讨论和展望部分将涵盖慢病毒靶向策略以及当前实现其微调的可能性。
