Receptor-targeting and intracellular disassembly of gene vectors

基因载体受到效率低和靶细胞特异性不足的限制。基因转移过程中的细胞瓶颈仍有待确定，以加快载体优化。在目前的项目中，最近建立的生化靶向策略将用于分析造血细胞中的基因转移过程。靶向将结合通过生物可逆性PEG化的联合收割机去靶向（屏蔽）和（再）靶向特异性细胞表面受体。该项目将重点关注造血细胞受体的已建立配体（转铁蛋白，抗CD 3或抗CD 117抗体）和来自研究网络合作的配体（如HSC特异性scFv）。我们将评估基于新型生物可降解聚合物的靶向聚合物复合物，并向我们的网络合作伙伴提供用于病毒载体修饰的去靶向/重靶向试剂。通过生物光学研究（流式细胞术，共聚焦显微镜），不同细胞途径的抑制剂的使用，以及细胞周期依赖性的分析，基因载体的进入途径和细胞内命运将与基因表达相关，以表征生产性进入途径。附加型载体将用于监测基因表达的维持。

Gene vectors are limited by low efficiency and insufficient target cell specificity. Cellular bottlenecks in the gene transfer process remain to be identified to speed up vector optimization. In the current project, a recently established biochemical targeting strategy will be applied for analysing the gene transfer process in hematopoietic cells. Targeting will combine detargeting (shielding) by bioreversible PEGylation and (re)targeting to specific cell surface receptors. The project will focus on established ligands of hematopoietic cell receptors (transferrin, anti-CD3 or anti-CD117 antibodies) and ligands derived from research network collaborations (such as HSC-specific scFvs). We will evaluate targeted polyplexes based on novel biodegradable polymers, and also provide detargeting/retargeting reagents for viral vector modification to our network partners. Entry pathways and intracellular fate of gene vectors by biooptical studies (flow cytometry, confocal microscopy), use of inhibitors for different cellular pathway, and analysis of cell cycle dependence will be correlated with gene expression to characterize the productive entry pathways. Episomal vectors will be applied for monitoring maintenance of gene expression.