蛋白质转接:优化因特因介导的GFP组件作为基因疗法开发的模型
Andrew Brovin1, Ekaterina Minskaia1, Matvei Sabantsev1
1Gene Therapy Department, Research Center for Translational Medicine, Sirius University of Science and Technology, Sirius, Russia.
Frontiers in bioengineering and biotechnology
|December 5, 2024
概括
由腺相关病毒 (AAV) 输送的基于蛋白质的蛋白质转接,显示出对大型基因疾病的基因治疗有前途. 这种NpuDnaE蛋白质变体在细胞系中组装蛋白质方面取得了高效率.
科学领域:
- 分子生物学分子生物学
- 基因治疗 基因治疗
- 生物技术是生物技术.
背景情况:
- 基于腺相关病毒 (AAV) 的基因疗法对于通过基因替代治疗遗传性疾病至关重要.
- 由于AAV (5千基) 的包装能力有限,这对治疗由大型基因 (大约5千基基因) 引起的遗传疾病构成了重大挑战. 5000个综合征). 这样一来,我们就有了5000个综合征.
- 目前的策略包括切断基因版本,载体过载和碎片化遗传物质传递.
研究的目的:
- 通过AAV提供的基于蛋白质的蛋白质转接技术的有效性,用于组装大型蛋白质.
- 在基因疗法应用中确定最佳的蛋白质变体和条件,以实现高效的蛋白质组装.
- 解决AAV包装容量的局限性,用于治疗大型基因相关遗传性疾病.
主要方法:
- 使用来自 DnaE 组 (Ssp,Npu,Ava) 的分裂整体来进行蛋白质转接.
- 采用绿色光蛋白 (GFP) 作为评估蛋白质组装效率的模型系统.
- 在HEK293和ARPE19细胞系中进行了实验,包括通过流细胞计分析的等离子体DNA转染和AAV5转导.
主要成果:
- 成功的蛋白质组装需要特定的氨基酸残留物围绕着蛋白质的分裂部位,而不仅仅是C+1.1的囊蛋白.
- 这种NpuDnaE中蛋白变体表现出N和C端片段相互作用的最高动力学.
- 在使用NpuDnaE的优化条件下,在80%的转染HEK293细胞和55%的AAV5转化细胞中实现了GFP组装,HEK293的效率高于ARPE19.
结论:
- 因特因介导蛋白质转接是克服AAV包装限制的大型基因疗法的可行策略.
- 在基因疗法环境中,NpuDnaE整蛋白显示出有效的蛋白质组合的显著潜力.
- 这些发现为开发用于与大基因突变相关的遗传性疾病的基因疗法提供了基础.
相关概念视频
Tagging and Fusion Proteins
6.6K
Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
6.6K
Gene Therapy
25.2K
Gene therapy is a technique where a gene is inserted into a person’s cells to prevent or treat a serious disease. The added gene may be a healthy version of the gene that is mutated in the patient, or it could be a different gene that inactivates or compensates for the patient’s disease-causing gene. For example, in patients with severe combined immunodeficiency (SCID) due to a mutation in the gene for the enzyme adenosine deaminase, a functioning version of the gene can be...
25.2K
Conservative Site-specific Recombination and Phase Variation
5.9K
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
The recognition sites for Cre recombinase called LoxP...
5.9K


