Graceffa Valeria
Cellular Health and Toxicology Research Group (CHAT), Institute of Technology Sligo, Ash Ln, Bellanode, Sligo, Ireland.
Centre for Mathematical Modelling and Intelligent Systems for Health and Environment (MISHE), Institute of Technology Sligo, Ash Ln, Bellanode, Sligo, Ireland.
Sci Rep. 2022 Mar 8;12(1):3752. doi: 10.1038/s41598-022-07750-y.
Cystinosis is a rare disease, caused by a mutation in the gene cystinosin and characterised by the accumulation of cystine crystals. Advantages of biomaterial-mediated gene delivery include reduced safety concerns and the possibility to cure organs that are difficult to treat using systemic gene transfer methods. This study developed novel fibrin hydrogels for controlled, localised gene delivery, for the treatment of cystinosis. In the first part, fabrication parameters (i.e., DNA, thrombin, and aprotinin concentrations) were optimised, using a Design of Experiment (DOE) methodology. DOE is a statistical engineering approach to process optimisation, which increases experimental efficiency, reduces the number of experiments, takes into consideration interactions between different parameters, and allows the creation of predictive models. This study demonstrated the utility of DOE to the development of gene delivery constructs. In the second part of the study, primary fibroblasts from a patient with cystinosis were seeded on the biomaterials. Seeded cells expressed the recombinant CTNS and showed a decrease in cystine content. Furthermore, conditioned media contained functional copies of the recombinant CTNS. These were taken up by monolayer cultures of non-transfected cells. This study described a methodology to develop gene delivery constructs by using a DOE approach and ultimately provided new insights into the treatment of cystinosis.
胱氨酸贮积症是一种罕见疾病，由胱氨酸转运蛋白基因的突变引起，其特征是胱氨酸晶体的积累。生物材料介导的基因递送的优点包括降低安全隐患，以及有可能治愈那些使用全身基因转移方法难以治疗的器官。本研究开发了新型纤维蛋白水凝胶用于可控的局部基因递送，以治疗胱氨酸贮积症。在第一部分中，使用实验设计（DOE）方法优化了制备参数（即DNA、凝血酶和抑肽酶浓度）。DOE是一种用于工艺优化的统计工程方法，可提高实验效率、减少实验次数、考虑不同参数之间的相互作用，并允许创建预测模型。本研究证明了DOE在基因递送构建体开发中的实用性。在研究的第二部分中，将来自一名胱氨酸贮积症患者的原代成纤维细胞接种到生物材料上。接种的细胞表达重组CTNS，并显示胱氨酸含量降低。此外，条件培养基中含有重组CTNS的功能性拷贝。这些被未转染细胞的单层培养物摄取。本研究描述了一种使用DOE方法开发基因递送构建体并最终为胱氨酸贮积症治疗提供新见解的方法。
