中国神经再生研究(英文版) ›› 2021, Vol. 16 ›› Issue (2): 357-361.doi: 10.4103/1673-5374.290902

• 原著:周围神经损伤修复保护与再生 • 上一篇    下一篇

基因修饰白细胞浓缩液用于中度脊髓损伤小型猪模型的个性化离体基因治疗

  

  • 出版日期:2021-02-15 发布日期:2020-12-04
  • 基金资助:

    这项研究得到了俄罗斯科学基金会(第16-15-00010号);喀山联邦大学俄罗斯政府竞争性增长计划的支持

Gene-modified leucoconcentrate for personalized ex vivo gene therapy in a mini pig model of moderate spinal cord injury

Rustem R. Islamov1, *, Farid V. Bashirov1, Mikhail E. Sokolov1, Andrei A. Izmailov1, Filip O. Fadeev1, Vage A. Markosyan1, Maria A. Davleeva1, Olga V. Zubkova2, Maxim M. Smarov2, Denis Yu. Logunov2, Boris S. Naroditskyi2, Ilnur I. Salafutdinov3, Albert A. Rizvanov3,  *, Ramil G. Turaev4#br#   

  1. 1 Kazan State Medical University, Kazan, Russia;  2 Gamaleya Research Institute of Epidemiology and Microbiology, Moscow, Russia;  3 Kazan Federal University, Kazan, Russia;  4 The Republican Blood Center of the Ministry of Health of the Republic of Tatarstan, Kazan, Russia
  • Online:2021-02-15 Published:2020-12-04
  • Contact: Albert A. Rizvanov, PhD, Dr.Sci, rizvanov@gmail.com; Rustem R. Islamov, MD, Dr.Sci., rustem.islamov@gmail.com.
  • Supported by:
    This study was supported by the Russian Science Foundation (No. 16-15-00010; to RRI). AAR was supported by the Russian Government Program of Competitive Growth of Kazan Federal University. 

摘要:

作者既往研究证实过表达重组神经营养因子基因修饰的脐带血单个核细胞有用于神经退行性疾病、神经创伤和脑卒中的细胞介导基因治疗的潜力。实验首次在脊髓损伤小型猪模型中,以外周血制备的基因修饰白细胞浓缩液用于个性化离体基因治疗。实验以携带增强绿色荧光蛋白报告基因的嵌合腺病毒载体(Ad5/35F)转导从小型猪外周血中获得白细胞浓缩液。取血后第2天,建立中度脊髓损伤小型猪模型,并于术后4h进行静脉输注白细胞浓缩液。 白细胞浓缩液治疗后1周,荧光显微镜观察到挫伤脊髓中表达增强绿色荧光蛋白的白细胞。在脾中,观察到位于淋巴滤泡中的增强绿色荧光蛋白阳性细胞群。白细胞浓缩液的体外流式细胞术和荧光显微镜观察也证实了增强绿色荧光蛋白标记白细胞的存在。实验获得了一种简单、有效的方法,在塑料血袋中成功制备腺病毒载体介导的Ad5/35F基因修饰的白细胞浓缩液,其有望用于脊髓损伤的治疗。

https://orcid.org/0000-0002-9427-5739 (Albert A. Rizvanov); 

https://orcid.org/0000-0002-6632-4636 (Rustem R. Islamov)

关键词: 脊髓损伤, 嵌合Ad5 / 35F病毒, 增强绿色荧光蛋白, 基因修饰的白细胞浓缩物, 小型猪, 外周血, 个性化离体基因治疗, 塑料血袋

Abstract: We previously demonstrated that gene-modified umbilical cord blood mononuclear cells overexpressing a combination of recombinant neurotrophic factors are a promising therapeutic approach for cell-mediated gene therapy for neurodegenerative diseases, neurotrauma, and stroke. In this study, using a mini pig model of spinal cord injury, we proposed for the first time the use of gene-modified leucoconcentrate prepared from peripheral blood in the plastic blood bag for personalized ex vivo gene therapy. Leucoconcentrate obtained from mini pig peripheral blood was transduced with a chimeric adenoviral vector (Ad5/35F) that carried an enhanced green fluorescent protein (EGFP) reporter gene in the plastic blood bag. The day after blood donation, the mini pigs were subjected to moderate SCI and four hours post-surgery they were intravenously autoinfused with gene-modified leucoconcentrate. A week after gene-modified leucoconcentrate therapy, fluorescent microscopy revealed EGFP-expressing leucocytes in spinal cord at the site of contusion injury. In the spleen the groups of EGFP-positive cells located in the lymphoid follicles were observed. In vitro flow cytometry and fluorescent microscopy studies of the gene-modified leucoconcentrate samples also confirmed the production of EGFP by leucocytes. Thus, the efficacy of leucocytes transduction in the plastic blood bag and their migratory potential suggest their use for temporary production of recombinant biologically active molecules to correct certain pathological conditions. This paper presents a proof-of-concept of simple, safe and effective approach for personalized ex vivo gene therapy based on gene-modified leucoconcentrate autoinfusion. The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee (approval No. 5) on May 27, 2014.

Key words: chimeric Ad5/35F virus, enhanced green fluorescent protein, gene-modified leucoconcentrate, mini pig, peripheral blood, personalized ex vivo gene therapy, plastic blood bag, spinal cord injury