2. Academic Validation
  3. Enhanced delivery of CRISPR/Cas9 system based on biomimetic nanoparticles for hepatitis B virus therapy

Enhanced delivery of CRISPR/Cas9 system based on biomimetic nanoparticles for hepatitis B virus therapy

  • J Control Release. 2024 Oct:374:293-311. doi: 10.1016/j.jconrel.2024.08.019.
Kexin Wu 1,  Miao He 2,  Binli Mao 3,  Yangchen Xing 1,  Shiqi Wei 1,  Dongjun Jiang 1,  Shunyao Wang 4,  Asma A Alkuhali 5,  Jinjun Guo 6,  Zongjie Gan 1,  Man Li 7,  Xiaosong Li 8,  Huali Chen 9
Affiliations

Affiliations

  • 1 College of Pharmacy, Chongqing Medical University, Chongqing 400016, PR China.
  • 2 Laboratory Animal Center, Chongqing Medical University, Chongqing 400016, PR China.
  • 3 Western (Chongqing) Collaborative Innovation Center for Intelligent Diagnostics and Digital Medicine, Chongqing National Biomedicine Industry Base, Chongqing 401329, PR China; Department of Blood Transfusion, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, PR China.
  • 4 Clinical Molecular Medicine Testing Center, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, PR China.
  • 5 Department of Physiology and Pathophysiology, School of Basic Medical Science, Xi'an Jiaotong University, Shanxi 710049, PR China.
  • 6 Bishan Hospital of Chongqing, Bishan Hospital of Chongqing Medical University, Chongqing 400016, PR China.
  • 7 Key Laboratory of Drug-Targeting and Drug Delivery System of the Education Ministry, Sichuan University, Sichuan 610041, PR China.
  • 8 Clinical Molecular Medicine Testing Center, The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, PR China; Western (Chongqing) Collaborative Innovation Center for Intelligent Diagnostics and Digital Medicine, Chongqing National Biomedicine Industry Base, Chongqing 401329, PR China. Electronic address: lixiaosong@cqmu.edu.cn.
  • 9 College of Pharmacy, Chongqing Medical University, Chongqing 400016, PR China. Electronic address: chenhuali@cqmu.edu.cn.
PMID: 39151831 DOI: 10.1016/j.jconrel.2024.08.019
Abstract

The persistent presence of covalently closed circular DNA (cccDNA) in hepatocyte nuclei poses a significant obstacle to achieving a comprehensive cure for hepatitis B virus (HBV). Current applications of CRISPR/Cas9 for targeting and eliminating cccDNA have been confined to in vitro studies due to challenges in stable cccDNA expression in animal models and the limited non-immunogenicity of delivery systems. This study addresses these limitations by introducing a novel non-viral gene delivery system utilizing Gemini Surfactant (GS). The developed system creates stable and targeted CRISPR/Cas9 nanodrugs with a negatively charged surface through modification with red blood cell membranes (RBCM) or hepatocyte membranes (HCM), resulting in GS-pDNA@Cas9-CMs complexes. These GS-pDNA complexes demonstrated complete formation at a 4:1 w/w ratio. The in vitro transfection efficiency of GS-pDNA-HCM reached 54.61%, showing homotypic targeting and excellent safety. Additionally, the study identified the most effective single-guide RNA (sgRNA) from six sequences delivered by GS-pDNA@Cas9-HCM. Using GS-pDNA@Cas9-HCM, a significant reduction of 96.47% in in vitro HBV cccDNA and a 52.34% reduction in in vivo HBV cccDNA were observed, along with a notable decrease in other HBV-related markers. The investigation of GS complex uptake by AML-12 cells under varied time and temperature conditions revealed clathrin-mediated endocytosis (CME) for GS-pDNA and caveolin-mediated endocytosis (CVME) for GS-pDNA-HCM and GS-pDNA-RBCM. In summary, this research presents biomimetic gene-editing nanovectors based on GS (GS-pDNA@Cas9-CMs) and explores their precise and targeted clearance of cccDNA using CRISPR/Cas9, demonstrating good biocompatibility both in vitro and in vivo. This innovative approach provides a promising therapeutic strategy for advancing the cure of HBV.

Keywords

Antiviral therapy; CRISPR/Cas9; Cell membrane; Gemini surfactant; Gene delivery; Hepatitis B virus.

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