Systemic delivery of specific and efficient CRISPR/Cas9 system targeting HPV16 oncogenes using LL-37 antimicrobial...

Area of Science:

• Oncology
• Molecular Biology
• Gene Therapy

Background:

• Human papillomavirus (HPV) type 16 is a primary cause of cervical cancer.
• CRISPR/Cas9 gene editing offers a promising novel therapeutic strategy for cancer treatment.

Purpose of the Study:

• To design and evaluate CRISPR/Cas9 gene editing targeting HPV16 oncogenes (E5, E6, E7) and the p97 promoter.
• To assess the efficacy of CRISPR/Cas9 delivery using Lipofectamine 2000 and LL-37 peptide in tumor cells.
• To investigate the therapeutic potential of CRISPR/Cas9 gene editing against pre-existing HPV16-induced tumors in a mouse model.

Main Methods:

• In silico design of guide RNA (gRNA) sequences for CRISPR/Cas9 targeting HPV16.
• Delivery of recombinant vectors into C3, TC1, and HeLa cells using Lipofectamine 2000 and LL-37 peptide.
• Western blot analysis of cell cycle proteins (p21, p53, Rb) and immunohistochemistry (IHC) for cleaved caspase-3 and mitotic index in tumor tissues.
• Evaluation of tumor size reduction and survival in C57BL/6 mice treated with gene editing vectors and cisplatin.

Main Results:

• The E6+E7 targeting CRISPR/Cas9 treatment demonstrated significant tumor reduction in mice.
• This group exhibited a high percentage of cleaved caspase-3 positive cells (45.75%) and a low mitotic index (2-3), indicating effective tumor cell apoptosis.
• The LL-37 antimicrobial peptide proved effective in overcoming CRISPR/Cas9 delivery challenges, marking a novel application.

Conclusions:

• CRISPR/Cas9-mediated gene editing is an effective, specific, and non-toxic approach for targeting pre-existing HPV16-induced tumors.
• The study highlights the potential of LL-37 peptide as a delivery enhancer for CRISPR/Cas9 gene therapy.
• Precise gene therapy using CRISPR/Cas9 presents a bright outlook for future cancer patient treatment.