Initial Steps for the Development of a Phage-Mediated Gene Replacement Therapy Using CRISPR-Cas9 Technology

Area of Science:

• Gene therapy
• Molecular biology
• Oncology

Background:

• p53 gene (TP53) replacement therapy shows promise for cancer treatment.
• Viral vectors face limitations in delivering large CRISPR-Cas9 systems for TP53 gene editing.
• Targeted delivery of CRISPR-Cas9 is crucial for effective cancer gene therapy.

Purpose of the Study:

• To investigate the feasibility of using a bacteriophage-based vector for CRISPR-Cas9 delivery in lung adenocarcinoma.
• To assess the efficacy of a tumor-targeted bacteriophage in delivering the CRISPR-Cas9 transgene.
• To demonstrate the potential of bacteriophages in TP53 replacement therapy.

Main Methods:

• Production of a tumor-targeted bacteriophage carrying a CRISPR-Cas9 transgene cassette.
• Quantitative PCR and colony-forming assays to evaluate vector titers.
• Western blot analysis and immunofluorescence staining to confirm in vitro cell transduction and p53 expression loss.

Main Results:

• The engineered bacteriophage successfully packaged a ~10 kb CRISPR-Cas9 genome without impacting particle viability.
• Cas9 expression was detected efficiently and specifically in human lung adenocarcinoma cells.
• Incorporation of p53 gRNA into the phage construct led to a loss of p53 protein expression.

Conclusions:

• Engineered bacteriophages are effective vectors for delivering large CRISPR-Cas9 payloads for cancer gene therapy.
• This phage-based system demonstrates potential for targeted TP53 replacement therapy in lung cancer.
• Bacteriophage vectors offer a viable alternative to viral vectors for complex gene editing applications.