p53 gene (TP53) replacement therapy has shown promising results in cancer gene therapy.\nHowever, it has been hampered, mostly because of the gene delivery vector of choice. CRISPR-Cas9\ntechnology (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)\ncan knock out the mutated TP53 (mutTP53), but due to its large size, many viral vectors are not\nsuitable or require implemented strategies that lower the therapeutic effciency. Here, we introduced a\nbacteriophage or phage-based vector with the ability to target cancer cells and aimed to investigate the\nfeasibility of using this vector to deliver CRISPR-Cas9 transgene in human lung adenocarcinoma cells.\nFirst, we produced a tumour-targeted bacteriophage carrying a CRISPR-Cas9 transgene cassette.\nNext, we investigated any negative impact on vector titers via quantitative polymerase chain\nreaction (qPCR) and colony-forming agar plate. Last, we combined Western blot analysis and\nimmunofluorescence staining to prove cell transduction in vitro. We showed that the tumour-targeted\nbacteriophage can package a large-size vector genome, ~10 kb, containing the CRISPR-Cas9 sequence\nwithout any negative impact on the active or total number of bacteriophage particles. Then,wedetected\nexpression of the Cas9 in human lung adenocarcinoma cells in a targeted and effcient manner. Finally,\nwe proved loss of p53 protein expression when a p53 gRNA was incorporated into the CRISPR-Cas9\nphage DNA construct. These proof-of-concept findings support the use of engineered bacteriophage\nfor TP53 replacement therapy in lung cancer.
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