Next Article in Journal
Epigenetic Regulation of Alternative mRNA Splicing in Dilated Cardiomyopathy
Previous Article in Journal
Family Caregiver Strain and Challenges When Caring for Orthopedic Patients: A Systematic Review
Previous Article in Special Issue
p53 CRISPR Deletion Affects DNA Structure and Nuclear Architecture
Open AccessArticle

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

Phage Therapy Group, Department of Brain Sciences, Imperial College London, London W12 0NN, UK
*
Authors to whom correspondence should be addressed.
J. Clin. Med. 2020, 9(5), 1498; https://doi.org/10.3390/jcm9051498
Received: 6 April 2020 / Revised: 4 May 2020 / Accepted: 14 May 2020 / Published: 16 May 2020
(This article belongs to the Special Issue CRISPR, Cancer, and p53)
p53 gene (TP53) replacement therapy has shown promising results in cancer gene therapy. However, it has been hampered, mostly because of the gene delivery vector of choice. CRISPR-Cas9 technology (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) can knock out the mutated TP53 (mutTP53), but due to its large size, many viral vectors are not suitable or require implemented strategies that lower the therapeutic efficiency. Here, we introduced a bacteriophage or phage-based vector with the ability to target cancer cells and aimed to investigate the feasibility of using this vector to deliver CRISPR-Cas9 transgene in human lung adenocarcinoma cells. First, we produced a tumour-targeted bacteriophage carrying a CRISPR-Cas9 transgene cassette. Next, we investigated any negative impact on vector titers via quantitative polymerase chain reaction (qPCR) and colony-forming agar plate. Last, we combined Western blot analysis and immunofluorescence staining to prove cell transduction in vitro. We showed that the tumour-targeted bacteriophage can package a large-size vector genome, ~10 kb, containing the CRISPR-Cas9 sequence without any negative impact on the active or total number of bacteriophage particles. Then, we detected expression of the Cas9 in human lung adenocarcinoma cells in a targeted and efficient manner. Finally, we proved loss of p53 protein expression when a p53 gRNA was incorporated into the CRISPR-Cas9 phage DNA construct. These proof-of-concept findings support the use of engineered bacteriophage for TP53 replacement therapy in lung cancer. View Full-Text
Keywords: bacteriophage; gene therapy; CRISPR-Cas9; p53; lung cancer; tumour suppressor gene replacement therapy bacteriophage; gene therapy; CRISPR-Cas9; p53; lung cancer; tumour suppressor gene replacement therapy
Show Figures

Figure 1

MDPI and ACS Style

Yang Zhou, J.; Suwan, K.; Hajitou, A. Initial Steps for the Development of a Phage-Mediated Gene Replacement Therapy Using CRISPR-Cas9 Technology. J. Clin. Med. 2020, 9, 1498.

Show more citation formats Show less citations formats
Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Article Access Map by Country/Region

1
Search more from Scilit
 
Search
Back to TopTop