Bacteriophage Mediates Efficient Gene Transfer in Combination with Conventional Transfection Reagents

Abstract

The development of commercially available transfection reagents for gene transfer applications has revolutionized the field of molecular biology and scientific research. However, the challenge remains in ensuring that they are efficient, safe, reproducible and cost effective. Bacteriophage (phage)-based viral vectors have the potential to be utilized for general gene transfer applications within research and industry. Yet, they require adaptations in order to enable them to efficiently enter cells and overcome mammalian cellular barriers, as they infect bacteria only; furthermore, limited progress has been made at increasing their efficiency. The production of a novel hybrid nanocomplex system consisting of two different nanomaterial systems, phage vectors and conventional transfection reagents, could overcome these limitations. Here we demonstrate that the combination of cationic lipids, cationic polymers or calcium phosphate with M13 bacteriophage-derived vectors, engineered to carry a mammalian transgene cassette, resulted in increased cellular attachment, entry and improved transgene expression in human cells. Moreover, addition of a targeting ligand into the nanocomplex system, through genetic engineering of the phage capsid further increased gene expression and was effective in a stable cell line generation application. Overall, this new hybrid nanocomplex system (i) provides enhanced phage-mediated gene transfer; (ii) is applicable for laboratory transfection processes and (iii) shows promise within industry for large-scale gene transfer applications.

A century of bacteriophage research and applications: impacts on biotechnology, health, ecology and the economy!

“…Now the favour is returned with novel molecular biotechniques such as clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9 being used to modify industrial biotechnologically important bacterial strains, such as Bacillus subtilis and E. coli , to increase their phage resistance during large‐scale fermentation processes . Also, viral metagenomics, complete phage genome sequencing, phage‐mediated efficient gene transfer and molecular characterization of phage lytic enzymes (endolysins) are now routinely studied in view of improved applications of phages to combat pathogens and spoilage bacteria in the medical and agri‐food sector …”

“…Now the favour is returned with novel molecular biotechniques such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 being used to modify industrial biotechnologically important bac- characterization of phage lytic enzymes (endolysins) are now routinely studied in view of improved applications of phages to combat pathogens and spoilage bacteria in the medical and agri-food sector. [62][63][64][65][66]…”

Systemically targeted cancer immunotherapy and gene delivery using transmorphic particles

“…These factors contribute significantly to poor uptake and induction of gene expression observed in bacteriophage vectors. Previous studies on the AAVP have explored a number of strategies to enhance the relatively low transduction efficiency when compared to conventional mammalian viruses (Kia et al , 2013 ; Przystal et al , 2013 ; Yata et al , 2014 , 2015 ; Donnelly et al , 2015 ; Tsafa et al , 2016 , 2020 ; Campbell et al , 2018 ). Furthermore, alternative studies have explored removing parts of the phage genome, albeit at the cost of packaging efficiency (Chasteen et al , 2006 ).…”

Characterization of a novel lytic bacteriophage from an industrial Escherichia coli fermentation process and elimination of virulence using a heterologous CRISPR–Cas9 system

“…As the most abundant biological entity on the planet, bacteriophages play an important ecological role, and have also been exploited for the development of many modern technologies, including gene transfer and treatment of bacterial infections [ 1 , 9 , 10 , 12 ]. On the other hand, owing to their ability to cause rapid lytic infections of bacterial cultures in a matter of minutes, the presence of bacteriophage in a modern industrial fermentation facility can be a serious problem, resulting in reduced product quality, loss in production capacity or asset utilization, and financial losses to the business.…”