Polymyxin B Treatment Improves Bactofection Efficacy and Reduces Cytotoxicity

Abstract

Improvements to bacterial vectors have resulted in non-viral gene therapy vehicles that are easily prepared and can achieve high levels of transfection efficacy. However, these vectors are plagued by potential cytotoxicity and immunogenicity, prompting means of attenuation to reduce unwanted biological outcomes while maintaining transfection efficiency. In this study, listeriolysin O (LLO) producing Escherichia coli BL21(DE3) strains were pre-treated with polymyxin B (PLB), a pore-forming antibiotic, and tested as a delivery vector for gene transfer to a murine RAW264.7 macrophage cell line using a 96-well high-throughput assay. PLB treatment resulted in statistically significant higher levels of gene delivery and lower cytotoxicity. The results suggest a fine balance between bacterial cellular damage, heightened gene and protein release, and increased mammalian cell gene delivery. Overall, the approach presented provides a simple and effective way to enhance bacterial gene delivery while simultaneously reducing unwanted outcomes as a function of using a biological vector.

Hybrid biosynthetic gene therapy vector development and dual engineering capacity

“…Previously, we identified a positive correlation between bacterial cell wall weakening and gene delivery (12). To investigate this relationship further and demonstrate the biological engineering potential of the hybrid device, we introduced and conditionally expressed bacteriophage ΦX174 LyE.…”

“…In particular, Escherichia coli, a rod-shaped, Gram-negative bacterium that is ∼2 μm in length and 0.5 μm in diameter, has been identified as an ideal gene carrier due to simple and scalable culture protocols, a rapid growth rate, and a wealth of established molecular biology manipulation techniques (11). Nonpathogenic strains of E. coli have exhibited minimal toxicity and immunogenicity (12)(13)(14)(15), further supported by in vivo and in vitro delivery of plasmid (12-14, 16, 17) and larger DNA constructs (18,19), shRNA (20), and immunogenic peptides (17,21,22). …”

Influence of molecular weight upon mannosylated bio-synthetic hybrids for targeted antigen presenting cell gene delivery

“…A single E. coli strain was selected as the bacterial vector based upon previous optimization studies [3, 7–9]. This particular bacterial strain was engineered to deliver a mammalian expression reporter plasmid (pCMV-Luc) with assistance of a chromosomal-located listeriolysin O (LLO) expression cassette driven by an inducible T7 promoter [17] at the clpP location [8].…”

“…YWT7- hly (BL21[DE3] background strain containing a chromosomally located copy of listeriolysin O [ hly ]) was selected as the bacterial vector for all gene delivery experiments [3, 7–9]. A luciferase reporter plasmid driven by a cytomegalovirus promoter (pCMV-Luc; Elim Biopharmaceuticals) was utilized during positive control transfection experiments and introduced to YWT7- hly via electroporation prior to bacterial or hybrid vector transfections.…”

Biomaterials at the interface of nano- and micro-scale vector–cellular interactions in genetic vaccine design

“…Biosafety issues have been addressed through various means including antibiotic pre-treatment 109 and auxotroph-conferring genetic manipulations. 100, 130–132 As a result, it is likely that additional engineering will allow the generation of safe and effective vectors.…”