Low endotoxin E. coli strain-derived plasmids reduce rAAV vector-mediated immune responses both in vitro and in vivo

Abstract

The major challenge of recombinant adeno-associated virus (rAAV) vectors is host immunological barriers. Compared to the neutralizing antibody and the cytotoxic T lymphocyte response, the host immune responses induced by unsatisfactory rAAV manufacturing were largely ignored previously. rAAV vector production usually requires large amounts of plasmid DNAs. The DNA are commonly isolated from the DH5a bacterial strain, which contains lipopolysaccharide (LPS) contamination. LPS, also named endotoxin, in plasmid DNA is intractable, and residual endotoxin in the subsequent rAAV vectors may result in substantial host immune response. Recently, a ClearColi K12 bacterial strain is commercially available, with genetically modified LPS that does not trigger endotoxic response in mammalian cells. Here, we produced rAAV-DJ vectors by plasmids yielded from either DH5a or ClearColi K12 bacterial strains. Our data indicated that the ClearColi K12 strain had satisfactory protection for the rAAV inverted terminal repeat (ITR) sequence. As expected, the ClearColi K12derived rAAV-DJ vectors had lower endotoxin levels. The physical and biological equivalency of the purified viral stocks were confirmed by electron micrographs, Coomassie blue staining, and transduction assays. Most importantly, the ClearColi K12-derived rAAV-DJ vectors triggered reduced nuclear factor-kappa B (NF-kB) signaling pathway both in cell cultures in vitro and in C57BL/6 mice retinas in vivo. We believe that the use of the ClearColi K12 bacterial strain could eliminate the LPS in the purified vector stock at the source. Our data indicate its promising use in future clinical development.

Improved gene therapy for MFRP deficiency-mediated retinal degeneration by knocking down endogenous bicistronic Mfrp and Ctrp5 transcript

“…Subretinal injection was performed in 4-week-old WT mice or 14-day-old rd6 mice via transvitreal approach under a surgical microscope as previously described. 22 Intravitreal injection was executed in 4-week-old WT mice. Briefly, mice were anesthetized with pentobarbitone and the ocular surface was anesthetized with proparacaine hydrochloride (0.5%).…”

“… 20 , 21 Among these capsid-modified vectors, rAAVDJ-3M (N498S, Y706F, and Y732F) showed the highest efficiency in the mouse and human liver ductal organoid. Since both we 22 and others 23 recently documented that rAAVDJ vectors possessed robust transgene expression in photoreceptors and the retinal pigment epithelium, we would like to compare, side-by-side, the transduction capacity of rAAVDJ-3M vectors with that of their parental rAAVDJ vectors through subretinal and intravitreal injection.…”

Improved endotoxin removal using ecofriendly detergents for intensified plasmid capture

“…Endotoxins, also known as lipopolysaccharides (LPS), are a major constituent of the outer cell wall of gram-negative bacteria and are naturally released during E.coli cultivation. 13 Although the quality and yield of plasmid DNA is largely defined by the host E.coli strain and plasmid construct design, 14 optimization of fermentation conditions and media selection are key to maximizing the ratio of plasmid to impurities (endotoxins, RNA). 13 Endotoxins are considered a critical process impurity, can be a risk to patient safety and are subject to strict control and limits.…”

Ultramicro and ultrasensitive detection of lipopolysaccharides based on triple-signal amplification via ultrafast ATRP and an ultramicroelectrode

“…[4][5][6] According to the US Food and Drug Administration (FDA) guidelines, LPS in intraocular delivered gene therapy drugs should not exceed 0.5 EU per mL per eye (∼500 pg per mL per eye). 7 10 pg mL −1 of LPS is the cut-off value to distinguish between healthy individuals and Gramnegative bacteremia patients in a clinical setting. 8 Characteristic symptoms of sepsis will occur in humans and chimpanzees following an exposure to only 1-5 ng per kg (0.001-0.005 pg mg −1 ) LPS.…”