Long-term Transgene Expression from Plasmid DNA Gene Therapy Vectors Is Negatively Affected by CpG Dinucleotides

Abstract

CpG-reduced, CMV-based plasmid DNA constructs encoding human alpha-galactosidase A and factor IX were injected into C57Bl/6, BALB/c, and CD1 mice using hydrodynamics-based delivery of plasmid DNA (pDNA), and gene expression was monitored for 6 months. Linearized and supercoiled pDNAs were compared for their abilities to support long-term expression and to generate immune responses to the transgene product. In all mouse strains supercoiled CpG-reduced pDNA encoding alpha-galactosidase A and factor IX generated higher and more sustained levels of circulating gene product than their supercoiled CpG-replete analogs. Linearizing supercoiled CpG-reduced pDNA did not significantly increase levels of circulating gene product beyond levels supercoiled CpG-reduced pDNA could achieve. Linearizing supercoiled CpG-replete pDNA vectors significantly increased expression compared to their supercoiled CpG-replete analogs, but the increase was short-lived or subtherapeutic. Regardless of vector, liver depot expression did not elicit significant antibody responses to human alpha-galactosidase A or factor IX. Taken together, these data suggest that a clinically acceptable hydrodynamics-based approach targeting the liver combined with CpG-reduced pDNA vectors may represent a viable option for individuals with hemophilia, a lysosomal storage disease, or other disease in which prolonged depot expression of a therapeutic protein from the liver is desirable.

Ultrasound-assisted non-viral gene transfer to the salivary glands

“…Firstgeneration plasmid vectors, such as the pGL3 plasmid, used in our experiments contain unmethylated C-phosphate-G (CpG) and other sequences of bacterial origin, which have been shown to trigger premature silencing of the episomal plasmid DNA. Advancements in plasmid gene transfer technologies such as CpGreduced 32 or 'minicircle' 33,34 gene transfer constructs have been shown to express for months after non-viral gene transfer. With non-viral gene transfer to the salivary glands now a practicable reality, perfecting the plasmid vector to allow gene expression for clinically-relevant durations, perhaps 6 months between boosters, is a paramount translational goal.…”

Persistent episomal transgene expression in liver following delivery of a scaffold/matrix attachment region containing non-viral vector

“…35 In similar reports, 36,37 it was verified that CpG dinucleotides could negatively affect transgene expression from pDNA in vivo, even with linearization of the transgene expression plasmid before hydrodynamic injection. Finally, other groups have reported the negative effect of CpG methylation in vitro, 38 and recently the extensive methylation of CpG islands of a CMV promoter-driven adenoviral vector following delivery into the muscles of mice was shown, with methylation levels similar to our results.…”

Improved anti‐cancer effect of interferon gene transfer by sustained expression using CpG‐reduced plasmid DNA

“…However, the low and transient nature of the induction of inflammatory cytokines after the injection of naked pDNA by the hydrodynamics-based procedure suggests that sustained gene expression from the pGZB vector is due not only to a reduction of the inflammatory response, but also to other mechanisms that have so far not been investigated. 33 Transcriptional repression of endogenous genes is often associated with a higher frequency of methylated cytosine residues in the 5 0 flanking region of the genes: promoter or enhancer. 34,35 Moreover, CpG methylation has been reported to be associated with the absence of integrated viral gene expression 36 and it has been suggested that de novo methylation of foreign DNA represents a cellular defense mechanism against the transcription of a foreign gene.…”