Minimal doses of a sequence-optimized transgene mediate high-level and long-term EPO expression in vivo: challenging CpG-free gene design

Abstract

Advanced gene delivery techniques can be combined with rational gene design to further improve the efficiency of plasmid DNA (pDNA)-mediated transgene expression in vivo. Herein, we analyzed the influence of intragenic sequence modifications on transgene expression in vitro and in vivo using murine erythropoietin (mEPO) as a transgene model. A single electro-gene transfer of an RNA-and codon-optimized mEPOopt gene into skeletal muscle resulted in a 3-to 4-fold increase of mEPO production sustained for 41 year and triggered a significant increase in hematocrit and hemoglobin without causing adverse effects. mEPO expression and hematologic levels were significantly lower when using comparable amounts of the wild type (mEPOwt) gene and only marginal effects were induced by mEPODCpG lacking intragenic CpG dinucleotides, even at high pDNA amounts. Corresponding with these observations, in vitro analysis of transfected cells revealed a 2-to 3-fold increased (mEPOopt) and 50% decreased (mEPODCpG) erythropoietin expression compared with mEPOwt, respectively. RNA analyses demonstrated that the specific design of the transgene sequence influenced expression levels by modulating transcriptional activity and nuclear plus cytoplasmic RNA amounts rather than translation. In sum, whereas CpG depletion negatively interferes with efficient expression in postmitotic tissues, mEPOopt doses o0.5 mg were sufficient to trigger optimal long-term hematologic effects encouraging the use of sequence-optimized transgenes to further reduce effective pDNA amounts.

Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression

“…Our results are very encouraging, since high expressers with an expression level increase of 2.6-fold to 15-fold showed no detrimental effect on solubility (JNK1, JNK3, p38a) or function (JNK1, JNK3 and CDC2). This positive effect of gene optimization on protein expression resulting in functional protein was also demonstrated in a recent publication by some of the authors [50], where a single electro-gene transfer of an RNA- and codon optimized EPO gene into skeletal muscle resulted in a 3- to 4-fold increase of EPO production over mice treated with non-optimized EPO genes, sustaining for >1 year and triggering a significant increase in hematocrit and hemoglobin without causing adverse effects [50]. Furthermore in addition to the mechanistic insights of overexpression in the stable system described for MIP1-α, the study provides supporting mechanistic insights of overexpression in a transient system [50].…”

Characterization of transgene expression and pDNA distribution of the suctioned kidney in mice

“…Several studies have reported that removal of CG dinucleotides from pDNA can achieve long-term transgene expression by avoiding the inflammatory response (Kosovac et al, 2011;Pringle et al, 2012). Therefore, we confirmed the duration of transgene expression of a CpG free plasmid encoding a secreted luciferase protein, Lucia, at the serum level.…”

Interplay of Promoter Usage and Intragenic CpG Content: Impact on GFP Reporter Gene Expression

“…The latter can be due to various effects, such as increasing mRNA stability 58 or increasing de novo mRNA synthesis of transgenes. 19,25 For hgfp genes with modified CpG content in the open reading frame, we have previously shown, using T7 polymerase-driven transcription in the cytoplasm of mammalian cells, that modulation of the CpG content does not affect mRNA stability or translational efficiency. 18 To avoid the spreading of repressive epigenetic marks surrounding the integration locus into the proximal transgene and to prevent loss of the transgene through the outgrowth of transgene-lacking cells, transgene expression is optimally conducted under selection pressure.…”

“…14,[22][23][24] In contrast, it was shown that CpG dinucleotides in the transgene open reading frame can provide improved and long-term transgene expression in mouse tissue. 25 These conflicting observations are suggested to be due to the use of different promoters and transgenes, which vary in codon quality or CpG content, and are flanked by diverse noncoding regions or cis-acting sequence elements. Moreover, different delivery systems were used in these studies, resulting in an episomal state or the random integration of multiple gene copies into the host cell genome.…”