Segmented poly(A) tails significantly reduce recombination of plasmid DNA without affecting mRNA translation efficiency or half-life

Abstract

Extensive research in the past decade has brought mRNA closer to the clinical realization of its therapeutic potential. One common structural feature for all cellular messenger RNAs is a poly(A) tail, which can either be brought in cotranscriptionally via the DNA template (plasmid- or PCR-based) or added to the mRNA in a post-transcriptional enzymatic process. Plasmids containing poly(A) regions recombine in E. coli, resulting in extensive shortening of the poly(A) tail. Using a segmented poly(A) approach, we could significantly reduce recombination of plasmids in E. coli without any negative effect on mRNA half-life and protein expression. This effect was independent of the coding sequence. A segmented poly(A) tail is characterized in that it consists of at least two A-containing elements, each defined as a nucleotide sequence consisting of 40-60 adenosines, separated by a spacer element of different length. Furthermore, reducing the spacer length between the poly(A) segments resulted in higher translation efficiencies compared to homogeneous poly(A) tail and reduced recombination (depending upon the choice of spacer nucleotide). Our results demonstrate the superior potential of segmented poly(A) tails compared to the conventionally used homogeneous poly(A) tails with respect to recombination of the plasmids and the resulting mRNA performance (half-life and translational efficiency).

Keywords: mRNA therapeutics; plasmid recombination; poly(A) tail; transcript therapy.

FIGURE 1.

Schematic representation of all combinations of poly(A) modifications tested in the current study. Different homo- or heteropolymeric poly(A) stretches were inserted downstream from the gene of interest (GOI).

FIGURE 2.

Quantification of poly(A) tail recombination for A120 and segmented poly(A) tails of poly(A)_{3 × 40_6} and poly(A)_{2 × 60_6}. (n) Total number of clones of d2EGFP, luciferase, and hEPO sequences tested with a particular poly(A) format.

FIGURE 3.

Determination of d2EGFP protein expression and mRNA quantification of different poly(A)-containing d2EGFP mRNAs post-transfection in A549 cells. (A) Mean MFI (median fluorescence intensity) at 4-, 24-, 48-, and 72-h post-transfection, measured by FACS in A549 cells. (B) d2EGFP mRNA quantification in A549 cells. (C) mRNA productivity was calculated by dividing the mean MFI (FACS data; A) by the mRNA amounts (real-time PCR data; B) and normalizing these ratios to those observed with poly(A)₁₂₀ construct. Values represent mean ± SD of three replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (*) P < 0.5, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001.

FIGURE 4.

Determination of luciferase expression and mRNA quantification of different poly(A)-containing luciferase mRNA 24-h post-transfection in A549 cells. (A) Luciferase activity, measured as relative light units (RLU: arbitrary units) in protein lysates from A549 cells transfected with different poly(A)-containing luciferase RNA measured 24-h post-transfection. (B) Luciferase mRNA quantification in A549 cells. (C) mRNA productivity was calculated by dividing the luciferase expression values (RLU; A) by the mRNA amounts (real-time PCR data; B) and normalizing these ratios to those observed with poly(A)₁₂₀ construct. Values represent mean ± SD of six replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (*) P < 0.5, (**) P < 0.01, (***) P < 0.001, (****) P < 0.0001.

FIGURE 5.

Quantification of secreted human erythropoietin protein levels as measured via ELISA in supernatants from HEK293 cells transfected either with poly(A)_120- or poly(A)_2×60-containing EPO mRNA at 24 h (A), 48 h (B), and 72 h (C) post-transfection. Values represent mean ± SD of three replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (**) P < 0.01, (***) P < 0.001, n = 3.

FIGURE 6.

Relative quantification of CFTR protein in 16HBE14o- lysates as measured via western blot. (A) 16HBE14o- cells were transfected either with poly(A)₁₂₀- or poly(A)_{2×60_6}-containing hCFTR mRNA and protein lysates analyzed at 24 and 48 h post-transfection. (B) Densitometry analysis of western blot images. Values represent mean ± SD of two replicates. Statistical significance was assessed by paired t-test with P-values: ns = P > 0.5.

FIGURE 7.

Determination of luciferase expression and mRNA quantification of different poly(A)-containing luciferase mRNA at 24 h post-transfection in A549 cells. (A) Luciferase activity, measured as relative light units (RLU: arbitrary units), in protein lysates from A549 cells transfected with different poly(A)-containing luciferase mRNA. (B) Luciferase mRNA quantification in A549 cells transfected with different poly(A)-containing luciferase mRNA. (C) mRNA productivity was calculated by dividing the luciferase expression values (RLU; A) by the mRNA amounts (real-time PCR data; B) and normalizing these ratios to those observed with poly(A)₁₂₀ construct. Values represent mean ± SD of six replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (*) P < 0.5, (**) P < 0.01, (****) P < 0.0001.

FIGURE 8.

Determination of luciferase expression and mRNA quantification of different poly(A)-containing luciferase mRNA at 24 h post-transfection in A549 cells. (A) Luciferase activity, measured as relative light units (RLU: arbitrary units), in protein lysates from A549 cells transfected with different poly(A)-containing luciferase mRNA. (B) Luciferase mRNA quantification in A549 cells transfected with different poly(A) containing luciferase mRNA. (C) mRNA productivity was calculated by dividing the luciferase expression values (RLU; A) by the mRNA amounts (real-time PCR data; B) and normalizing these ratios to those observed with poly(A)₁₂₀ construct. Values represent mean ± SD of six replicates. Statistical significance was assessed by two-way ANOVA test with P-values: (*) P < 0.5, (****) P < 0.0001.

FIGURE 9.

Quantification of poly(A) tail recombination rate for segmented poly(A) tails with a single nucleotide spacer. (n) Total number of clones of luciferase tested with a particular poly(A) format.