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ACE-tRNA DNA minivectors exhibit better bioavailability, lower innate immune response induction, and better biostability as compared to pDNA. ( A ) 16HBE14o-cells containing the genomically encoded SGG PTC reporter were cultured and transfected either in the presence of the antimitotic agent aphidicolin or the DMSO vehicle with ACE-tRNA CEDT or pDNA (n = 3). NLuc-PTC suppression was used to demonstrate levels of PTC rescue for the various vectors ( B ) HeLa cells were transfected with ACE-tRNA MC, CEDT, or pDNA and after 6 hours in culture, were lysed, and assayed for cyclic GMP-AMP <t>(cGAMP)</t> levels by competitive ELISA (n = 5). The level of cGAMP present demonstrates activation of the cGAS-STING innate immune response pathway. ( C ) ACE-tRNAs encoded as either MC, CEDT, or pDNA (P) were subjected to incubation in human blood serum at 37 °C for the times indicated. After treatment vector DNA was purified by phenol/chloroform extraction, precipitated, and the amount of residual intact DNA was assessed by agarose gel electrophoresis and band densitometry (n = 3). Band densitometry data was fit to a one phase decay using non-linear regression, which is shown on the graph. Data are presented as the mean ± SEM with significance determined by one-way ANOVA and Tukey’s post-hoc test, where ** ** p < 0.01 and **** p < 0.0001.
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ACE-tRNA DNA minivectors exhibit better bioavailability, lower innate immune response induction, and better biostability as compared to pDNA. ( A ) 16HBE14o-cells containing the genomically encoded SGG PTC reporter were cultured and transfected either in the presence of the antimitotic agent aphidicolin or the DMSO vehicle with ACE-tRNA CEDT or pDNA (n = 3). NLuc-PTC suppression was used to demonstrate levels of PTC rescue for the various vectors ( B ) HeLa cells were transfected with ACE-tRNA MC, CEDT, or pDNA and after 6 hours in culture, were lysed, and assayed for cyclic GMP-AMP (cGAMP) levels by competitive ELISA (n = 5). The level of cGAMP present demonstrates activation of the cGAS-STING innate immune response pathway. ( C ) ACE-tRNAs encoded as either MC, CEDT, or pDNA (P) were subjected to incubation in human blood serum at 37 °C for the times indicated. After treatment vector DNA was purified by phenol/chloroform extraction, precipitated, and the amount of residual intact DNA was assessed by agarose gel electrophoresis and band densitometry (n = 3). Band densitometry data was fit to a one phase decay using non-linear regression, which is shown on the graph. Data are presented as the mean ± SEM with significance determined by one-way ANOVA and Tukey’s post-hoc test, where ** ** p < 0.01 and **** p < 0.0001.

Journal: bioRxiv

Article Title: Anticodon Edited Transfer RNAs (ACE-tRNAs) Encoded as Therapeutic Nonviral Minimal DNA Vectors

doi: 10.1101/2025.09.06.674645

Figure Lengend Snippet: ACE-tRNA DNA minivectors exhibit better bioavailability, lower innate immune response induction, and better biostability as compared to pDNA. ( A ) 16HBE14o-cells containing the genomically encoded SGG PTC reporter were cultured and transfected either in the presence of the antimitotic agent aphidicolin or the DMSO vehicle with ACE-tRNA CEDT or pDNA (n = 3). NLuc-PTC suppression was used to demonstrate levels of PTC rescue for the various vectors ( B ) HeLa cells were transfected with ACE-tRNA MC, CEDT, or pDNA and after 6 hours in culture, were lysed, and assayed for cyclic GMP-AMP (cGAMP) levels by competitive ELISA (n = 5). The level of cGAMP present demonstrates activation of the cGAS-STING innate immune response pathway. ( C ) ACE-tRNAs encoded as either MC, CEDT, or pDNA (P) were subjected to incubation in human blood serum at 37 °C for the times indicated. After treatment vector DNA was purified by phenol/chloroform extraction, precipitated, and the amount of residual intact DNA was assessed by agarose gel electrophoresis and band densitometry (n = 3). Band densitometry data was fit to a one phase decay using non-linear regression, which is shown on the graph. Data are presented as the mean ± SEM with significance determined by one-way ANOVA and Tukey’s post-hoc test, where ** ** p < 0.01 and **** p < 0.0001.

Article Snippet: The total protein in the supernatant was quantified by BCA assay (Pierce), and the 2’-5’/3’-5’ cyclic-GMP-AMP (cGAMP) levels were assessed by competitive ELISA according to manufacturer instructions (Invitrogen, Ref EIAGAMP).

Techniques: Cell Culture, Transfection, Competitive ELISA, Activation Assay, Incubation, Plasmid Preparation, Purification, Extraction, Agarose Gel Electrophoresis