Tissue specificity and age-related dynamics of CAG promoter activity in rAAV-mediated gene therapy

INTRODUCTION. Artificial promoters based on the chicken beta-actin promoter (CAG, CBA, or CB) are frequently used in recombinant adeno-associated virus (rAAV)-based gene therapy products. However, data on the activity and age-related activity dynamic of these promoters remain quite limited. In this study, we quantitatively assessed CAG promoter activity in various organs of postnatal mice to determine its suitability for the development of rAAV-mediated gene therapy.

AIM. Evaluation of the relative CAG promoter activity and its dynamics in mouse organs from 3 to 12 weeks of life.

MATERIALS AND METHODS. Two-day-old ICR (CD-1) mice were injected once with rAAV9 carrying a cassette for SMN expression under the control of the CAG promoter. At 3, 6, and 12 weeks after administration, total DNA and RNA were isolated from organ samples (brain, spinal cord, liver, lungs, heart, quadriceps femoris muscle). The content of rAAV genomes and SMN mRNA in nucleic acid preparations were determined by quantitative PCR. Relative promoter activity was calculated as the ratio of the SMN mRNA concentration to the concentration of rAAV genomes, normalized on the RNA:DNA ratio in the organ. Data were analyzed using the geometric mean and regression analysis.

RESULTS. CAG promoter activity varies significantly in different organs. At week 3 after injection, the highest values were observed for the quadriceps femoris muscle, 4.7–9.7 times higher than those in other organs. It was also found that in the brain and lungs, promoter activity decreased 4.7-fold (p=0.0094) and 5.2-fold (p=0.0039), respectively, from 3 to 12 weeks after injection, while in other tissues no significant changes in activity were observed.

CONCLUSIONS. The CAG promoter is poorly suited for transgene expression in the lung and brain cells due to promoter silencing and is suboptimal for expression in the liver cells because of relatively low activity; these findings should be taken into account in the development of gene therapy products.

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