AUTHOR=Hollidge Bradley S. , Carroll Hayley B. , Qian Randolph , Fuller Madison L. , Giles April R. , Mercer Andrew C. , Danos Olivier , Liu Ye , Bruder Joseph T. , Smith Jared B. 

TITLE=Kinetics and durability of transgene expression after intrastriatal injection of AAV9 vectors

JOURNAL=Frontiers in Neurology

VOLUME=Volume 13 - 2022

YEAR=2022

URL=https://www.frontiersin.org/journals/neurology/articles/10.3389/fneur.2022.1051559

DOI=10.3389/fneur.2022.1051559

ISSN=1664-2295

ABSTRACT=Understanding the kinetics and durability of AAV-mediated transgene expression in the brain is essential for conducting basic neuroscience studies as well as for developing gene therapy approaches for CNS diseases. Here, we characterize and compare the temporal profile of transgene expression after bilateral injections into the mouse striatum of rAAV9 encoding GFP under the control of either a ubiquitous promoter (CAG), or the neuron-specific human synapsin (hSyn) and CamKII promoters. GFP protein expression with the CAG promoter was highest at three weeks, and then decreased to stable levels at three and six months. Surprisingly, GFP mRNA levels continued to increase from three weeks to three months, despite GFP protein expression decreasing during this time. GFP protein expression with hSyn increased more slowly, reaching a maximum at three months, which was equivalent to protein expression levels from CAG at that time point. Importantly, transgene expression driven by the hSyn promoter at six months was not silenced as previously reported, and GFP mRNA was continuing to rise even at the final six-month time point. Thus, hSyn as a promoter for transgene expression demonstrates long-term durability but may require more time after vector administration to achieve steady-state levels. Because CAG had the highest GFP protein expression in our comparison, which was at three weeks post administration, the early kinetics of transgene expression from CAG was examined (one, two, five and ten days after injection). This analysis showed that GFP protein expression and GFP mRNA increased during the first three weeks after administration. Interestingly, vector DNA rapidly decreased ten-fold over the first three weeks following injection as it assembled into stable circular episomes and concatemers. Surprisingly, the processing of vector genomes into circular episomes and concatemers was continually dynamic up to three months after injection. These results provide novel insight into the dynamic processing of vector genomes and promoter-specific temporal patterns of transgene expression in the brain.