A2 - AAV Vectors - Virology and Vectorology
67: Novel AAV8 and AAVrh39 ITRs and ITR-Proximal Regions Significantly Enhance Transgene Expression through Enhancer/Promoter-Like Activities
Type: Oral Abstract Session
Presentation Details
Session Title: AAV Vector Biology and Development I
Advancements in adeno-associated virus (AAV)-based gene therapy engineering have greatly increased within the past decade. These trends have been mainly within the scopes of capsid engineering and regulatory cassette design. However, there has been an underwhelming amount of research focused on exploring genetic elements of wildtype AAV and their roles in transcription. Previous research has demonstrated that inverted terminal repeats (ITRs), the last viral elements in AAV vectors, have promoter-like activities. Furthermore, ITR-proximal regions (IPRs) at the 3’ ends of wildtype AAV genomes also show tissue-dependent enhancer-like activities. These results have shown the potential for native elements within the AAV genome to further improve gene therapy vectors. To study the different contributions of AAV genetic elements towards vector transduction, we tested the ITRs and IPRs of AAV8 and AAVrh.39 for their potential to act as enhancers and/or promoters in vitro and in vivo. As previously reported, we found that the ITRs of AAV2, AAV8, and AAVrh.39 (ITR2, ITR8, and ITRrh.39 respectively) have very weak promoter-like activities on their own. However, we found that the IPRs of AAV8 and AAVrh.39 (IPR8 and IPRrh.39, respectively) have a stronger impact on transcription, conferring four-fold greater activity than achieved by the human phosphoglycerate kinase (hPGK) promoter. In addition, while AAV2’s IPR (IPR2) only showed liver-specific activity, IPRrh39 was equally active in HEK293 and cultured hepatocytes. We next produced vectors carrying ITRs and IPRs from AAV8 or AAVrh.39. Vector production and sequencing of the vectors by third-generation sequencing showed that the these elements are fully compatible with standard triple-plasmid transfection production schemes using Rep proteins from AAV2. Surprisingly, we found that AAV vectors carrying ITR8 and ITRrh.39 showed a >10-fold enhancement in mouse liver transduction over vectors that carried conventional ITR2. We are currently working toward understanding the mechanisms that underpin ITR and IPR function. In conclusion, we demonstrate that there are still facets of AAV biology that remain to be uncovered and further studied in order to improve gene therapy vectors. G.G. and P.T. are corresponding authors
Suk Namkung1, Elisabet Mandon1,2, Jialing Yang Liang1, Mengtian Cui1, Fang Zhang1, Jackson McGowan1, Mitchell Yip1, Thomas Leland1, Sophia Liu1, Tapan Sharma1, Dan Wang1, Guangping Gao1, Phillip Tai1
1Horae Gene Therapy Center, UMass Chan Medical School, Worcester, MA,2RNA Therapeutics Institute, UMass Chan Medical School, Worcester, MA"
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