I4 - Molecular and Cellular Methods (Including assess vector integration, genome integrity, and outcomes)
348: Characterization of AAV Vectors in Tissues Transduced with Conventional and Novel ITRs Show Differences in ITR Recombination, Episome Structures, and Transgene Expression
Type: Oral Abstract Session
Presentation Details
Session Title: AAV Vector Integration
The inverted terminal repeat (ITR) sequences that were originally isolated from the genomic termini of adeno-associated virus serotype 2 (AAV2) have been obligatory for producing recombinant (r)AAVs. The ITRs are also essential for driving intramolecular recombination to form stable circular episomes in the host cell. It has been reported that ITRs contribute to transgene expression via their promoter-like activities. However, the consequences of vector genome circularization on transgene expression have not been extensively explored. We serendipitously found a vector design that when transduced into mouse livers, showed a complete lack of transgene expression. Through Hirt DNA extractions and nanopore sequencing of episomal vector species in transduced tissues, we revealed that the episomes completely lacked the ITRs. Some species even included deletions of transgene segments. AAV-GPseq of the vector preparation showed that the vectors were intact and homogenous, suggesting that the lack of ITRs in episomes extracted from transduced tissues were not due to truncations found in vector preparations. We hypothesize that recombination to form episomes is error prone and stochastic. This premise offers another facet to poor transduction seen in certain vector designs. To understand how transgene expression is impacted by ITRs post-circularization, we are developing a novel platform that permits the examination of different episomal forms (e.g., monomers, dimers/concatemers), and the characterization of ITR structures post-transduction. Using third-generation, single molecule, real-time (SMRT) sequencing, this study aims to reveal, 1) the fate of ITRs post-transduction and circularization, and 2) outcomes of recombination between ITRs of different AAV serotypes that yield distinct transgene expression profiles. This work also uncovers concepts that contrasts with common knowledge regarding the retention and conformation of ITRs post-transduction in vivo. These results shed light on an unexplored property of vector genome processing and has paradigm-shifting impact for AAV vector biology and the field of gene therapy. In addition, our findings open the door to new avenues of research aimed at improving the transgene expression and stability of rAAV episomes in order to increase the efficacy of rAAV-based gene therapies. G.G. and P.T. are corresponding authors
Suk Namkung1, Elisabet Mandon1,2, Jialing Yang Liang1, Fang Zhang1, Mitchell Yip1, Tapan Sharma1, Jackson McGowan1, Sophia Liu1, Thomas Leland1, Mengtian Cui3, Dan Wang1,2, Phillip Tai1, Guangping Gao1
1Horae Gene Therapy Center, UMass Chan Medical School, Worcester, MA,2RNA Therapeutics Institute, UMass Chan Medical School, Worcester, MA,3Horae Gene Therapy Center, Umass Medical School, Worcester, MA"
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