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Adeno-Associated Virus Serotype-Specific Inverted Terminal Repeat Sequence Role in Vector Transgene Expression.
Lauriel F Earley1, Laura M Conatser1,2, Victoria M Lue1,2
1Gene Therapy Center, University of North Carolina, Chapel Hill, North Carolina.
Human Gene Therapy
|January 10, 2020
Summary
Adeno-associated virus (AAV) inverted terminal repeats (ITRs) possess inherent transcriptional activity. This study reveals varying promoter strengths across AAV serotypes, impacting gene expression and vector design.
Area of Science:
- Molecular Biology
- Gene Therapy
- Virology
Background:
- Adeno-associated viral vectors (AAV) are crucial for gene delivery, with 96% of their genome replaced by therapeutic genes.
- The remaining 145 bp adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences are essential for vector function but possess inherent transcriptional activity.
- This intrinsic ITR promoter activity, particularly from AAV serotype 2 (ITR2), can interfere with intended transgene expression in gene therapy applications.
Purpose of the Study:
- To comprehensively analyze and compare the promoter activity of adeno-associated virus (AAV) inverted terminal repeat (ITR) sequences from various serotypes.
- To investigate the in vitro and in vivo transcriptional activity of AAV ITRs across different cell lines and in a mouse model.
- To identify potential transcriptional start sites within the ITR sequences and their relationship to promoter activity.
Main Methods:
- Compared the transcriptional activity of AAV ITRs from serotypes 1-4, 6, and 7.
- Evaluated ITR promoter activity across multiple cell lines in vitro and in a mouse model in vivo.
- Utilized next-generation sequencing to identify transcriptional start sites within the ITR sequences.
Main Results:
- All tested AAV ITRs exhibited promoter activity both in vitro and in vivo.
- In vitro, AAV ITRs were classified into three expression levels: high (AAV2, AAV3), intermediate (AAV4), and low (AAV1, AAV6), with consistent activity across cell lines.
- AAV ITR7 showed cell-type dependent transcriptional activity, and next-generation sequencing identified multiple transcriptional start sites within the ITRs, primarily in the Rep binding element.
Conclusions:
- The serotype of the adeno-associated virus (AAV) inverted terminal repeat (ITR) sequence significantly influences its inherent promoter activity.
- AAV ITRs can independently affect transgene expression cassettes, irrespective of the chosen external promoter.
- Understanding AAV ITR promoter activity is critical for optimizing gene expression and minimizing off-target effects in AAV-based gene therapy.
