# Mechanistic Studies of AAP and Capsid Assembly of AAV Vectors

> **NIH NIH R01** · OREGON HEALTH & SCIENCE UNIVERSITY · 2022 · $729,095

## Abstract

SUMMARY
Adeno-associated viruses (AAV) have become increasingly popular as vectors for human gene therapy.
However, the requirement for high vector doses needed to achieve therapeutic efficacy poses a challenge to
cost-effective manufacturing of AAV vectors for clinical use. The fundamental process of AAV vector
production is virion assembly, the process by which 60 VP capsid protein subunits form an icosahedral protein
shell followed by AAV vector genome packaging. Unfortunately, this basic process remains poorly understood
and there is an urgent need to elucidate the process and mechanisms underlying AAV vector production. Such
an understanding also could potentially uncover the key to production of high titer and high-quality AAV
vectors. In this regard, there was a paradigm-shifting discovery in 2010 that the AAV cap gene expresses
assembly-activating protein (AAP), a previously unidentified non-structural protein that promotes capsid
assembly. Unexpectedly, studies on AAV capsid assembly from our group and others in the post-AAP
discovery era have convincingly demonstrated that the AAV capsid assembly process is not conserved among
different AAV serotypes, that is, knowledge of AAV capsid assembly built in the pre-AAP discovery era through
studies using AAV2 is not translatable to capsid assembly of other AAV serotypes. Our preliminary data has
challenged the long-standing dogma of AAV capsid assembly in the nucleolus and revealed significant
serotype-dependent heterogeneity in the capsid assembly process. In addition, there is a growing appreciation
for additional roles that AAP plays beyond promoting capsid assembly. Here, in order to advance our
understanding of AAV vectors and improve their effective utilization in gene therapy, we seek to thoroughly
understand the AAV capsid assembly process and the multifaceted roles of AAP in virion assembly of AAV
vectors, using robust approaches including high-throughput mutagenesis, directed evolution, barcoding,
proximity-based labeling based on BioID, and state-of-the-art bottom-up and top-down mass spectrometry.
Furthermore, we will explore various potential strategies to enhance the yield and quality of AAV vectors
through manipulation of various pathways we will identify in the project. Thus, the specific aims of this project
are: (Aim1) To comprehensively understand AAP biology in the process of virion assembly of AAV vectors that
varies across different AAV serotypes; (Aim 2) To identify host cell proteins involved in AAV capsid assembly;
and (Aim 3) To explore novel strategies to enhance the yield and quality of AAV vectors by manipulating the
process of virion assembly. Our project will not only address fundamental questions about the mechanism of
virion assembly of AAV vectors, but also substantially further our understanding of AAV-host interactions in
general. The project also has potential to discover novel strategies to improve vector production.

## Key facts

- **NIH application ID:** 10302279
- **Project number:** 5R01AI143667-03
- **Recipient organization:** OREGON HEALTH & SCIENCE UNIVERSITY
- **Principal Investigator:** Hiroyuki Nakai
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $729,095
- **Award type:** 5
- **Project period:** 2019-12-01 → 2024-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10302279

## Citation

> US National Institutes of Health, RePORTER application 10302279, Mechanistic Studies of AAP and Capsid Assembly of AAV Vectors (5R01AI143667-03). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10302279. Licensed CC0.

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