# In vivo PET imaging of novel engineered AAVs informs capsid design

> **NIH NIH R01** · STANFORD UNIVERSITY · 2022 · $685,392

## Abstract

Delivery of gene and protein therapy to the brain has traditionally been extremely limited. Using a directed
evolution approach to viral capsid engineering and selection, the Gradinaru group at Caltech identified specific
peptides that, when displayed on the surface of modified capsids, enhanced neuronal transduction compared
to the conventionally-used adeno-associated virus AAV9, following intravenous (IV) injection in mice. The brain
uptake of these novel AAVs studied here by PET imaging by the Ferrara and Gambhir groups at Stanford
reaches an extraordinary temporal-peak spatial-maximum of ~35% ID/cc at 4 h post-injection in mouse
models. Our quantitative analysis yields a 50-fold enhancement in the brain receptor affinities as compared
with earlier AAVs. Recent selections at Caltech have provided additional capsids that transduce the mouse
brain with reduced expression in the liver, spleen, kidneys, and lungs. However, strain and species differences
in the blood-brain barrier (BBB) and transport of these capsids (e.g. high in most mouse and rat strains but low
in BALB/c mice) raise questions as to the nature of transport in primates (including humans). In order to
understand species/strain differences and facilitate future translational development of these therapies, we
propose novel combined positron emission tomography (PET) imaging techniques that non-invasively assess
the pharmacokinetics of the AAV over the first days after injection and the resulting gene expression over
months or potentially years. We plan to address key issues by assessing receptor binding, transcytosis and
neutralizing antibody (NAb) effects across species. There are several innovative aspects to our approach.
First, the radioactive tag used for tracking the capsid is based on a multichelator, increasing the signal-to-noise
ratio and allowing us to assess binding to the brain endothelium (key for effective BBB crossing) over the first
minutes and hours after injection. Second, PET analysis of AAVs as nanometer-scale therapeutics allows us to
non-invasively estimate accumulation and clearance. Our data suggest receptor-mediated accumulation of the
engineered AAVs on the brain endothelium over the first few minutes after injection. Third, for real-time
reporting on gene transduction, we include a dual reporter system. The HSV1-sr39tk reporter gene has low
background in the peripheral tissues with the reporter probe [18F]FHBG. The pyruvate kinase M2 (PKM2)
reporter gene has a low background level in the brain and is imaged with [18F]DASA-23, a tracer that freely
crosses the BBB. For preliminary studies in rodents we add optical capsid tags and reporter genes to assess
cell-specific uptake. Our resulting specific aims are to: 1) validate and apply imaging to assess receptor binding
affinity, transcytosis, clearance and transduction across organ systems and 2) image new variants of AAV9
across species to gain insight into the impact of capsid structure. We hypothesize tha...

## Key facts

- **NIH application ID:** 10400047
- **Project number:** 5R01EB028646-04
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** Katherine W Ferrara
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $685,392
- **Award type:** 5
- **Project period:** 2019-08-01 → 2025-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10400047

## Citation

> US National Institutes of Health, RePORTER application 10400047, In vivo PET imaging of novel engineered AAVs informs capsid design (5R01EB028646-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10400047. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*