# Simplified and agile AAV production by integrating rapid DNA synthesis with continuous AAV collection

> **NIH FDA R01** · GENERAL ELECTRIC GLOBAL RESEARCH CTR · 2021 · $396,583

## Abstract

PROJECT SUMMARY/ABSTRACT
Adeno-associated virus (AAV) gene therapies for ultra-rare disorders utilize complex and expensive raw materi-
als and procedures that complicate clinical preparation. The high cost of existing AAV therapies (e.g., $850,000
for Luxterna) reflects the high costs of goods (e.g. DNA) and procedures used in the vector manufacturing pro-
cess. Solutions are needed that provide near-personalized scale-out, and agile AAV production for one to a few
patients at a time, at significantly reduced cost. Our objectives to reduce complexity and cost are three-fold:
integrating (1) rapidly-produced synthetic DNA as raw material for cell transfection, (2) a single-use static bio-
reactor that enables high cell density filterless perfusion and (3) continuous virus collection in which AAV is dis-
placed from the bioreactor during perfusion, and concentrated using affinity capture resins that bind AAV sur-
face proteins.
 First, we will leverage rolling circle amplification (RCA DNA) for synthetic DNA production to replace cur-
rent reliance on cGMP plasmids, accelerating DNA production time from >4 months to <4 days and reducing
DNA production costs ~10-fold by removing bacterial fermentation and downstream purification processes.
RCA DNA is produced cell-free and endotoxin-free, can produce gram-quantities of tandemly repeated DNA in a
day. We have demonstrated proof-of-concept for AAV production using triple transfection of RCA DNA into
HEK293 cells, resulting in titers comparable to standard plasmid transfection.
 Second, we will leverage a low shear filterless static perfusion bioreactor that supports up to 30x106 cells per
mL to develop high density transfection protocols that reduce both transfection reagent usage and cell culture
medium consumption during virus production for a further ~3 to 10-fold relative cost reduction.
 Third, we will design a continuous AAV production/collection protocol using the low shear filterless static
perfusion bioreactor. We will establish methods for high titer recovery of secreted AAV (post-transfection with
synthetic RCA DNA) via a continuous perfusion method that collects secreted virus while maintaining producer
cells in the bioreactor. The secreted AAV will be collected by affinity capture resins that bind AAV capsid pro-
teins.
 Our approach will enable continuous media harvest of AAV from high-density transfected cells by exploiting
the simplicity of RCA-based DNA production, minimizing media costs through high density cell culture, and uti-
lizing a continuous virus collection procedure that provides a higher cumulative AAV yield than obtained in cur-
rent batch-based lytic processes.

## Key facts

- **NIH application ID:** 10263265
- **Project number:** 5R01FD007228-02
- **Recipient organization:** GENERAL ELECTRIC GLOBAL RESEARCH CTR
- **Principal Investigator:** Brian Michael Davis
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** FDA
- **Fiscal year:** 2021
- **Award amount:** $396,583
- **Award type:** 5
- **Project period:** 2020-09-10 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10263265

## Citation

> US National Institutes of Health, RePORTER application 10263265, Simplified and agile AAV production by integrating rapid DNA synthesis with continuous AAV collection (5R01FD007228-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10263265. Licensed CC0.

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