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

Award Number: R01FD007228
ORGANIZATION: FOOD AND DRUG ADMINISTRATION
OPDIV: FDA
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/10/2020
PERIOD OF PERFORMANCE END DATE: 08/31/2023

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


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2024 ( Subtotal = -$300 )
2024	2022	GENERAL ELECTRIC COMPANY	1 RESEARCH CIR	NISKAYUNA	NY	12309	SCHENECTADY	USA	Food and Drug Administration Research	000	3	4/27/2024	NON-COMPETING CONTINUATION	-$300
														Subtotal = -$300

Issue Date FY: 2022 ( Subtotal = $380,074 )
2022	2022	GENERAL ELECTRIC COMPANY	1 RESEARCH CIR	NISKAYUNA	NY	12309	SCHENECTADY	USA	Food and Drug Administration Research	000	3	8/31/2022	NON-COMPETING CONTINUATION	$380,074
														Subtotal = $380,074

Issue Date FY: 2021 ( Subtotal = $396,583 )
2021	2021	GENERAL ELECTRIC COMPANY	1 RESEARCH CIR	NISKAYUNA	NY	12309	SCHENECTADY	USA	Food and Drug Administration Research	001	1	4/2/2021	NEW	$373,923
2021	2021	GENERAL ELECTRIC COMPANY	1 RESEARCH CIR	NISKAYUNA	NY	12309	SCHENECTADY	USA	Food and Drug Administration Research	002	2	8/20/2021	NON-COMPETING CONTINUATION	$396,583
2021	2021	GENERAL ELECTRIC COMPANY	1 RESEARCH CIR	NISKAYUNA	NY	12309	SCHENECTADY	USA	Food and Drug Administration Research	000	1	3/29/2021	NEW	-$373,923
														Subtotal = $396,583

Issue Date FY: 2020 ( Subtotal = $373,923 )
2020	2020	GENERAL ELECTRIC COMPANY	1 RESEARCH CIR	NISKAYUNA	NY	12309	SCHENECTADY	USA	Food and Drug Administration Research	000	1	9/14/2020	NEW	$373,923
														Subtotal = $373,923

Grand Total All Awards = $1,150,280

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

Award Number: R01FD007228

ORGANIZATION: FOOD AND DRUG ADMINISTRATION

OPDIV: FDA

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 09/10/2020

PERIOD OF PERFORMANCE END DATE: 08/31/2023

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