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Using highly expressed circular RNAs to substantially enhance protein expression yields in mammalian cells

Award Number: R43GM139433
ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 08/01/2020
PERIOD OF PERFORMANCE END DATE: 04/30/2021

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Using highly expressed circular RNAs to substantially enhance protein expression yields in mammalian cells - SUMMARY Large-scale production of recombinant proteins in mammalian cells is the process for manufacturing protein biologics as medical therapies, for obtaining sufficient quantities of proteins for large-scale screens, and for creating protein bioconjugates. Many proteins must be made in mammalian cells (as opposed to bacteria) if the protein requires specific patterns of glycosylation, disulfide bond formation, or other modifications that can only be formed in mammalian cells. However, protein production in mammalian cells is much less efficient than protein production in bacterial cells. This is largely because the mRNA encoding the transgene is only a small fraction of the total cellular mRNA in mammalian cells. In contrast, in bacterial cells, the transgene mRNA can account for up to 50% of total cellular mRNA. In this application, we describe a strategy to increase transcript levels in mammalian cells by 50-100-fold compared to current levels. This quantum leap in transcript expression levels could fundamentally change protein production in mammalian cells. To do this, we will use the novel “Tornado” technology for expressing transgenes as circular RNAs. These circular RNAs are exceptionally stable and can accumulate to exceptionally high levels compared to corresponding linear mRNAs. Chimerna scientists have generated key proof-of-concept data demonstrating the ability to express RNA circles containing inserts similar in size to protein-coding open reading frames. In order to develop a fundamentally new way to express proteins in mammalian cells at high levels, the specific aims of this proposal are: (1) To optimize protein expression from Tornado-derived circular RNAs. Circular RNAs can encode proteins if they contain an internal ribosome entry site (IRES). The goal of this aim is to characterize the optimal IRES, insert size, and cell line suitable for protein translation using Tornado-expressed circular RNA. We will systematically test each of these features and characterize protein output. (2) To compare protein output from Tornado-derived circular RNA and linear mRNAs. In this aim, we will compare protein output for cytosolic proteins and secreted proteins. We will directly compare linear to Tornado-encoded circular RNA and determine if the high-level circular RNA production achieved using the Tornado system results in increased protein production in cultured cells. Together, these experiments will allow us to test the idea that genetically encoded circular RNAs can serve as a new platform for high-level protein expression in mammalian cells. This expression system could have a major effect on biomedical research and protein manufacturing by reducing costs for protein manufacturing, increasing protein yields and simplifying protein expression.


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: 2021 ( Subtotal = $0 )
2021	2020	CHIMERNA THERAPEUTICS, INC.	3960 BROADWAY	NEW YORK	NY	10032	NEW YORK	USA	Biomedical Research and Research Training	001	1	8/29/2021	NEW	$0
2021	2020	CHIMERNA THERAPEUTICS, INC.	3960 BROADWAY	NEW YORK	NY	10032	NEW YORK	USA	Biomedical Research and Research Training	000	1	7/19/2021	NEW	$0
														Subtotal = $0

Issue Date FY: 2020 ( Subtotal = $225,000 )
2020	2020	CHIMERNA THERAPEUTICS INC.	455 MAIN ST APT 9B	NEW YORK	NY	10044	NEW YORK	USA	Biomedical Research and Research Training	000	1	7/31/2020	NEW	$225,000
														Subtotal = $225,000

Grand Total All Awards = $225,000

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Using highly expressed circular RNAs to substantially enhance protein expression yields in mammalian cells

Award Number: R43GM139433

ORGANIZATION: NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 08/01/2020

PERIOD OF PERFORMANCE END DATE: 04/30/2021

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