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The Impact of Nucleotide Modification Patterns on Therapeutic Small Interfering RNA Activity in the Central Nervous System

Award Number: F31NS122493
ORGANIZATION: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS
PERIOD OF PERFORMANCE START DATE: 12/19/2021
PERIOD OF PERFORMANCE END DATE: 09/18/2024

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The Impact of Nucleotide Modification Patterns on Therapeutic Small Interfering RNA Activity in the Central Nervous System - PROJECT SUMMARY Huntington’s Disease (HD) is caused by a CAG trinucleotide repeat expansion in exon 1 of the Huntingtin (HTT) gene that produces mutant HTT mRNA and protein. Expression of mutant HTT leads to progressive neurodegeneration via mechanisms that are poorly understood. Because HD is a genetically-defined disease, it is an ideal candidate for study and therapeutic intervention by small interfering RNAs (siRNAs) – which incorporate into the RNA-induced silencing complex (RISC) to target and degrade disease-causing genes. With the development of a divalent (di)-siRNA chemical architecture, siRNAs can be delivered throughout the central nervous system (CNS) of rodents and non-human primates. To ensure stability in the CNS, di-siRNAs require chemical modifications on every nucleotide. However, chemical modifications can affect siRNA activity and cellular localization – limiting the utility and flexibility of di-siRNAs in the CNS. The most common nucleotide modifications in siRNA replace the 2′OH of the ribose with 2′-Fluoro (2′F) or 2′-O- Methyl (2′OMe). Recent work suggests that incorporation of 2′OMe and 2′F at certain nucleotide positions may hinder siRNA loading into RISC or target binding/cleavage by RISC, and may alter the nuclear-to-cytoplasmic localization of siRNAs. Yet, the limited scope of this work has made it difficult to identify general design parameters for efficacious, compartment-specific siRNA. With guidance from Drs. Anastasia Khvorova (siRNA chemistry), Neil Aronin (HD), Phillip Zamore (RNA biochemistry) and Athma Pai (RNA sequencing), this proposal will systematically assess the impact of modification patterns on siRNA efficacy and cellular localization in the CNS to optimize siRNAs as an HD therapy and research tool for dissecting HD pathology. Aim 1 will characterize how siRNA chemical modifications impact RISC loading in vivo and target binding and cleavage in vitro. To measure how modifications alter RISC loading, a pool of differentially-modified siRNAs will be injected into the CNS of mice, RISC will be pulled down and loaded siRNAs will be sequenced. To determine the effect of siRNA chemical modifications on RISC-target interactions, target binding and cleavage kinetics will be measured for a panel of modified siRNAs using single-molecule total internal reflection fluorescence microscopy. Mechanistic insight into how modifications impact siRNA efficacy in the CNS will provide a framework with which to design optimized siRNAs to treat HD and other CNS diseases. Aim 2 will use the same modified siRNA pool from Aim 1 to identify optimal modification patterns for enhanced nuclear localization of siRNA in the CNS. These data will be used to design efficacious chemically-modified di-siRNAs targeting nuclear or cytoplasmic-only HTT RNA. These di-siRNA will then be injected into YAC128 HD mice and the effect on motor deficits, neurodegeneration, and striatal mRNA expression will be assessed. These results will provide valuable insight into the biology of HD and determine the potential of nuclear RNA-targeting siRNAs as a therapeutic paradigm for repeat expansion disorders with underlying RNA toxicity.


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: 2025 ( Subtotal = -$4,046 )
2025	2024	UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL	55 LAKE AVE N	WORCESTER	MA	01655	WORCESTER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	000	3	7/25/2025	NON-COMPETING CONTINUATION	-$4,046
														Subtotal = -$4,046

Issue Date FY: 2024 ( Subtotal = $26,672 )
2024	2024	UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL	55 LAKE AVE N	WORCESTER	MA	01655	WORCESTER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	000	3	12/20/2023	NON-COMPETING CONTINUATION	$25,662
2024	2024	UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL	55 LAKE AVE N	WORCESTER	MA	01655	WORCESTER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	001	3	5/7/2024	NON-COMPETING CONTINUATION	$1,010
2024	2024	UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL	55 LAKE AVE N	WORCESTER	MA	01655	WORCESTER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	002	3	6/10/2024	NON-COMPETING CONTINUATION	$0
														Subtotal = $26,672

Issue Date FY: 2023 ( Subtotal = $32,448 )
2023	2023	UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL	55 LAKE AVE N	WORCESTER	MA	01655	WORCESTER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	000	2	1/4/2023	NON-COMPETING CONTINUATION	$31,506
2023	2023	UNIVERSITY OF MASSACHUSETTS MEDICAL SCHOOL	55 LAKE AVE N	WORCESTER	MA	01655	WORCESTER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	001	2	2/8/2023	NON-COMPETING CONTINUATION	$942
														Subtotal = $32,448

Issue Date FY: 2022 ( Subtotal = $31,506 )
2022	2022	UNIVERSITY OF MASSACHUSETTS	55 LAKE AVE NORTH	WORCESTER	MA	01655	WORCESTER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	002	1	5/6/2022	NEW	$717
2022	2022	UNIVERSITY OF MASSACHUSETTS	55 LAKE AVE NORTH	WORCESTER	MA	01655	WORCESTER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	001	1	12/15/2021	NEW	$0
2022	2022	UNIVERSITY OF MASSACHUSETTS	55 LAKE AVE NORTH	WORCESTER	MA	01655	WORCESTER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	000	1	12/15/2021	NEW	$30,790
2022	2022	UNIVERSITY OF MASSACHUSETTS	55 LAKE AVE NORTH	WORCESTER	MA	01655	WORCESTER	USA	Extramural Research Programs in the Neurosciences and Neurological Disorders	003	1	6/10/2022	NEW	-$1
														Subtotal = $31,506

Grand Total All Awards = $86,580

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The Impact of Nucleotide Modification Patterns on Therapeutic Small Interfering RNA Activity in the Central Nervous System

Award Number: F31NS122493

ORGANIZATION: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: FELLOWSHIP/SCHOLARSHIP/STUDENT LOANS

PERIOD OF PERFORMANCE START DATE: 12/19/2021

PERIOD OF PERFORMANCE END DATE: 09/18/2024

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