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Development, Optimization and Preclinical Modeling of Hematopoietic Stem Cell Gene Editing for the Treatment of RAG1 Immunodeficiency

Award Number: R01AI155796
ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 06/08/2021
PERIOD OF PERFORMANCE END DATE: 05/31/2026

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Development, Optimization and Preclinical Modeling of Hematopoietic Stem Cell Gene Editing for the Treatment of RAG1 Immunodeficiency - PROJECT SUMMARY Hematopoietic Stem/Progenitor Cells (HSPC) gene therapy has provided clinical benefits in several patients affected by a variety of genetic diseases, some of which already reached market authorization for selected indications. However, the use of semi-randomly integrating vectors poses the risk of insertional mutagenesis and ectopic/unregulated transgene expression. These issues become even more relevant when the affected gene needs to be highly express to exert its function and when its activity directly impacts genome stability, such as the case for Recombination-Activating 1 (RAG1) gene. RAG1 is express in a high but tightly regulated manner in differentiating lymphocyte precursors, where it directs the VDJ recombination process required for assembly the T- and B-cell receptors, and its inactivating mutations are one of the most frequent causes of severe- combined immunodeficiency (SCID). While the high risk of genomic damage due to unregulated RAG1 expression has so far hampered the use of viral vectors to treat RAG1 deficiencies, there is a need to develop novel and effective therapeutic approaches, especially for patients who lacks a compatible HSPC donor or are not eligible for allogeneic transplant. The long-term goal of our proposal is to address this unmet medical need and develop an effective novel treatment directed at restoring both function and expression control of the RAG1 gene on autologous patient derived HSC. Our central hypothesis is that gene repair strategies that preserve physiologic expression control represent a safe and effective approach for treating RAG1 deficiencies. We reported that by tailoring culture conditions and gene delivery vehicles, it is possible to partially overcome the biologic barriers that constrain gene editing in the most primitive and clinically relevant HSPC subsets (Genovese, Nature 2014; Schiroli, Science-Translational-Medicine 2017). Within this project we will capitalize our previous achievements to i) directly fix RAG1 mutations, ii) improve efficiency of current HSPC gene editing protocols and iii) investigate non-genotoxic conditioning on suitable mouse models. Functional correction of the engineered RAG1 gene will be stringently assessed on patient derived cells, by exploiting state-of-the-art in vitro T cell differentiation assay and in vivo xenotransplantation experiments. We will take advantage of our recently optimized gene editing procedure and barcoding technology (BAR-seq, Ferrari et al, Nat. Biotech. 2020) to maximize editing efficiency while reducing cellular toxicity on the treated HSPC, thus increasing the yield of long- term engrafting lymphoid cells. To support the rational for clinical testing, we will assess correction of the disease phenotype by limiting amounts of functional HSPC in two RAG1 murine models and test efficacy of emerging immunotoxin conditioning regimens to reduce transplant toxicity and increase lymphoid reconstitution. Overall, this project will contribute to the development of an innovative treatment approach for RAG1 deficiencies and position homology-based gene editing as a standard for precise HSC engineering, providing for safer and more efficacious therapeutic strategies with broad applicability in hematology.


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 = $442,500 )
2025	2025	CHILDREN'S HOSPITAL CORPORATION, THE	300 LONGWOOD AVE	BOSTON	MA	02115	SUFFOLK	USA	Allergy and Infectious Diseases Research	000	6	5/26/2025	NON-COMPETING CONTINUATION	$442,500
														Subtotal = $442,500

Issue Date FY: 2024 ( Subtotal = $443,901 )
2024	2024	CHILDREN'S HOSPITAL CORPORATION, THE	300 LONGWOOD AVE	BOSTON	MA	02115	SUFFOLK	USA	Allergy and Infectious Diseases Research	004	5	6/4/2024	NON-COMPETING CONTINUATION	$442,500
2024	2023	DANA-FARBER CANCER INSTITUTE, INC.	450 BROOKLINE AVE	BOSTON	MA	02215	SUFFOLK	USA	Allergy and Infectious Diseases Research	002	3	2/9/2024	NON-COMPETING CONTINUATION	-$1
2024	2023	DANA-FARBER CANCER INSTITUTE, INC.	450 BROOKLINE AVE	BOSTON	MA	02215	SUFFOLK	USA	Allergy and Infectious Diseases Research	000	3	11/22/2023	NON-COMPETING CONTINUATION	-$441,099
2024	2023	CHILDREN'S HOSPITAL CORPORATION, THE	300 LONGWOOD AVE	BOSTON	MA	02115	SUFFOLK	USA	Allergy and Infectious Diseases Research	001	4	11/22/2023	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$442,500
2024	2023	CHILDREN'S HOSPITAL CORPORATION, THE	300 LONGWOOD AVE	BOSTON	MA	02115	SUFFOLK	USA	Allergy and Infectious Diseases Research	003	4	2/9/2024	CHANGE OF GRANTEE / TRAINING INSTITUTION / AWARDING INSTITUTION	$1
														Subtotal = $443,901

Issue Date FY: 2023 ( Subtotal = $441,100 )
2023	2023	DANA-FARBER CANCER INSTITUTE, INC.	450 BROOKLINE AVE	BOSTON	MA	02115	SUFFOLK	USA	Allergy and Infectious Diseases Research	000	3	5/17/2023	NON-COMPETING CONTINUATION	$441,100
														Subtotal = $441,100

Issue Date FY: 2022 ( Subtotal = $441,100 )
2022	2022	DANA-FARBER CANCER INSTITUTE, INC.	450 BROOKLINE AVE	BOSTON	MA	02115	SUFFOLK	USA	Allergy and Infectious Diseases Research	000	2	5/12/2022	NON-COMPETING CONTINUATION	$441,100
														Subtotal = $441,100

Issue Date FY: 2021 ( Subtotal = $441,100 )
2021	2021	DANA-FARBER CANCER INSTITUTE, INC.	450 BROOKLINE AVE	BOSTON	MA	02115	SUFFOLK	USA	Allergy and Infectious Diseases Research	000	1	6/8/2021	NEW	$441,100
														Subtotal = $441,100

Grand Total All Awards = $2,209,701

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Development, Optimization and Preclinical Modeling of Hematopoietic Stem Cell Gene Editing for the Treatment of RAG1 Immunodeficiency

Award Number: R01AI155796

ORGANIZATION: NATIONAL INSTITUTE OF ALLERGY & INFECTIOUS DISEASES

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

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

PERIOD OF PERFORMANCE START DATE: 06/08/2021

PERIOD OF PERFORMANCE END DATE: 05/31/2026

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