TY - JOUR
T1 - CRISPR editing of CCR5 and HIV-1 facilitates viral elimination in antiretroviral drug-suppressed virus-infected humanized mice
AU - Dash, Prasanta K.
AU - Chen, Chen
AU - Kaminski, Rafal
AU - Su, Hang
AU - Mancuso, Pietro
AU - Sillman, Brady
AU - Zhang, Chen
AU - Liao, Shuren
AU - Sravanam, Sruthi
AU - Liu, Hong
AU - Waight, Emiko
AU - Guo, Lili
AU - Mathews, Saumi
AU - Sariyer, Rahsan
AU - Mosley, R. Lee
AU - Poluektova, Larisa Y.
AU - Caocci, Maurizio
AU - Amini, Shohreh
AU - Gorantla, Santhi
AU - Burdo, Tricia H.
AU - Edagwa, Benson
AU - Gendelman, Howard E.
AU - Khalili, Kamel
N1 - Funding Information:
We acknowledge Dr. Siddappa Byrareddy of University of Nebraska Medical Center (UNMC) for providing the facility and support for the droplet digital PCR (ddPCR) assay and Dr. Jennifer Gordon of Excision BioTherapeutics for critical reading of the manuscript. We acknowledge Mahmudul Hasan and Milankumar Patel for providing sincere help in the preparation of the graphical abstract. This study was supported by University of Nebraska Foundation donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship, and the Frances and Louie Blumkin and Harriet Singer research endowments. The research received support from NIH grants T32NS105594, 5R01MH121402, 1R01Al158160, R01DA054535, R01NS126089, R01 AI145542, R01NS36126, R01MH115860, 1R33DA041018, and 2R01NS034239 (UNMC), and T32M-H079785, P30MH092177, and UM1AI164568 (Temple University Medical School). This publication’s contents are the sole responsibility of the authors as detailed. Temple University Medical School is responsible for the creation of the CRISPR constructs for both HIV-1 and CCR5, sequencing analysis and validation in cell culture, packaging in adeno-associated virus (AAV) vectors and transfer to UNMC for application to where the LASER ART formulations were created and applied, and the animal model studies, tissues harvesting, gene editing evaluations, and VOS performed. The studies related to viral gene editing and expression were conducted in a blinded manner. The mouse breeding, the humanization of the mice, the flow cytometric monitoring of the generated human cell immunocytes, the HIV-1 infection and virological monitoring, the development and LASER ART characterization, the monitoring of posttreatment viral suppression, the checks of animal well being and antiretroviral drug levels, the protocol generation for the experiments and the Institutional Animal Care and Use Committee procedures used for each of the animal studies that included detection of viral DNA and RNA by nested real-time qPCR, ddPCR, and RNAscope together with the in vivo viral outgrowth assay were performed at UNMC.
Funding Information:
ACKNOWLEDGMENTS. We acknowledge Dr. Siddappa Byrareddy of University of Nebraska Medical Center (UNMC) for providing the facility and support for the droplet digital PCR (ddPCR) assay and Dr. Jennifer Gordon of Excision BioTherapeutics for critical reading of the manuscript. We acknowledge Mahmudul Hasan and Milankumar Patel for providing sincere help in the preparation of the graphical abstract. This study was supported by University of Nebraska Foundation donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship, and the Frances and Louie Blumkin and Harriet Singer research endowments. The research received support from NIH grants T32NS105594, 5R01MH121402, 1R01Al158160, R01DA054535, R01NS126089, R01 AI145542, R01NS36126, R01MH115860, 1R33DA041018, and 2R01NS034239 (UNMC), and T32MH079785, P30MH092177, and UM1AI164568 (Temple University Medical School). This publication’s contents are the sole responsibility of the authors as detailed. Temple University Medical School is responsible for the creation of the CRISPR constructs for both HIV-1 and CCR5, sequencing analysis and validation in cell culture, packaging in adeno-associated virus (AAV) vectors and transfer to UNMC for application to where the LASER ART formulations were created and applied, and the animal model studies, tissues harvesting, gene editing evaluations, and VOS performed. The studies related to viral gene editing and expression were conducted in a blinded manner.The mouse breeding, the humanization of the mice, the flow cytometric monitoring of the generated human cell immunocytes, the HIV-1 infection and virological monitoring, the development and LASER ART characterization, the monitoring of posttreatment viral suppression, the checks of animal well being and antiretroviral drug levels, the protocol generation for the experiments and the Institutional Animal Care and Use Committee procedures used for each of the animal studies that included detection of viral DNA and RNA by nested real-time qPCR, ddPCR, and RNAscope together with the in vivo viral outgrowth assay were performed at UNMC.
Publisher Copyright:
Copyright © 2023 the Author(s). Published by PNAS.
PY - 2023/5/9
Y1 - 2023/5/9
N2 - Treatment of HIV-1ADA-infected CD34+ NSG-humanized mice with long-acting ester prodrugs of cabotegravir, lamivudine, and abacavir in combination with native rilpivirine was followed by dual CRISPR-Cas9 C-C chemokine receptor type five (CCR5) and HIV-1 proviral DNA gene editing. This led to sequential viral suppression, restoration of absolute human CD4+ T cell numbers, then elimination of replication-competent virus in 58% of infected mice. Dual CRISPR therapies enabled the excision of integrated proviral DNA in infected human cells contained within live infected animals. Highly sensitive nucleic acid nested and droplet digital PCR, RNAscope, and viral outgrowth assays affirmed viral elimination. HIV-1 was not detected in the blood, spleen, lung, kidney, liver, gut, bone marrow, and brain of virus-free animals. Progeny virus from adoptively transferred and CRISPR-treated virus-free mice was neither detected nor recovered. Residual HIV-1 DNA fragments were easily seen in untreated and viral-rebounded animals. No evidence of off-target toxicities was recorded in any of the treated animals. Importantly, the dual CRISPR therapy demonstrated statistically significant improvements in HIV-1 cure percentages compared to single treatments. Taken together, these observations underscore a pivotal role of combinatorial CRISPR gene editing in achieving the elimination of HIV-1 infection.
AB - Treatment of HIV-1ADA-infected CD34+ NSG-humanized mice with long-acting ester prodrugs of cabotegravir, lamivudine, and abacavir in combination with native rilpivirine was followed by dual CRISPR-Cas9 C-C chemokine receptor type five (CCR5) and HIV-1 proviral DNA gene editing. This led to sequential viral suppression, restoration of absolute human CD4+ T cell numbers, then elimination of replication-competent virus in 58% of infected mice. Dual CRISPR therapies enabled the excision of integrated proviral DNA in infected human cells contained within live infected animals. Highly sensitive nucleic acid nested and droplet digital PCR, RNAscope, and viral outgrowth assays affirmed viral elimination. HIV-1 was not detected in the blood, spleen, lung, kidney, liver, gut, bone marrow, and brain of virus-free animals. Progeny virus from adoptively transferred and CRISPR-treated virus-free mice was neither detected nor recovered. Residual HIV-1 DNA fragments were easily seen in untreated and viral-rebounded animals. No evidence of off-target toxicities was recorded in any of the treated animals. Importantly, the dual CRISPR therapy demonstrated statistically significant improvements in HIV-1 cure percentages compared to single treatments. Taken together, these observations underscore a pivotal role of combinatorial CRISPR gene editing in achieving the elimination of HIV-1 infection.
KW - CCR5 targeting
KW - CRISPR-Cas9
KW - HIV-1
KW - humanized mice
KW - long-acting ART
UR - http://www.scopus.com/inward/record.url?scp=85158002311&partnerID=8YFLogxK
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U2 - 10.1073/pnas.2217887120
DO - 10.1073/pnas.2217887120
M3 - Article
C2 - 37126704
AN - SCOPUS:85158002311
SN - 0027-8424
VL - 120
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 19
M1 - e2217887120
ER -