TY - JOUR
T1 - CRISPR-Cas9 Mediated Exonic Disruption for HIV-1 Elimination
AU - Herskovitz, Jonathan
AU - Hasan, Mahmudul
AU - Patel, Milankumar
AU - Blomberg, Wilson R.
AU - Cohen, Jacob D.
AU - Machhi, Jatin
AU - Shahjin, Farah
AU - Mosley, R. Lee
AU - McMillan, Jo Ellyn
AU - Kevadiya, Bhavesh D.
AU - Gendelman, Howard E.
N1 - Funding Information:
The work was supported by the University of Nebraska Foundation, which includes donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship and individual donor support from the Frances and Louie Blumkin Foundation and from Harriet Singer. The research received support from National Institutes of Health grants T32 NS105594, 5R01MH121402, 1R01Al158160, R01 DA054535, PO1 DA028555, R01 NS126089, R01 NS36126, PO1 MH64570, P30 MH062261, and 2R01 NS034239. The authors thank Drs. Beat Bornhauser (U. of Zurich) and Won-Bin Young (U. of Pittsburgh) for kindly providing plasmids used in this project. We want to thank Edward Makarov and Dr. Santhi Gorantla for their help with the ddPCR. Some of the figure panels were made with BioRender.com. The following reagent was obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: NIH ARP 776, 349, 165, 11919. Gratitude is extended to Nicholas Conoan (UNMC Electron Microscopy Core) for his technical assistance. Sanger sequencing was performed with the help of UNMC Genomics Core through Ronald J. Redder. Genomics Core receives partial support from the National Institute for General Medical Science (NIGMS) INBRE- P20GM103427-19 grant as well as The Fred & Pamela Buffett Cancer Center Support Grant - P30 CA036727. This publication's contents are the sole responsibility of the authors and do not necessarily represent the official views of the NIH or NIGMS. We are also thankful to Evan A. Schroder for technical support. A final word of appreciation is directed to Daniel Chadash and the engineering team at Genome Compiler for making open access cloning software freely available online. DNA Sanger sequence chromatograms and spreadsheets used for Inference of CRISPR Edits v2 (ICEv2), along with algorithm results and raw flow cytometry data are uploaded to Mendeley Data repository are publicly accessible at doi:10.17632/phyy89w9c2.1.
Funding Information:
The work was supported by the University of Nebraska Foundation, which includes donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship and individual donor support from the Frances and Louie Blumkin Foundation and from Harriet Singer. The research received support from National Institutes of Health grants T32 NS105594, 5R01MH121402, 1R01Al158160, R01 DA054535, PO1 DA028555, R01 NS126089, R01 NS36126, PO1 MH64570, P30 MH062261, and 2R01 NS034239. The authors thank Drs. Beat Bornhauser (U. of Zurich) and Won-Bin Young (U. of Pittsburgh) for kindly providing plasmids used in this project. We want to thank Edward Makarov and Dr. Santhi Gorantla for their help with the ddPCR. Some of the figure panels were made with BioRender.com. The following reagent was obtained through the NIH HIV Reagent Program, Division of AIDS, NIAID, NIH: NIH ARP 776, 349, 165, 11919. Gratitude is extended to Nicholas Conoan (UNMC Electron Microscopy Core) for his technical assistance. Sanger sequencing was performed with the help of UNMC Genomics Core through Ronald J. Redder. Genomics Core receives partial support from the National Institute for General Medical Science (NIGMS) INBRE- P20GM103427-19 grant as well as The Fred & Pamela Buffett Cancer Center Support Grant - P30 CA036727. This publication's contents are the sole responsibility of the authors and do not necessarily represent the official views of the NIH or NIGMS. We are also thankful to Evan A. Schroder for technical support. A final word of appreciation is directed to Daniel Chadash and the engineering team at Genome Compiler for making open access cloning software freely available online.
Publisher Copyright:
© 2021 The Author(s)
PY - 2021/11
Y1 - 2021/11
N2 - Background: A barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high HIV-1 mutation rate leads to viral diversity, immune evasion, and consequent antiretroviral drug resistance. While CRISPR-spCas9 can eliminate latent proviral DNA, its efficacy is limited by HIV strain diversity and precision target cell delivery. Methods: A library of guide RNAs (gRNAs) designed to disrupt five HIV-1 exons (tat1-2/rev1-2/gp41) was constructed. The gRNAs were derived from a conseensus sequence of the transcriptional regulator tat from 4004 HIV-1 strains. Efficacy was affirmed by gRNA cell entry through transfection, electroporation, or by lentivirus or lipid nanoparticle (LNP) delivery. Treated cells were evaluated for viral excision by monitoring HIV-1 DNA, RNA, protein, and progeny virus levels. Findings: Virus was reduced in all transmitted founder strains by 82 and 94% after CRISPR TatDE transfection or lentivirus treatments, respectively. No recorded off-target cleavages were detected. Electroporation of TatDE ribonucleoprotein and delivery of LNP TatDE gRNA and spCas9 mRNA to latently infected cells resulted in up to 100% viral excision. Protection against HIV-1-challenge or induction of virus during latent infection, in primary or transformed CD4+ T cells or monocytes was achieved. We propose that multi-exon gRNA TatDE disruption delivered by LNPs enables translation for animal and human testing. Interpretation: These results provide “proof of concept’ for CRISPR gRNA treatments for HIV-1 elimination. The absence of full-length viral DNA by LNP delivery paired with undetectable off-target affirms the importance of payload delivery for effective viral gene editing. Funding: The work was supported by the University of Nebraska Foundation, including donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship, and individual donor support from the Frances and Louie Blumkin Foundation and from Harriet Singer. The research received support from National Institutes of Health grants T32 NS105594, 5R01MH121402, 1R01Al158160, R01 DA054535, PO1 DA028555, R01 NS126089, R01 NS36126, PO1 MH64570, P30 MH062261, and 2R01 NS034239.
AB - Background: A barrier to HIV-1 cure rests in the persistence of proviral DNA in infected CD4+ leukocytes. The high HIV-1 mutation rate leads to viral diversity, immune evasion, and consequent antiretroviral drug resistance. While CRISPR-spCas9 can eliminate latent proviral DNA, its efficacy is limited by HIV strain diversity and precision target cell delivery. Methods: A library of guide RNAs (gRNAs) designed to disrupt five HIV-1 exons (tat1-2/rev1-2/gp41) was constructed. The gRNAs were derived from a conseensus sequence of the transcriptional regulator tat from 4004 HIV-1 strains. Efficacy was affirmed by gRNA cell entry through transfection, electroporation, or by lentivirus or lipid nanoparticle (LNP) delivery. Treated cells were evaluated for viral excision by monitoring HIV-1 DNA, RNA, protein, and progeny virus levels. Findings: Virus was reduced in all transmitted founder strains by 82 and 94% after CRISPR TatDE transfection or lentivirus treatments, respectively. No recorded off-target cleavages were detected. Electroporation of TatDE ribonucleoprotein and delivery of LNP TatDE gRNA and spCas9 mRNA to latently infected cells resulted in up to 100% viral excision. Protection against HIV-1-challenge or induction of virus during latent infection, in primary or transformed CD4+ T cells or monocytes was achieved. We propose that multi-exon gRNA TatDE disruption delivered by LNPs enables translation for animal and human testing. Interpretation: These results provide “proof of concept’ for CRISPR gRNA treatments for HIV-1 elimination. The absence of full-length viral DNA by LNP delivery paired with undetectable off-target affirms the importance of payload delivery for effective viral gene editing. Funding: The work was supported by the University of Nebraska Foundation, including donations from the Carol Swarts, M.D. Emerging Neuroscience Research Laboratory, the Margaret R. Larson Professorship, and individual donor support from the Frances and Louie Blumkin Foundation and from Harriet Singer. The research received support from National Institutes of Health grants T32 NS105594, 5R01MH121402, 1R01Al158160, R01 DA054535, PO1 DA028555, R01 NS126089, R01 NS36126, PO1 MH64570, P30 MH062261, and 2R01 NS034239.
KW - CRISPR delivery
KW - Clustered regularly interspaced short palindromic repeat
KW - Human immunodeficiency virus type one
KW - Latent infection
KW - RNA loaded Lipid nanoparticles (rLNP)
KW - Viral eradication
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U2 - 10.1016/j.ebiom.2021.103678
DO - 10.1016/j.ebiom.2021.103678
M3 - Article
C2 - 34774454
AN - SCOPUS:85119412522
VL - 73
JO - EBioMedicine
JF - EBioMedicine
SN - 2352-3964
M1 - 103678
ER -