CRISPR-Cas9 Mediated Exonic Disruption for HIV-1 Elimination.
CRISPR-Cas Systems
Cell Line
Conserved Sequence
Exons
Fluorescent Antibody Technique
Gene Editing
Gene Targeting
Genes, Reporter
Genetic Therapy
Genetic Vectors
/ administration & dosage
Genome, Viral
HIV Infections
/ therapy
HIV-1
/ genetics
Humans
Liposomes
Macrophages
/ metabolism
Nanoparticles
Proviruses
/ genetics
RNA Interference
RNA, Messenger
/ administration & dosage
rev Gene Products, Human Immunodeficiency Virus
/ genetics
tat Gene Products, Human Immunodeficiency Virus
/ genetics
RNA, Guide, CRISPR-Cas Systems
CRISPR delivery
Clustered regularly interspaced short palindromic repeat
Human immunodeficiency virus type one
Latent infection
RNA loaded Lipid nanoparticles (rLNP)
Viral eradication
Journal
EBioMedicine
ISSN: 2352-3964
Titre abrégé: EBioMedicine
Pays: Netherlands
ID NLM: 101647039
Informations de publication
Date de publication:
Nov 2021
Nov 2021
Historique:
received:
06
09
2021
revised:
04
10
2021
accepted:
22
10
2021
pubmed:
15
11
2021
medline:
3
3
2022
entrez:
14
11
2021
Statut:
ppublish
Résumé
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. A library of guide RNAs (gRNAs) designed to disrupt five HIV-1 exons (tat 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. 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. 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.
Sections du résumé
BACKGROUND
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
METHODS
A library of guide RNAs (gRNAs) designed to disrupt five HIV-1 exons (tat
FINDINGS
RESULTS
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
CONCLUSIONS
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
BACKGROUND
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.
Identifiants
pubmed: 34774454
pii: S2352-3964(21)00472-2
doi: 10.1016/j.ebiom.2021.103678
pmc: PMC8633974
pii:
doi:
Substances chimiques
Lipid Nanoparticles
0
Liposomes
0
RNA, Messenger
0
rev Gene Products, Human Immunodeficiency Virus
0
tat Gene Products, Human Immunodeficiency Virus
0
RNA, Guide, CRISPR-Cas Systems
0
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
103678Subventions
Organisme : NINDS NIH HHS
ID : R01 NS036126
Pays : United States
Organisme : NIGMS NIH HHS
ID : P20 GM103427
Pays : United States
Organisme : NIMH NIH HHS
ID : P30 MH062261
Pays : United States
Organisme : NIAID NIH HHS
ID : R01 AI158160
Pays : United States
Organisme : NIMH NIH HHS
ID : R01 MH121402
Pays : United States
Organisme : NCI NIH HHS
ID : P30 CA036727
Pays : United States
Organisme : NIMH NIH HHS
ID : P01 MH064570
Pays : United States
Organisme : NINDS NIH HHS
ID : R01 NS126089
Pays : United States
Organisme : NINDS NIH HHS
ID : R01 NS034239
Pays : United States
Organisme : NIDA NIH HHS
ID : P01 DA028555
Pays : United States
Organisme : NIDA NIH HHS
ID : R01 DA054535
Pays : United States
Organisme : NINDS NIH HHS
ID : T32 NS105594
Pays : United States
Organisme : NIGMS NIH HHS
ID : P30 GM106397
Pays : United States
Informations de copyright
Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.
Déclaration de conflit d'intérêts
Declaration of Competing Interest J.H., M.H., and H.E.G. are named inventors on provisional patents for the CRISPR therapy described in this report (62/985,392; 62/986,216). J.H., M.H., B.K., and H.E.G hold a patent on a virus-like particle-based delivery for HIV-1 CRISPR therapeutics (Docket No. 19040PCT; Serial No. PCT/US2020/016126; International Publication No. WO 2020/160418 A1). H.E.G is a member of the scientific advisory board at Longevity Biotech and a co-founder of Exavir Therapeutics, Inc.
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