Abstract
Disruption of CCR5 or CXCR4, the main human immunodeficiency virus type 1 (HIV-1) co-receptors, has been shown to protect primary human CD4+ T cells from HIV-1 infection. Base editing can install targeted point mutations in cellular genomes, and can thus efficiently inactivate genes by introducing stop codons or eliminating start codons without double-stranded DNA break formation. Here, we applied base editors for individual and simultaneous disruption of both co-receptors in primary human CD4+ T cells. Using cytosine base editors we observed premature stop codon introduction in up to 89% of sequenced CCR5 or CXCR4 alleles. Using adenine base editors we eliminated the start codon in CCR5 in up to 95% of primary human CD4+ T cell and up to 88% of CD34+ hematopoietic stem and progenitor cell target alleles. Genome-wide specificity analysis revealed low numbers of off-target mutations that were introduced by base editing, located predominantly in intergenic or intronic regions. We show that our editing strategies prevent transduction with CCR5-tropic and CXCR4-tropic viral vectors in up to 79% and 88% of human CD4+ T cells, respectively. The engineered T cells maintained functionality and overall our results demonstrate the effectiveness of base-editing strategies for efficient and specific ablation of HIV co-receptors in clinically relevant cell types.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 130-144 |
| Number of pages | 15 |
| Journal | Molecular Therapy |
| Volume | 30 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 5 2022 |
Bibliographical note
Funding Information:This work was supported by the Bill and Melinda Gates foundation (M.J.O. and D.R.L.). M.J.O is also supported by the Saint Baldrick's Foundation, Kidz1st Fund, and the Chambers Family Innovation Fund. D.R.L. and G.A.N. are funded by U01 AI142756, RM1 HG009490, R01 EB022376, R35 GM118062, the Howard Hughes Medical Institute, and the St. Jude Collaborative Research Consortium. G.A.N. is supported by a Helen Hay Whitney Postdoctoral Fellowship. J.T. receives support from the Edmund Wallace Tulloch and Anna Marie Tulloch Chair in Stem Cell Biology, Genetics and Genomics. E.V. C.C. and A.G. are supported by the grant Agence Nationale de la Recherche (AAV-Chem) and the EuroNanoMed grant CELLUX. E.V. also receives research funding (CRISPR screen Action) from the Canceropôle Provence-Alpes-côte d'Azur, the French National Cancer Institute (INCa) and the Provence -Alpes-côte d'Azur Region. T.I.C. receives sponsored research support from Cellectis S.A. (ZVS20170614, ZVK20180202, ZVK2019031801) and Miltenyi Biotec (LV4502282793). C.M. is supported by the European Union's Horizon 2020 research and innovation program under the Marie Skłodowska–Curie grant agreement no. 765269. We gratefully acknowledge Shengdar Tsai and Cicera Lazzarotto from St. Jude Children's Research Hospital for advice regarding CIRCLE-seq parameters, as well as Jordana Henderson from Trilink BioTechnologies for mRNA synthesis. F.K. performed cell experiments, flow cytometry, targeted sequencing experiments, and analysis. G.A.N. performed CIRCLE-seq experiments, produced Cas9 protein variants, and optimized editor mRNAs. F.K. and G.A.N. analyzed CIRCLE-seq and sequencing data. C.R.E. A.N.McE. S.C.N. B.S. S.P.B. and C.J.F. performed and analyzed HSC and CFU assays and qRT-PCR. Y.F. C.M. K.S. K.L.H. and B.R.B. performed and analyzed cell activation studies and T.I.C. produced and titrated CCR5-tropic pseudotyped viral vector. E.V. C.C. and A.G.-G. produced and titrated CXCR4-tropic pseudotyped viral vector. A.McC. advised on design of mRNA constructs. F.K. G.A.N. and M.J.O. designed the research. M.J.O. and D.R.L. supervised the research. F.K. and M.J.O. wrote the manuscript with input from all authors. D.R.L. M.J.O. and J.T. provided funding support. D.R.L. is a consultant and equity owner of Beam Therapeutics, Prime Medicine, and Pairwise Plants, companies that use genome editing. D.R.L. and G.A.N. have filed patent applications relating to the use of genome editors. T.I.C. has sponsored research collaboration with Cellectis and Miltenyi Biotec. The remaining authors declare no competing interests. A.M. is a scientific advisory board member at mCureX Therapeutics, Inc. an mRNA company and a consultant for TriLink BioTechnologies.
Funding Information:
This work was supported by the Bill and Melinda Gates foundation (M.J.O. and D.R.L.). M.J.O is also supported by the Saint Baldrick’s Foundation, Kidz1st Fund, and the Chambers Family Innovation Fund . D.R.L. and G.A.N. are funded by U01 AI142756 , RM1 HG009490 , R01 EB022376 , R35 GM118062 , the Howard Hughes Medical Institute , and the St. Jude Collaborative Research Consortium. G.A.N. is supported by a Helen Hay Whitney Postdoctoral Fellowship . J.T. receives support from the Edmund Wallace Tulloch and Anna Marie Tulloch Chair in Stem Cell Biology , Genetics and Genomics. E.V., C.C., and A.G. are supported by the grant Agence Nationale de la Recherche (AAV-Chem) and the EuroNanoMed grant CELLUX. E.V. also receives research funding (CRISPR screen Action) from the Canceropôle Provence-Alpes-côte d’Azur, the French National Cancer Institute (INCa) and the Provence -Alpes-côte d’Azur Region. T.I.C. receives sponsored research support from Cellectis S.A. ( ZVS20170614 , ZVK20180202 , ZVK2019031801 ) and Miltenyi Biotec ( LV4502282793 ). C.M. is supported by the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska–Curie grant agreement no. 765269 . We gratefully acknowledge Shengdar Tsai and Cicera Lazzarotto from St. Jude Children’s Research Hospital for advice regarding CIRCLE-seq parameters, as well as Jordana Henderson from Trilink BioTechnologies for mRNA synthesis.
Publisher Copyright:
© 2021 The American Society of Gene and Cell Therapy
Keywords
- Base editing
- CCR5
- CRISPR/Cas9
- CXCR4
- HIV
- HSC
PubMed: MeSH publication types
- Journal Article
- Research Support, N.I.H., Extramural
- Research Support, Non-U.S. Gov't
