Disruption of HIV-1 co-receptors CCR5 and CXCR4 in primary human T cells and hematopoietic stem and progenitor cells using base editing

Knipping, Friederike; Newby, Gregory A.; Eide, Cindy R.; McElroy, Amber N.; Nielsen, Sarah C.; Smith, Kyle D.; Fang, Yongxing; Cornu, Tatjana I.; Costa, Caroline; Gutiérrez‐Guerrero, Alejandra; Bingea, Samuel P.; Feser, Colby J.; Steinbeck, Benjamin; Hippen, Keli L.; Blazar, Bruce R.; McCaffrey, Anton P.; Mussolino, Claudio; Verhoeyen, Els; Tolar, Jakub; Liu, David R.; Osborn, Mark J.

doi:10.1016/j.ymthe.2021.10.026

Cited by 25 publications

(19 citation statements)

References 73 publications

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“…This exploration identifies key limitations and drivers of functional EV-mediated delivery, including a potential mechanism of receptor binding-mediated delivery enhancement to T cells. We validate these technologies by demonstrating a therapeutically relevant capability—delivering Cas9 ribonucleoprotein complex (RNP) to ablate the gene encoding the HIV co-receptor CXCR4 27,28 in primary human CD4 + T cells.…”

Section: Main Textmentioning

confidence: 91%

Bioengineering multifunctional extracellular vesicles for targeted delivery of biologics to T cells

Stranford

Simons

Berman

et al. 2022

Preprint

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Genetically modifying T cells can enable applications ranging from cancer immunotherapy to HIV treatment, yet delivery of T cell-targeted therapeutics remains challenging. Extracellular vesicles (EVs) are nanoscale particles secreted by all cells that naturally encapsulate and transfer proteins and nucleic acids, making them an attractive and clinically-relevant platform for engineering biocompatible delivery vehicles. We report a suite of technologies for genetically engineering cells to produce multifunctional EV vehicles—without employing chemical modifications that complicate biomanufacturing. We display high affinity targeting domains on the EV surface to achieve specific, efficient binding to T cells, identify a protein tag to confer active cargo loading into EVs, and display fusogenic glycoproteins to increase EV uptake and fusion with recipient cells. We demonstrate integration of these technologies by delivering Cas9-sgRNA complexes to edit primary human T cells. These approaches could enable targeting vesicles to a range of cells for the efficient delivery of cargo.

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Section: Main Textmentioning

confidence: 91%

Bioengineering multifunctional extracellular vesicles for targeted delivery of biologics to T cells

Stranford

Simons

Berman

et al. 2022

Preprint

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“…Each method has advantages and disadvantages, and depending on the intended application and target cell type, the right delivery method should be selected. These methods can be based on viral delivery [ 51 , 52 , 53 , 54 ] or lipid vector delivery of the CRISPR/Cas9 system [ 55 ], but genome editing can also be facilitated through fusion of the Cas9 protein to cell-penetrating peptides [ 56 ], or by directly electroporating the CRISPR/Cas9 system into cells of interest [ 27 , 55 , 57 , 58 ]. Interestingly, recently, the mechanism HIV-1 employs to target CD4 + T cells has been repurposed as a tool to direct viral particles containing the Cas9 system specifically to cells expressing CD4, i.e., T cells [ 59 ], opening up new potential avenues with this addition to the CRISPR/Cas9 toolbox [ 60 ], e.g., in vivo gene editing of CD4 + T cells.…”

Section: Gene Editing Basics—mode Of Action and Different Toolsmentioning

confidence: 99%

“…Nonetheless, it was shown that dual knockout of CXCR4 and CCR5 is feasible in primary human CD4 + T cells [ 54 , 98 ]. Furthermore, knocking out both chemokine receptors did not impact survival and proliferation [ 54 , 98 ], and allowed T cells to retain their in vitro cytokine production [ 27 ]. Importantly, double knockout also conferred in vitro resistance to HIV infection with both CXCR4- and CCR5-tropic HIV strains [ 27 , 54 , 98 ].…”

Section: Simultaneous Deletion Of Ccr5 and Cxcr4 Via Crispr/cas9-medi...mentioning

confidence: 99%

“…One way of increasing the donor pool would be through genetic modification. Several gene editing tools have been employed in the battle against HIV, including Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system [ 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ]. While each tool has their advantages and disadvantages, the main benefit of using the CRISPR/Cas system is the limited off-target effects [ 28 , 29 ] and broad genome targeting capacity [ 30 ].…”

Section: Introductionmentioning

confidence: 99%

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Closing the Door with CRISPR: Genome Editing of CCR5 and CXCR4 as a Potential Curative Solution for HIV

Heeren¹

2022

BioTech

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Human immunodeficiency virus (HIV) infection can be controlled by anti-retroviral therapy. Suppressing viral replication relies on life-long medication, but anti-retroviral therapy is not without risks to the patient. Therefore, it is important that permanent cures for HIV infection are developed. Three patients have been described to be completely cured from HIV infection in recent years. In all cases, patients received a hematopoietic stem cell (HSC) transplantation due to a hematological malignancy. The HSCs were sourced from autologous donors that expressed a homozygous mutation in the CCR5 gene. This mutation results in a non-functional receptor, and confers resistance to CCR5-tropic HIV strains that rely on CCR5 to enter host cells. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one of the methods of choice for gene editing, and the CRISPR/Cas system has been employed to target loci of interest in the context of HIV. Here, the current literature regarding CRISPR-mediated genome editing to render cells resistant to HIV (re)-infection by knocking out the co-receptors CCR5 and CXCR4 is summarized, and an outlook is provided regarding future (research) directions.

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“…Using cytosine base editors, they introduced premature stop codons in the CCR5 and CXCR4 genes and mutated the CCR5 start codon using adenine base editors. The efficiency of edits detected in primary CD4 + T cells and HSPCs was high, around 90%, albeit biallelic KO was lower ( Knipping et al., 2022 ). Unlike NHEJ-mediated gene KO, base editing produces much fewer off-target mutations because of the lack of dsDNA breaks, offering precise DNA modification as in HDR-related editing, and, therefore, can be considered a promising technology.…”

Section: Perspectives In Crispr/cas Anti-hiv Therapy and Concluding R...mentioning

confidence: 99%

Application of CRISPR/Cas Genomic Editing Tools for HIV Therapy: Toward Precise Modifications and Multilevel Protection

Maslennikova

Mazurov

2022

Front. Cell. Infect. Microbiol.

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Although highly active antiretroviral therapy (HAART) can robustly control human immunodeficiency virus (HIV) infection, the existence of latent HIV in a form of proviral DNA integrated into the host genome makes the virus insensitive to HAART. This requires patients to adhere to HAART for a lifetime, often leading to drug toxicity or viral resistance to therapy. Current genome-editing technologies offer different strategies to reduce the latent HIV reservoir in the body. In this review, we systematize the research on CRISPR/Cas-based anti-HIV therapeutic methods, discuss problems related to viral escape and gene editing, and try to focus on the technologies that effectively and precisely introduce genetic modifications and confer strong resistance to HIV infection. Particularly, knock-in (KI) approaches, such as mature B cells engineered to produce broadly neutralizing antibodies, T cells expressing fusion inhibitory peptides in the context of inactivated viral coreceptors, or provirus excision using base editors, look very promising. Current and future advancements in the precision of CRISPR/Cas editing and its delivery will help extend its applicability to clinical HIV therapy.

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Disruption of HIV-1 co-receptors CCR5 and CXCR4 in primary human T cells and hematopoietic stem and progenitor cells using base editing

Cited by 25 publications

References 73 publications

Bioengineering multifunctional extracellular vesicles for targeted delivery of biologics to T cells

Bioengineering multifunctional extracellular vesicles for targeted delivery of biologics to T cells

Closing the Door with CRISPR: Genome Editing of CCR5 and CXCR4 as a Potential Curative Solution for HIV

Application of CRISPR/Cas Genomic Editing Tools for HIV Therapy: Toward Precise Modifications and Multilevel Protection

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