Targeted adenovirus-mediated transduction of human T cells in vitro and in vivo

Freitag, Patrick C.; Kaulfuß, Meike; Flühler, Lea; Mietz, Juliane; Weiss, Fabian; Brücher, Dominik; Kolibius, Jonas; Hartmann, Karen Patricia; Smith, Sheena N.; Münz, Christian; Chijioke, Obinna; Plückthun, Andreas

doi:10.1016/j.omtm.2023.02.012

Cited by 7 publications

(9 citation statements)

References 52 publications

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“…4b). To achieve high transduction efficiency, we delivered the HIV-1 specific gRNAs through novel T cell specific CD3-CD28-IL2-retargeted Adenovirus (Ad) vector 20,28 . Ad-CRISPR-HIV1 containing the SpCas9 and both selected HIV-1-specific gRNAs, –TAR and –p24, were tested in comparison to Ad-CRISPR-Con, which contained the two random nucleotides gRNAs –Con1 and –Con2 tested previously (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Adenoviruses (Ads) containing either the CRISPR-HIV1, CRISPR-Con or iRFP reporter expression cassettes were preincubated with CD3-, CD28- and IL-2-retargeting adapters in a 32.5-fold total molar excess over adenoviral fiber knob for 1.5 h on ice in PBS (10.8-fold molar excess of each single adapter). Production and purification of adapters retargeting adapters was performed as previously described 20 . 1×10 5 activated primary CD4 + T cells were seeded in a 96-well plate and transduced with 2×10 4 VP (virus particles)/cell of retargeting adapter-coated Ad-iRFP, Ad-CRISPR-HIV1 or Ad-CRISPR-Con.…”

Section: Methodsmentioning

confidence: 99%

“…4b). To achieve high transduction efficiency, we delivered the HIV-1 specific gRNAs through novel T cell specific CD3-CD28-IL2-retargeted Adenovirus (Ad) vector 20,28 .…”

Section: Hiv-1 Inhibition By Grna-tar/p24 Delivered By Cd3-cd28-il2-r...mentioning

confidence: 99%

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Targeting HIV-1 with CRISPR/Cas9 delivered by retargeted adenoviruses effectively suppresses viral replication

Klinnert,

Freitag,

Plückthun

et al. 2023

Preprint

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Integrated, intact, latent HIV-1 viruses in infected cells are the main obstacle to curing HIV-1 infections. Targeted inactivation of HIV-1 proviruses with CRISPR/Cas9 is a promising strategy to eradicate HIV-1. In addition, CRISPR/Cas9 is able to target replicating HIV-1 and could be used as a therapy during productive infection.Here, we combine the CRISPR/Cas9 system with a novel adenovirus (Ad) targeted delivery technology to test it as a therapeutic approach to inhibit HIV-1. First, we selected six HIV-1-specific gRNAs targeting the HIV-1 LTRs and thegaggene and tested their efficacy in inhibiting HIV-1 virion production in an HEK 293T cell co-transfection screen. The gRNA-TAR showed the most robust and potent inhibition of HIV-1 by >99% alone or in combination with the gRNA-p24, which induced a ∼1 kb deletion between both gRNA target sites in HIV-1 DNA. Delivery of this dual gRNA-TAR/p24 CRISPR/Cas9 system with CD3-CD28-IL2-retargeted Ads was highly effective, transducing 62.3±23.3% of cells and suppressing HIV-1 replication by 88.0±4.5% in primary CD4+T cells from three independent donors.Our dual gRNA-TAR/p24-CRISPR/Cas9-Ad strategy represents a novel therapeutic approach to effectively inhibit HIV-1 in a highly HIV-1 and T cell-specific manner.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Targeting HIV-1 with CRISPR/Cas9 delivered by retargeted adenoviruses effectively suppresses viral replication

Klinnert,

Freitag,

Plückthun

et al. 2023

Preprint

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“…Adapter-mediated targeting of a specific cell type requires a surface marker that can be used to redirect the binding of an adenoviral vector [37][38][39][40][41][42] . It is necessary for this surface marker to be expressed at sufficient levels to make the cell susceptible for transduction.…”

Section: Dc-sign Was Identified As Best Target For Mono-adapters Medi...mentioning

confidence: 99%

“…Our lab developed a gene-delivery system based on HAdV5 that targets the vector to specific cell types while simultaneously inhibiting the natural tropism. 37 Our technology has allowed for direct transduction of T cells, 38 fibroblasts, 39 or cancer cells 19,40 in vivo, while drastically reducing the considerable viral tropism to the liver by using shielded vectors. 41 We have achieved this by using stable trimerized protein clamps that bind with high avidity to the HAdV5 knob, covering the CAR epitope and effectively reducing natural transduction via CAR.…”

Section: Introductionmentioning

confidence: 99%

Dendritic cell targeting in lymph nodes with engineered modular adapters improves HAdV5 and HC-HAdV5 tumor vaccination by co-secretion of IL-2v and IL-21

Weiss,

Kolibius,

Freitag

et al. 2024

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Adenoviral vectors demonstrate encouraging clinical outcomes for B- and T-cell vaccines. With such approaches, multiple payloads can be delivered, beyond the antigen itself. Nevertheless, the human adenoviral vector serotype C5 (HAdV5) exhibits limited transduction efficiency to dendritic cells (DC), therefore necessitating very high viral loads. Targeting antigen-presenting cells (APC) has remained challenging. To solve this problem, we developed a versatile platform that employs modular retargeting adapters to enhance transduction of specific cell types, including challenging host cells. By rational design, we constructed a dual-adapter for DC-SIGN and CD11c and demonstrate successful targeting of HAdV5 to human and murine DCs. Our in vivo characterization highlights improved and specific transduction of DCs in draining lymph nodes. Moreover, a tumor vaccination study showcases the advantageous co-expression of T cell stimulatory cytokines (IL-2v or IL-21) locally in lymph nodes alongside a potent tumor antigen. Lymph node-directed gene therapy at significantly reduced vector loads circumvents potential systemic toxicity of stimulating payloads. Our proposed low-dosage DC-targeted vaccine offers an effective solution for patients and also minimizes potential adenovirus-related side-effects. The robust immunogenicity of HC-HAdV5, with its large coding capacity (37 kbp DNA), opens up exciting possibilities for future therapeutic combination strategies.

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