An evolved AAV variant enables efficient genetic engineering of murine T cells

Nyberg, William A.; Ark, Jonathan; To, Angela; Clouden, Sylvanie; Reeder, Gabriella C.; Muldoon, Joseph J.; Chung, Jing-Yi; Xie, William Haowei; Allain, Vincent; Steinhart, Zachary; Chang, Christopher; Talbot, Alexis; Kim, Sandy; Rosales, Alan; Havlik, L. Patrick; Pimentel, Harold; Asokan, Aravind; Eyquem, Justin

doi:10.1016/j.cell.2022.12.022

Cited by 29 publications

(12 citation statements)

References 54 publications

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“…2, B and C), reaching high knock-in repair efficiencies of greater than 50% as measured by mCherry reporter expression (table S1). Our results are in line with a recent report about high knock-in efficiency in mouse T cells using related technology ( 33 ). Sorted, repaired CD8 + mCherry + T cells expressed Prf1 in response to activation, whereas mock-repaired T cells carrying a stop codon in exon 3 showed similar knock-in efficiencies but did not express Prf1 (Fig.…”

Section: Resultssupporting

confidence: 93%

Precise CRISPR-Cas9 gene repair in autologous memory T cells to treat familial hemophagocytic lymphohistiocytosis

Li,

Wirtz,

Weber

et al. 2024

Sci. Immunol.

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Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, often fatal immune deficiency characterized by severe systemic hyperinflammation. Although allogeneic bone marrow transplantation can be curative, more effective therapies are urgently needed. FHL is caused by inactivating mutations in proteins that regulate cellular immunity. Here, we used an adeno-associated virus–based CRISPR-Cas9 system with an inhibitor of nonhomologous end joining to repair such mutations in potentially long-lived T cells ex vivo. Repaired CD8 memory T cells efficiently cured lethal hyperinflammation in a mouse model of Epstein-Barr virus–triggered FHL2, a subtype caused by perforin-1 ( Prf1 ) deficiency. Furthermore, repair of PRF1 and Munc13-4 ( UNC13D )—whose deficiency causes the FHL subtype FHL3—in mutant memory T cells from two critically ill patients with FHL restored T cell cytotoxicity. These results provide a starting point for the treatment of genetic T cell immune dysregulation syndromes with repaired autologous T cells.

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Section: Resultssupporting

confidence: 93%

Precise CRISPR-Cas9 gene repair in autologous memory T cells to treat familial hemophagocytic lymphohistiocytosis

Li,

Wirtz,

Weber

et al. 2024

Sci. Immunol.

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“…1d ). Compared with non-targeting retroviral vectors, precise knock-in at the TRAC locus has been demonstrated to improve CAR-T cell anti-tumour potency 2 , 29 . At 6 days after treatment, we observed that the frequency of edited cells was modestly higher with electroporation than with PERC (Fig.…”

Section: Resultsmentioning

confidence: 99%

Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes

et al. 2023

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CRISPR-mediated genome editing of primary human lymphocytes is typically carried out via electroporation, which can be cytotoxic, cumbersome and costly. Here we show that the yields of edited primary human lymphocytes can be increased substantially by delivering a CRISPR ribonucleoprotein mixed with an amphiphilic peptide identified through screening. We evaluated the performance of this simple delivery method by knocking out genes in T cells, B cells and natural killer cells via the delivery of Cas9 or Cas12a ribonucleoproteins or an adenine base editor. We also show that peptide-mediated ribonucleoprotein delivery paired with an adeno-associated-virus-mediated homology-directed repair template can introduce a chimaeric antigen receptor gene at the T-cell receptor α constant locus, and that the engineered cells display antitumour potency in mice. The method is minimally perturbative, does not require dedicated hardware, and is compatible with multiplexed editing via sequential delivery, which minimizes the risk of genotoxicity. The peptide-mediated intracellular delivery of ribonucleoproteins may facilitate the manufacturing of engineered T cells.

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“…Likewise, the in vivo optogenetic model presented in this paper depends on homeostatic proliferation to reconstitute the T lymphocyte population in TCRb knock out recipients. While reliance on homeostatic proliferation can be a drawback when investigating immune responses in vivo, adoptive transfer of MSCV-BLU-VIPR-transduced TCR transgenic, or Chimeric Antigen Receptor (CAR), T lymphocytes should circumvent this limitation [17][18][19] . Ultimately, BLU-VIPR enables the transformation of an off-the-shelf transgenic Cas model into an optogenetic gene perturbation-ready model.…”

Section: Discussionmentioning

confidence: 99%

Light induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo

Pulgarin,

Pelo,

Ferrandiz

et al. 2023

Preprint

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There is currently a lack of tools capable of perturbing genes in both a precise and spatiotemporal fashion. CRISPR’s ease of use and flexibility, coupled with light’s unparalleled spatiotemporal resolution deliverable from a controllable source, makes optogenetic CRISPR a well-suited solution for precise spatiotemporal gene perturbations. Here we present a new optogenetic CRISPR tool, BLU-VIPR, that diverges from prevailing split-Cas design strategies and instead focuses on optogenetic regulation of gRNA production. This simplifies spatiotemporal gene perturbation and works in vivo with cells previously intractable to optogenetic gene editing. We engineered BLU-VIPR around a new potent blue-light activated transcription factor and ribozyme-flanked gRNA. The BLU-VIPR design is genetically encoded and ensures precise excision of multiple gRNAs from a single mRNA transcript, allowing for optogenetic gene editing in T lymphocytes in vivo.

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An evolved AAV variant enables efficient genetic engineering of murine T cells

Cited by 29 publications

References 54 publications

Precise CRISPR-Cas9 gene repair in autologous memory T cells to treat familial hemophagocytic lymphohistiocytosis

Precise CRISPR-Cas9 gene repair in autologous memory T cells to treat familial hemophagocytic lymphohistiocytosis

Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes

Light induced expression of gRNA allows for optogenetic gene editing of T lymphocytes in vivo

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