Lectures and seminars OnkPat Friday Seminar: In vivo CRISPR engineering to reprogram human T cells

Host: Kasper Karlsson, Department of Oncology-Pathology.

Abstract

Engineered T cells, reprogrammed to express chimeric antigen receptors (CAR) or T cell receptors (TCR), have transformed cancer treatment and are being explored as therapeutics for autoimmune disorders and infectious diseases. Enhancing T cell function through genome editing—either by disrupting endogenous genes or precisely inserting DNA payloads—has shown significant promise. However, the ex vivo manufacturing process is lengthy, costly, and requires pre-conditioning, limiting the accessibility of these therapies. In vivo generation of CAR-T cells could overcome these barriers. In this presentation, I will demonstrate that stable and cell-specific transgene expression can be achieved through in vivo site-specific integration of large DNA payloads. We developed a two-vector system to deliver CRISPR-Cas9 ribonucleoproteins (RNPs) and a DNA donor template, using enveloped delivery vehicles (EDVs) and adeno-associated virus (AAVs), respectively. By integrating a CAR transgene into a T cell-specific locus we generated therapeutic levels of CAR-T cells in a humanized mouse model in vivo. These findings offer a promising pathway to more efficient, precise, and widely accessible T cell therapies.

Bio

William Nyberg, PhD, is a principal investigator at the Karolinska Institute since 2024. After finishing is PhD at the Karolinska Instiutute in 2019, he moved to the US and UCSF to conduct his postdoctoral training in cell therapy and genetical engineering. Building on scientific discoveries as a postdoc in Justin Eyquem’s research lab at UCSF, his lab focuses on reprogramming T cells in vivo using advanced genetical engineering for therapeutic purposes. This is done primarily with the use of CARs, but are also exploring the use of other synthetic receptors and therapeutic TCR sequences to develop new T cell therapies against cancers. In 2023, he was honored with the Excellence in Research Award by the ASGCT and joined the Karolinska Institute and Science for Life Laboratory in 2024 to pursue his research.

Relevant publications

In vivo engineering of murine T cells using the evolved adeno-associated virus variant Ark313.
Nyberg WA, Wang CH, Ark J, Liu C, Clouden S, Qualls A, Caryotakis S, Wells E, Simon K, Garza C, Bernard PL, Lopez-Ichikawa M, Li Z, Seo J, Kimmerly GR, Muldoon JJ, Chen PA, Li M, Liang HE, Kersten K, Rosales A, Kuhn N, Ye CJ, Gardner JM, Molofsky A, Ricardo-Gonzalez RR, Asokan A, Eyquem J
Immunity 2025 Feb;58(2):499-512.e7

An evolved AAV variant enables efficient genetic engineering of murine T cells.
Nyberg WA, Ark J, To A, Clouden S, Reeder G, Muldoon JJ, Chung JY, Xie WH, Allain V, Steinhart Z, Chang C, Talbot A, Kim S, Rosales A, Havlik LP, Pimentel H, Asokan A, Eyquem J
Cell 2023 Jan;186(2):446-460.e19

RASA2 ablation in T cells boosts antigen sensitivity and long-term function.
Carnevale J, Shifrut E, Kale N, Nyberg WA, Blaeschke F, Chen YY, Li Z, Bapat SP, Diolaiti ME, O'Leary P, Vedova S, Belk J, Daniel B, Roth TL, Bachl S, Anido AA, Prinzing B, Ibañez-Vega J, Lange S, Haydar D, Luetke-Eversloh M, Born-Bony M, Hegde B, Kogan S, Feuchtinger T, Okada H, Satpathy AT, Shannon K, Gottschalk S, Eyquem J, Krenciute G, Ashworth A, Marson A
Nature 2022 Sep;609(7925):174-182

SEED-Selection enables high-efficiency enrichment of primary T cells edited at multiple loci.
Chang CR, Vykunta VS, Lee JHJ, Li K, Kochendoerfer C, Muldoon JJ, Wang CH, Mazumder T, Sun Y, Goodman DB, Nyberg WA, Liu C, Allain V, Rothrock A, Ye CJ, Marson A, Shy BR, Eyquem J
Nat Biotechnol 2025 Feb;():