9/ Importantly, our optimized protocol did not lead to an increase of chromosomal rearrangements or loss of heterozygosity.

8/ SEED process is highly scalable! We demonstrated that it can be used in clinical-scale manufacturing, generating >2.5 billion fully edited T cells at two loci in just 7 days!

7/ We next wanted to fully reprogram CD4 T cells for TCR specificity, co-receptor swapping and MHC-I with six simultaneous modifications (3 KO & 3 KI). We achieved robust triple KI efficiency (>20%), which was enriched to >90% after SEED.

6/ We also tackled TCR mispairing, a major challenge in engineered TCR therapies.
➡️By integrating the epitope-edited TCR, we can identify and eliminate mispaired TCRs and SEED select for cells with proper receptor expression and function!

5/ To use SEED on TCR-based receptors (rTCR or HIT), we generated an epitope edited TCR. Using a pool KI screening technology, we identified TCR beta constant chain variants (here D112K) that evade GMP anti-TCR antibodies and enables TCR engineered T cells to be enriched to >95%.

4/ We show that SEED can be multiplexed, enriching for more than 90% purity of double CAR / CD47 KI T cells in a single negative selection step.

3/ SEED-Selection achieves >95% purity for single modifications. We show we can easily enrich for TRAC-targeted CAR T cells or B2M-targeted CD47 positive T cells through negative selection.

2/ How does it work? We developed a novel class of repair templates called Synthetic Exon Expression Disruptors (SEEDs). SEEDs link successful transgene integration to the disruption of an endogenous surface protein, allowing for easy negative selection.

7/ Finally, unlike many AAVs, we found that Ark313 is significantly de-targeted from both the liver and macrophages, reducing off-target effects.

6/ Achieving site-specific integration in vivo is a grail in gene therapy. Ark313 enables targeted integration of large DNA payloads in vivo! Using Homology Directed Repair and Homology-Independent Targeted Integration (HITI), we achieved in vivo KI in a cas9 expressing mouse!

5/ We also tested whether Ark313 could enhance cancer immunotherapy. Knockout of Regnase-1 using Ark313 led to improved T cell function and tumor control in a murine melanoma model.
🔥In vivo gene editing to enhance anti-tumor immunity!

4/ We validated Ark313 for in vivo CRISPR gene editing. A single injection of Ark313 delivering a sgRNA in Cas9-expressing mice resulted in up to 50% KO of the transduced T cells—including tissue-resident cells.

3/ With a single IV injection, Ark313 achieves gene delivery in up to 25% of T cells, in both circulating and tissues, including tissue resident memory! This unlocks new possibilities for studying T cell function in their natural niche. (🙏 to Roberto Ricardo Gonzalez and @arimolofskylab.bsky.social)

2/ We first show that Ark313 can efficiently transduce T cells in vivo, including naïve, memory and regulatory subsets, outperforming natural AAV serotypes (AAV5 and AAV6).