Justin Eyquem
@j-eyquem.bsky.social
Eyquem Lab _ Genome editing and Synthetic Immunologist at UCSF.
Investigator @Gladstone Institutes and Parker Institute for Cancer Immunotherapy
Investigator @Gladstone Institutes and Parker Institute for Cancer Immunotherapy
Thank you for your amazing work at MSK, you inspired a generation whole of junior scientists and helped so many of us transitioning to our dream jobs!
March 1, 2025 at 2:33 AM
Thank you for your amazing work at MSK, you inspired a generation whole of junior scientists and helped so many of us transitioning to our dream jobs!
10/ This was an awesome collab with the
@brshy.bsky.social Lab and led by Chris Chang. A huge shoutout to our amazing teams, collaborators and funders for making this possible!
@ucsfcancer.bsky.social @gladstoneinst.bsky.social @parkerici.bsky.social @pewhealth.bsky.social
@brshy.bsky.social Lab and led by Chris Chang. A huge shoutout to our amazing teams, collaborators and funders for making this possible!
@ucsfcancer.bsky.social @gladstoneinst.bsky.social @parkerici.bsky.social @pewhealth.bsky.social
February 5, 2025 at 7:03 PM
10/ This was an awesome collab with the
@brshy.bsky.social Lab and led by Chris Chang. A huge shoutout to our amazing teams, collaborators and funders for making this possible!
@ucsfcancer.bsky.social @gladstoneinst.bsky.social @parkerici.bsky.social @pewhealth.bsky.social
@brshy.bsky.social Lab and led by Chris Chang. A huge shoutout to our amazing teams, collaborators and funders for making this possible!
@ucsfcancer.bsky.social @gladstoneinst.bsky.social @parkerici.bsky.social @pewhealth.bsky.social
9/ Importantly, our optimized protocol did not lead to an increase of chromosomal rearrangements or loss of heterozygosity.
February 5, 2025 at 7:03 PM
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!
February 5, 2025 at 7:03 PM
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.
February 5, 2025 at 7:03 PM
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!
➡️By integrating the epitope-edited TCR, we can identify and eliminate mispaired TCRs and SEED select for cells with proper receptor expression and function!
February 5, 2025 at 7:03 PM
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!
➡️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%.
February 5, 2025 at 7:03 PM
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.
February 5, 2025 at 7:03 PM
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.
February 5, 2025 at 7:03 PM
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.
February 5, 2025 at 7:03 PM
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.
1/ T cell engineering is essential for next-gen cell therapies, but multiplexed genome edits result in heterogeneous mixtures of partially edited cells. SEED-Selection allows us to enrich for fully edited T cells through negative selection, leaving engineered cells untouched!
February 5, 2025 at 7:03 PM
1/ T cell engineering is essential for next-gen cell therapies, but multiplexed genome edits result in heterogeneous mixtures of partially edited cells. SEED-Selection allows us to enrich for fully edited T cells through negative selection, leaving engineered cells untouched!
10/ Excited to see how this tool will help the field of immunology and gene therapy!
Ark13 on addgene: addgene.org/200257/
📢Read the full paper here: authors.elsevier.com/sd/article/S...
💬Questions? Thoughts? Let’s discuss! ⬇️
Ark13 on addgene: addgene.org/200257/
📢Read the full paper here: authors.elsevier.com/sd/article/S...
💬Questions? Thoughts? Let’s discuss! ⬇️
Addgene: pArk313
Plasmid pArk313 from Dr. Justin Eyquem's lab contains the insert Ark313 Rep/Cap and is published in Cell. 2023 Jan 19;186(2):446-460.e19. doi: 10.1016/j.cell.2022.12.022. Epub 2023 Jan 12. This plasm...
addgene.org
February 4, 2025 at 8:50 PM
10/ Excited to see how this tool will help the field of immunology and gene therapy!
Ark13 on addgene: addgene.org/200257/
📢Read the full paper here: authors.elsevier.com/sd/article/S...
💬Questions? Thoughts? Let’s discuss! ⬇️
Ark13 on addgene: addgene.org/200257/
📢Read the full paper here: authors.elsevier.com/sd/article/S...
💬Questions? Thoughts? Let’s discuss! ⬇️
9/ This was a collaboration with the asokan Lab and led by @williamnyberg.bsky.social, Charlotte Wang
and Jon Ark. Huge thanks to our incredible team, collaborators and funders @parkerici.bsky.social @gladstoneinst.bsky.social
@ucsfcancer.bsky.social @pewhealth.bsky.social
and Jon Ark. Huge thanks to our incredible team, collaborators and funders @parkerici.bsky.social @gladstoneinst.bsky.social
@ucsfcancer.bsky.social @pewhealth.bsky.social
February 4, 2025 at 8:50 PM
9/ This was a collaboration with the asokan Lab and led by @williamnyberg.bsky.social, Charlotte Wang
and Jon Ark. Huge thanks to our incredible team, collaborators and funders @parkerici.bsky.social @gladstoneinst.bsky.social
@ucsfcancer.bsky.social @pewhealth.bsky.social
and Jon Ark. Huge thanks to our incredible team, collaborators and funders @parkerici.bsky.social @gladstoneinst.bsky.social
@ucsfcancer.bsky.social @pewhealth.bsky.social
8/ TL;DR—Ark313 enables in vivo T cell engineering:
✅High-efficiency transduction of circulating & tissue-resident T cells
✅Supports CRISPR editing in living mice
✅Enhances cancer immunotherapy via gene KO
✅Enables site-specific transgene integration in T cells
✅High-efficiency transduction of circulating & tissue-resident T cells
✅Supports CRISPR editing in living mice
✅Enhances cancer immunotherapy via gene KO
✅Enables site-specific transgene integration in T cells
February 4, 2025 at 8:50 PM
8/ TL;DR—Ark313 enables in vivo T cell engineering:
✅High-efficiency transduction of circulating & tissue-resident T cells
✅Supports CRISPR editing in living mice
✅Enhances cancer immunotherapy via gene KO
✅Enables site-specific transgene integration in T cells
✅High-efficiency transduction of circulating & tissue-resident T cells
✅Supports CRISPR editing in living mice
✅Enhances cancer immunotherapy via gene KO
✅Enables site-specific transgene integration in T cells
7/ Finally, unlike many AAVs, we found that Ark313 is significantly de-targeted from both the liver and macrophages, reducing off-target effects.
February 4, 2025 at 8:50 PM
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!
February 4, 2025 at 8:50 PM
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!
🔥In vivo gene editing to enhance anti-tumor immunity!
February 4, 2025 at 8:50 PM
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!
🔥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.
February 4, 2025 at 8:50 PM
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)
February 4, 2025 at 8:50 PM
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)