Azalea grew out of collaborative research conducted at the Innovative Genomics Institute in Jennifer Doudna’s lab and in Justin Eyquem’s lab as part of the Gladstone-UCSF Institute of Genomic Immunology.
@j-eyquem.bsky.social
@innovativegenomics.bsky.social
Genomic medicine has transformed lives through ex vivo cell therapies, now it’s time to make it scalable.
Today we unveil Azalea Therapeutics, making in vivo genomic medicine a reality without compromising precision or specificity, starting with T cells!
Exciting collaboration combining multiplex epigenetic reprogramming and site-specific payload integration!
The Weill Cancer Hub is fostering groundbreaking @ucsanfrancisco.bsky.social - @stanford-cancer.bsky.social collaborations to develop transformative cancer therapies. I’m grateful to co-lead one of the selected teams with @mfgrp.bsky.social to advance next-generation in vivo CAR T cell therapy!
Backed by a $100M gift from Joan and Sandy Weill, UCSF and @stanfordmedicine.bsky.social @stanford-cancer.bsky.social are launching Weill Cancer Hub West, a $200M initiative in team science to accelerate cancer research and improve care over the next decade.
tiny.ucsf.edu/rZIspe
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
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.
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!
Excited to share our latest work, now out in
@NatureBiotech
! We introduce SEED-Selection, a powerful method that enables high-efficiency enrichment of T cells edited at multiple loci in a single step 🧵👇
www.nature.com/articles/s41...
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! ⬇️
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
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
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).
1/ T cell engineering is critical for immunology research and therapy, but ex vivo manipulation is complex. We present Ark313, an AAV variant we previously evolved, to directly deliver genes to T cells in vivo—without the need for isolation and re-infusion!
🚨New paper alert! 🚨 Our latest work in
@cp-immunity.bsky.social shows that Ark313, an evolved AAV variant enables in vivo genetic engineering of murine T cells! 🧵👇
sciencedirect.com/science/arti...
Wonderful to see the #immunology community growing here.
The first starter pack is full (they cap at 150), so I created a second one. Let me know if you'd like to be added
go.bsky.app/PY5tCas
go.bsky.app/FmERUoD
Would love to be added! Thanks!!
