Sangamo Therapeutics : Zinc finger fusions and synthetic DNA donor engineering improve the performance of reprogrammed modular integrases at the TRAC locus
SGMO
Published on 05/12/2026 at 06:20 am EDT
Zinc finger fusions and synthetic DNA donor engineering improve the performance of reprogrammed modular integrases at the TRAC locus
Jessica E Davis, Friedrich Fauser, Sebastian Arangundy-Franklin, Lifeng Liu, Nicola J Schmidt, Luis Rodriguez, Danny F Xia, Nga Nguyen, Nicholas A Scarlott,Yuanyue Zhou, Lynn N Truong, Rakshaa Mureli, Irene Tan, Satria Sajuthi, Sarah J Hinkley, Bhakti N Kadam, Stephen Lam, Bryan Bourgeois, Emily Tait, Mohammad Qasim,Vishvesha Vaidya, Adeline Chen, Andrew Nguyen,Yuri R. Bendaña, David A. Shivak, Patrick Li, Andreas Reik, David E Paschon, Gregory D Davis and Jeffrey C Miller
Sangamo Therapeutics, Inc.
ASGCT, May 11-16, 2026
Fully programmable gene integration: the ideal genomic medicine approach
Large payload delivery
Irreversible integration: Integrates via
attachment sites (attP → attB)
DNA break-free
Cell type independence: Does not depend on DNA repair machinery
Images by Biorender 4
Bxb1 is a Large Serine Integrase with promising therapeutic potential
High activity in human cells, and in vivo mouse and NHP studies
Structural analysis and scanning mutagenesis studies predicted likely DNA-binding domains and locations of their target sites within attB
DNA binding by each domain hypothesized to occur in a modular fashion, guiding our approach to retargeting
Helix
Low off-target integration levels in human genome
Hairpin
Loop
High fidelity of cargo integrity after integration via cut and paste mechanism
Bxb1
5
Bacterial selections to generate each DNA-binding domain
Built archive of domains for ease of retargeting
Bacterial selections to generate each DNA-binding domain
Built archive of domains for ease of retargeting
Stack domains as monomers to target attB half-sites
Bacterial selections to generate each DNA-binding domain
Built archive of domains for ease of retargeting
Stack domains as monomers to target attB half-sites
Pair monomers and validate activity at full genomic attB target site
K562 genome
MINTTM is a trademark of Sangamo Therapeutics, Inc
Images by Biorender 8
TRAC intron 1 is an appealing retargeting challenge due to:
Small search window (1.9 kb) pushes capabilities of platform
Relevance for CAR-T engineering therapies
Using our MINT platform, we evolved Bxb1 DNA-binding domains that targeted a novel attB site here
9
We achieved full re-targeting of Bxb1 to TRAC, achieving 1% TI
Next step: increase activity further
localizing MINT reagents to their intended genomic target site
Screened a panel of ZF arrays and linkers with TRAC reagents
Best set of ZF fusion and linker combinations achieved
15% TI at the TRAC locus in K562 cells
relevant activity with TRAC reagents
eeBxb1 is an activity-increasing Bxb1 variant developed by
David Liu's team at Harvard*
loop, helix, hairpin
ee
V74A
V375I
E229K
*Pandey et al. (2024) Nature
Combining eeBxb1 with our TRAC reagents drove
comparable improvements as ZF fusions
Combining ZF fusions with activity-increasing mutations achieved ~34% TI at the TRAC in K562 cells
Achieved ~29% TI at the TRAC locus and up to 44% GFP expression
in primary human T cells using ZF fusions
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Images by Biorender
ZF fusions
increase TRAC
ZF fusions
increase TRAC
Unbiased genome-wide assay nominated off-targets
Activity-increasing mutations reduced specificity
ZF-fusions selectively increased the on-target signal
Validated top two off-target sites in K562 and T cells
Relative TI followed similar trends as nomination assay
No translocations observed across samples
Did observe a weak, local genomic inversion only with activity-increasing mutations
All assays performed in K562 cells
ZF-fused MINT reagents indicate a superior specificity profile
Only requires retargeted MINT reagents for activity
Removes need for wild-type Bxb1
Reduces off-targets
Donor engineering removed off-targets
1 and 2 for the most active TRAC reagents
K562 cells
Summary
MINT platform enables retargeting Bxb1
to therapeutically-relevant sites
34% TRAC TI and 29% AAVS1 TI in K562 cells
Used archive of pre-characterized Bxb1 DNA-binding domains to target two additional sites
Achieved high activity in T cells
29% integration at TRAC locus
44% GFP-expressing cells
ZF fusions improve activity & specificity
WT-free donor engineering further increases specificity
Accepted at Nature Biotech
Full preprint of work presented here:
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Disclaimer
Sangamo Therapeutics Inc. published this content on May 12, 2026, and is solely responsible for the information contained herein. Distributed via Public Technologies (PUBT), unedited and unaltered, on May 12, 2026 at 10:19 UTC.