Sangamo Therapeutics : Site-directed integration of large DNA sequences into endogenous sites in the human genome using engineered Modular Integrases (MINTS)

Friedrich Fauser, Sebastian Arangundy-Franklin, Jessica E Davis, Lifeng Liu, Nicholas A Scarlott, Nicola J Schmidt, Rakshaa Mureli, Danny F Xia, Luis Rodriguez, Mohammad Qasim,Vishvesha Vaidya, Sarah J Hinkley, Bhakti N Kadam, Nga Nguyen, Stephen Lam, Bryan Bourgeois,Adeline Chen,Andrew Nguyen, Patrick Li, David E Paschon, Gregory D Davis and Jeffrey C Miller

Poster #1680

Introduction

Bxb1-DNA interaction mapping

• The use of serine integrases as genome editing reagents has been hampered by the difficulty in their reprogramming to non-cognate sites.
• We have used a combination of structural modeling and experimental characterization of a series of systematic mutations to both Bxb1 and its target site to map critical Bxb1-DNA interactions.
• We conjectured that, if Bxb1 recognized its target sites in a modular fashion, we could reprogram its DNA target specificity by performing directed evolution on each domain in parallel using Bxb1 libraries that randomize the residues that make sequence- specific DNA contacts.

DNA recognition helix engineering changes Bxb1 specificity

• Next, we tested Bxb1 variant pairs against their chromosomal target sites.This resulted in variant pairs that substantially outperformed wild-type Bxb1.

• This demonstrates that the specificity of Bxb1 can be changed through engineering of the helix and hairpin peptide regions, and that high levels of efficiency can be achieved at endogenous sites in the human genome.

Retargeting Bxb1 to therapeutically relevant sites

• Encouraged by our success targeting endogenous human sites where wild-type Bxb1 is already active, we proceeded to the more difficult challenge of targeting desired regions of the human genome where wild-type Bxb1 is inactive, such as the AAVS1 locus, a well-established safe harbor, and the TRAC locus.

Bxb1 efficiently facilitates targeted integration in human cells

• We developed a directed evolution system in which a recombinase variant present in a library is enriched when it can restore the open reading frame of an antibiotic-resistance gene if it acts on a new target sequence.
• We carried out 64 separate selections using our library of Bxb1 helix variants and each possible DNA triplet sequence:

SATALKR wild-type Bxb1 residues 231-237

XXXXLXX helix variant library

• We characterized the targeting specificity of the wild-type helix (SATALKR) which yielded a preference for NAC DNA binding motifs (N = G/A/T/C) consistent with natural target sites (attB: GAC; attP:AAC and CAC).
• Many of the selected helices were able to targeted alternative DNA triplets with high molecular specificity, and the data suggests interaction patterns.

Details presented by Sebastian Arangundy-Franklin:

"192: DIRECTED EVOLUTION OF BXB1 FOR THE

DEVELOPMENT OF MODULAR INTEGRASES (MINTs)"

Thursday, May 9th 4:00-4:15 pm - Ballroom 3

AAV-mediated donor delivery

• During the phage lifecycle, integration occurs when one Bxb1 dimer binds to a phage attP site, and a second dimer binds an attB site in the bacterial host's genome.
• As an initial test of our wild-type Bxb1 constructs, we tested them in a "landing pad" cell line where we had first integrated the natural attB or attP target in human K562 cells using nuclease-mediated HDR.
• We observed the highest targeted integration efficiency in human K562 cells when targeting an attB landing pad within the AAVS1 locus.

Performance of Bxb1 variants at endogenous human sites

• We identified Bxb1 pseudo-sites in human K562 cells using both computational and experimental methods.We then generated a plasmid target library consisting of corresponding RD and ZD regions and tested evolved Bxb1 variants against it.

Conclusion

• We have demonstrated the direct reprogramming of the site-specific serine integrase Bxb1.
• We showed the therapeutic potential of this approach by retargeting Bxb1 to clinically relevant sites and utilizing therapeutically relevant delivery modalities.
• In addition to human therapeutics, the MINT platform has a variety of potential applications in biomanufacturing, drug discovery, agricultural biotech settings, and basic research.