Large Serine Recombinases for Targeted Gene Insertion

A large serine recombinase (LSR) recognizes matched DNA attachment sites and recombines donor DNA into the genome. Unlike nuclease-based insertion approaches, the LSR reaction does not create exposed double-strand breaks during insertion. ElevateBio has identified and characterized more than 100 active LSRs for targeted large gene insertion.

Not always. Direct LSRs can insert at naturally occurring genomic pseudo-sites that resemble an LSR’s cognate attachment sequence. When a program requires insertion at a preselected genomic locus, ElevateBio has separately demonstrated a landing-pad approach in which an attachment site is first installed at the target locus and then used for LSR-mediated cargo insertion. At TRAC in primary human T cells, this workflow achieved >90% TCR knockout via landing-pad insertion and >50% targeted CAR insertion across multiple donors, with insertion reaching up to 67%.

ElevateBio demonstrated greater than 50% targeted CD19 CAR insertion across multiple primary T-cell donors, with insertion reaching 67% in the highest-performing donor. The workflow used a landing pad installed at TRAC followed by LSR-mediated cargo insertion.

ElevateBio has demonstrated insertion of a 4.6 kb+ CD19-CAR DNA payload in primary human T cells using novel LSRs. ElevateBio has not disclosed the maximum payload size validated experimentally.

Different LSRs have different attachment-site preferences, activity levels, and genomic integration profiles. ElevateBio identified more than 30,000 LSR candidates and has experimentally identified more than 100 active LSRs, with several outperforming the literature benchmark Bxb1 in mammalian-cell assays. ElevateBio’s discovery pipeline also predicts the cognate attB and attP sites associated with candidate LSRs, providing the components needed for functional testing. A larger, more diverse collection increases the opportunity to identify a useful starting point for a therapeutic program.

They are two separate studies.
The landing-pad study installed an attachment site at TRAC and then used LSR-mediated insertion, achieving greater than 50% targeted CD19 CAR insertion across donors and up to 67%.
In a separate delivery study, ElevateBio compared electroporation with LNP delivery of LSR mRNA and CAR donor DNA. That study measured less than 20% CD19-CAR-positive cells with electroporation versus up to approximately 88% with LNP delivery.
The LNP and TRAC landing-pad workflows have not been demonstrated together in the disclosed data.

In ElevateBio’s disclosed active-learning study, protein engineering produced a single-mutant LSR with up to 2.4-fold greater activity than wildtype and a double-mutant with up to 3.4-fold greater activity. These are improvements in measured LSR activity in the experimental system, not demonstrated changes in clinical dose, efficacy, or safety.

Not in the disclosed data presented here. ElevateBio’s published LSR insertion results are from in vitro mammalian-cell studies, including primary human T cells. The chosen-locus landing-pad workflow is currently positioned for ex vivo applications.