JUL 26, 2023 4:45 PM EDT

SLEEK: A highly efficient transgene knock-in technology in clinically relevant cell types

C.E. Credits: P.A.C.E. CE Florida CE


Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.
Key Points:

  • SLEEK can generate multiplex-edited allogeneic T cells with near homogeneity.
  • SLEEK can be used to insert up to four transgene cargos in tandem in a single knock-in cassette.
  • SLEEK offers tunable expression of the transgene cargo through knock-in at other essential genes each with their own intrinsic endogenous promoter strengths.

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