Human Genome REWRITE for Off-the-Shelf Stem Cells Reveals an "Epigenetic Ghost"

Area of Science:

• Genomics and Gene Editing
• Immunology and Transplantation
• Stem Cell Biology

Background:

• Human leukocyte antigen (HLA) gene diversity presents a significant barrier to creating universally compatible cell therapies.
• Existing genome editing technologies face limitations in efficiently modifying large DNA segments required for complex genetic engineering.

Purpose of the Study:

• To develop a novel platform for large-scale, precise genome editing in human pluripotent stem cells (hPSCs).
• To engineer hPSCs with modified HLA loci to overcome immune rejection for allogeneic cell therapies.
• To investigate epigenetic phenomena associated with large-scale genetic modifications.

Main Methods:

• Development of REWRITE, a modular, scar-minimized platform for iterative genome writing of large synthetic constructs (>100 kb).
• Application of REWRITE to delete substantial portions of the endogenous HLA locus and install synthetic HLA haplotypes and antigen-processing genes in hPSCs.
• Analysis of epigenetic states, including the identification of 'epigenetic ghosts', and assessment of gene expression and immune compatibility in differentiated cell lineages.

Main Results:

• Successfully deleted 105-209 kb of the HLA locus and integrated synthetic constructs up to 100 kb.
• Discovered a persistent 'epigenetic ghost' phenomenon, where active states lingered after genetic removal, which was resolved by native DNA elements.
• Engineered cells demonstrated restored inducible expression, resistance to Natural Killer (NK) cell-mediated killing, and established T-cell tolerance, crucial for off-the-shelf therapies.

Conclusions:

• The REWRITE platform enables efficient, large-scale genome engineering in hPSCs, overcoming HLA polymorphism challenges.
• The findings reveal novel insights into epigenetic memory and its resolution during genome editing.
• This technology facilitates the creation of 'off-the-shelf' allogeneic cell therapies and offers potential for broader genomic programming applications.