High-fidelity PAMless base editing of hematopoietic stem cells to treat chronic granulomatous disease

Oct 16, 2024Science translational medicine

Precise gene editing of blood stem cells without DNA sequence limits to treat chronic granulomatous disease

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Abstract

Up to 70% correction of the X-CGD mutation c.676C>T was achieved using adenine base editor ABE8e-SpRY in human hematopoietic stem and progenitor cells.

The engineered PAMless Cas enzyme SpRY allows for more flexible editing of genetic mutations.
ABE8e-SpRY demonstrated efficiencies greater than three-and-one-half times those of previous CRISPR nuclease and donor template methods.
Minimal off-target DNA edits and transcriptome-wide RNA edits were observed in base-edited HSPCs.
Edited alleles remained stable after transplantation into immunodeficient mice.
These findings support the potential for a first-in-human clinical trial and may guide treatments for other inborn errors of immunity.

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X-linked chronic granulomatous disease (X-CGD) is an inborn error of immunity (IEI) resulting from genetic mutations in the cytochrome b-245 beta chain () gene. The applicability of base editors (BEs) to correct mutations that cause X-CGD is constrained by the requirement of Cas enzymes to recognize specific protospacer adjacent motifs (PAMs). Our recently engineered PAMless Cas enzyme, SpRY, can overcome the PAM limitation. However, the efficiency, specificity, and applicability of SpRY-based BEs to correct mutations in human hematopoietic stem and progenitor cells (HSPCs) have not been thoroughly examined. Here, we demonstrated that the adenine BE ABE8e-SpRY can access a range of target sites in HSPCs to correct mutations causative of X-CGD. For the prototypical X-CGD mutationc.676C>T, ABE8e-SpRY achieved up to 70% correction, reaching efficiencies greater than three-and-one-half times higher than previous CRISPR nuclease and donor template approaches. We profiled potential off-target DNA edits, transcriptome-wide RNA edits, and chromosomal perturbations in base-edited HSPCs, which together revealed minimal off-target or bystander edits. Edited alleles persisted after transplantation of the base-edited HSPCs into immunodeficient mice. Together, these investigational new drug-enabling studies demonstrated efficient and precise correction of an X-CGD mutation with PAMless BEs, supporting a first-in-human clinical trial (NCT06325709) and providing a potential blueprint for treatment of other IEI mutations. CYBB CYBB

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High-fidelity PAMless base editing of hematopoietic stem cells to treat chronic granulomatous disease

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