Development of a Beta-globin Gene Replacement Strategy as a Therapeutic Approach for β-Thalassemia

Kirby Wallace, Ph.D. – Graphite Bio; Christopher Bandoro, Ph.D. – Graphite Bio; Aishwarya Churi, Ph.D. – Graphite Bio; James Partridge, Ph.D. – Graphite Bio; Rajiv Sharma, Ph.D. – Graphite Bio; William Matern, Ph.D. – Graphite Bio; Sebastian Treusch, Ph.D. – Graphite Bio; Daniel Dever, Ph.D. – Graphite Bio

Abstract Text: β-thalassemia is a genetic disorder characterized by reduced production of β-globin, a protein necessary for functional, oxygen-carrying hemoglobin (HbA), and severe anemia. Over 300 mutations in the β-globin (HBB) gene are known. A gene editing platform using homology directed repair (HDR) that replaces the HBB gene to achieve a normal or trait phenotype with HbA expression similar to healthy individuals is an ideal strategy. The challenge is achieving high levels of gene replacement that results in high HbA expression.

We set out to develop a DNA donor that can replace a dysfunctional with a functional HBB gene but avoids sequence homology of the insert. As introns are required for HBB expression, we hypothesized that incorporation of non-homologous introns would result in physiological HbA production. We screened 39 DNA donors containing various heterologous introns and polyadenylation signals by knocking them into primary CD34+ hematopoietic stem and progenitor cells (HSPCs) leveraging our UltraHDR® platform that uses CRISPR-Cas9 and AAV6 to deliver the DNA template. HSPCs were then differentiated into RBCs in vitro and evaluated for β-globin expression. We identified two heterologous intron DNA donors that resulted in high β-globin expression. Using HSPCs from sickle cell disease patients as a therapeutically relevant model, we investigated if these two DNA donors can replace the dysfunctional HBB gene and found that gene replacement restored HbA expression. In summary, we developed a precise HBB gene replacement strategy that is efficient and results in physiological HbA production, offering a potential differentiated approach for treating β-thalassemia.