High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition.

Therapeutic applications of nuclease-based genome editing would benefit from improved methods for transgene integration via homology-directed repair (HDR). To improve HDR efficiency, we screened six small-molecule inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key protein in the alternative repair pathway of non-homologous end joining (NHEJ), which generates genomic insertions/deletions (INDELs). From this screen, we identified AZD7648 as the most potent compound. The use of AZD7648 significantly increased HDR (up to 50-fold) and concomitantly decreased INDELs across different genomic loci in various therapeutically relevant primary human cell types. In all cases, the ratio of HDR to INDELs markedly increased, and, in certain situations, INDEL-free high-frequency (>50%) targeted integration was achieved. This approach has the potential to improve the therapeutic efficacy of cell-based therapies and broaden the use of targeted integration as a research tool.

Conjugate prodrug AN-233 induces fetal hemoglobin expression in sickle erythroid progenitors and ß-YAC transgenic mice.

Pharmacologic induction of fetal hemoglobin (HbF) is an effective strategy for treating sickle cell disease (SCD) by ameliorating disease severity. Hydroxyurea is the only FDA-approved agent that induces HbF, but significant non-responders and requirement for frequent monitoring of blood counts for drug toxicity limit clinical usefulness. Therefore, we investigated a novel prodrug conjugate of butyric acid (BA) and δ-aminolevulinate (ALA) as a potential HbF inducing agent, using erythroid precursors and a preclinical ß-YAC mouse model. We observed significantly increased γ-globin gene transcription and HbF expression mediated by AN-233 in K562 cells. Moreover, AN-233 stimulated mild heme biosynthesis and inhibited expression of heme-regulated eIF2α kinase involved in silencing γ-globin expression. Studies using primary erythroid precursors generated from sickle peripheral blood mononuclear cells verified the ability of AN-233 to induce HbF, increase histone H3 and H4 acetylation levels at the γ-globin promoter and reduce erythroid precursor sickling by 50%. Subsequent drug treatment of ß-YAC transgenic mice confirmed HbF induction in vivo by AN-233 through an increase in the percentage of HbF positive red blood cells and HbF levels measured by flow cytometry. These data support the potential development of AN-233 for the treatment of SCD.

Mechanisms of NRF2 activation to mediate fetal hemoglobin induction and protection against oxidative stress in sickle cell disease.

IMPACT STATEMENT: Sickle cell disease (SCD) is a group of inherited blood disorders caused by mutations in the human ß-globin gene, leading to the synthesis of abnormal hemoglobin S, chronic hemolysis, and oxidative stress. Inhibition of hemoglobin S polymerization by fetal hemoglobin holds the greatest promise for treating SCD. The transcription factor NRF2, is the master regulator of the cellular oxidative stress response and activator of fetal hemoglobin expression. In animal models, various small chemical molecules activate NRF2 and ameliorate the pathophysiology of SCD. This review discusses the mechanisms of NRF2 regulation and therapeutic strategies of NRF2 activation to design the treatment options for individuals with SCD.