Long-term combination therapy with metformin and oxymetholone in a Fanconi anemia mouse model.

Fanconi anemia (FA) is a disease caused by defective deoxyribonucleic acid (DNA) repair that manifests as bone marrow failure, cancer predisposition, and developmental defects. We previously reported that monotherapy with either metformin (MET) or oxymetholone (OXM) improved peripheral blood (PB) counts and the number and functionality of bone marrow hematopoietic stem progenitor cells (HSPCs) number in Fancd2-/- mice. To evaluate whether the combination treatment of these drugs has a synergistic effect to prevent bone marrow failure in FA, we treated cohorts of Fancd2-/- mice and wildtype controls with either MET alone, OXM alone, MET+OXM, or placebo diet from age 3 weeks to 18 months. The OXM treated animals showed modest improvements in blood parameters including platelet count (p = .01) and hemoglobin levels (p < .05). In addition, the percentage of quiescent hematopoietic stem cell (HSC) (LSK [Lin-Sca+c-Kit+]) was significantly increased (p = .001) by long-term treatment with MET alone. The combination of metformin and oxymetholone did not result in a significant synergistic effect in any hematopoietic parameter. Gene expression analysis of liver tissue from these animals showed that some of the expression changes caused by Fancd2 deletion were partially normalized by metformin treatment. Importantly, no adverse effects of the individual or combination therapies were observed, despite the long-term administration. We conclude that androgen therapy is not a contraindication to concurrent metformin administration in clinical trials. HIGHLIGHTS: Long-term coadministration of metformin in combination with oxymetholone is well tolerated by Fancd2-/- mice. Hematopoietic stem cell quiescence in mutant mice was enhanced by treatment with metformin alone. Metformin treatment caused a partial normalization of gene expression in the livers of mutant mice.

Long-term combination therapy with Metformin and Oxymetholone in a Fanconi Anemia mouse model.

Fanconi Anemia (FA) is a disease caused by defective DNA repair which manifests as bone marrow failure, cancer predisposition, and developmental defects. Mice containing inactivating mutations in one or more genes in the FA pathway partially mimic the human disease. We previously reported that monotherapy with either metformin (MET) or oxymetholone (OXM) improved peripheral blood (PB) counts and the number and functionality of bone marrow (BM) hematopoietic stem progenitor cells (HSPCs) number in Fancd2-/- mice. To evaluate whether the combination treatment of these drugs has a synergistic effect to prevent bone marrow failure in FA, we treated cohorts of Fancd2-/- mice and wild-type controls with either MET alone, OXM alone, MET+OXM or placebo diet. Both male and female mice were treated from age 3 weeks to 18 months. The OXM treated animals showed modest improvements in blood parameters including platelet count (p=0.01) and hemoglobin levels (p<0.05). In addition, the percentage of quiescent HSC (LSK) was significantly increased (p=0.001) by long-term treatment with MET alone. However, the absolute number of progenitors, measured by LSK frequency or CFU-S, was not significantly altered by MET therapy. The combination of metformin and oxymetholone did not result in a significant synergistic effect on any parameter. Male animals on MET+OXM or MET alone were significantly leaner than controls at 18 months, regardless of genotype. Gene expression analysis of liver tissue from these animals showed that some of the expression changes caused by Fancd2 deletion were partially normalized by metformin treatment. Importantly, no adverse effects of the individual or combination therapies were observed, despite the long-term administration.

Induced Liver Regeneration Enhances CRISPR/Cas9-Mediated Gene Repair in Tyrosinemia Type 1.

The efficiency of gene repair by homologous recombination in the liver is enhanced by CRISP/Cas9 incision near the mutation. In this study, we explored interventions designed to further enhance in vivo hepatocyte gene repair in a model of hereditary tyrosinemia. A two-AAV system was employed: one virus carried a Staphylococcus pyogenes Cas9 (SpCas9) expression cassette and the other harbored a U6 promoter-driven sgRNA and a fragment of fumarylacetoacetate hydrolase (Fah) genomic DNA as the homologous recombination donor. In neonatal mice, a gene correction frequency of ∼10.8% of hepatocytes was achieved. The efficiency in adult mice was significantly lower at ∼1.6%. To determine whether hepatocyte replication could enhance the targeting frequency, cell division was induced with thyroid hormone T3. This more than doubled the gene correction efficiency to 3.5% (p < 0.005). To determine whether SpCas9 delivery was rate limiting, the gene repair AAV was administered to SpCas9 transgenic mice. However, this did not significantly enhance gene repair. Finally, we tested whether the Fanconi anemia (FA) DNA repair pathway was important in hepatocyte gene repair. Gene correction frequencies were significantly lower in neonatal mice lacking the FA complementation group A (Fanca) gene. Taken together, we conclude that pharmacological induction of hepatocyte replication along with manipulation of DNA repair pathways could be a useful strategy for enhancing in vivo gene correction.