Correction of Fanconi Anemia Mutations Using Digital Genome Engineering

Area of Science:

• Genetics and Genomics
• Hematology
• Gene Therapy

Background:

• Fanconi anemia (FA) is a rare genetic disorder characterized by DNA repair pathway defects, leading to bone marrow failure and cancer.
• Allogeneic hematopoietic cell transplant (HCT) is curative but limited by donor availability and risks like graft-versus-host disease (GvHD).
• Current gene-editing strategies for autologous HCT are inefficient in FA cells due to inherent DNA repair deficiencies.

Purpose of the Study:

• To investigate the efficacy of base editors (BEs) for correcting Fanconi anemia mutations in patient-derived cells.
• To establish a gene-editing approach that circumvents the need for DNA double-strand breaks (DSBs) and donor DNA.
• To demonstrate the potential of this 'digital' genome editing for autologous stem cell therapy in FA.

Main Methods:

• Utilized cytosine and adenine base editors (BEs) for precise C:G > T:A and A:T > C:G base transitions in FANCA genes.
• Applied digital genome editing to primary Fanconi anemia patient cells, avoiding DSB induction and homology-directed repair (HDR).
• Assessed phenotypic rescue by measuring resistance to Mitomycin C (MMC) and restored FANCA protein expression and FA pathway function.

Main Results:

• Successfully corrected FANCA mutations in primary FA patient cells using base editors.
• Demonstrated phenotypic rescue, including resistance to Mitomycin C (MMC), and restored FANCA protein expression.
• Confirmed functional FA pathway restoration through FANCD2 monoubiquitination induction.

Conclusions:

• Base editor-mediated digital genome editing offers an efficient strategy for correcting FA mutations without inducing DSBs.
• This approach enables the potential use of gene-corrected autologous hematopoietic stem and progenitor cells (HSPCs) for FA treatment.
• Digital genome editing provides a promising alternative to allogeneic HCT, mitigating risks associated with donor limitations and DSB induction.