Novel HDAd/EBV Reprogramming Vector and Highly Efficient Ad/CRISPR-Cas Sickle Cell Disease Gene Correction

Area of Science:

• Stem cell biology
• Gene therapy
• Molecular genetics

Background:

• Sickle cell disease (SCD) is a debilitating genetic disorder.
• Patient-specific induced pluripotent stem cells (iPSCs) offer a potential therapeutic avenue.
• Efficient and rapid generation of gene-corrected iPSCs is crucial for clinical application.

Purpose of the Study:

• To develop a rapid and efficient method for generating gene-corrected, patient-specific iPSCs for sickle cell disease therapy.
• To utilize a novel helper-dependent adenovirus/Epstein-Barr virus (HDAd/EBV) hybrid reprogramming vector for iPSC generation.
• To assess the efficiency and safety of CRISPR/Cas9 gene editing in patient-derived iPSCs.

Main Methods:

• Developed a novel HDAd/EBV hybrid vector (rCLAE-R6) for episomal delivery of six reprogramming factors.
• Reprogrammed keratinocytes from SCD patients into iPSCs with high efficiency.
• Corrected the sickle mutation in iPSCs using CRISPR/Cas9 delivered via adenovirus and nucleoporation with a single-stranded oligodeoxynucleotide (ssODN) template.

Main Results:

• Achieved footprint-free iPSC generation with high efficiency using the HDAd/EBV vector.
• Obtained correction efficiencies of up to 67.9% for the sickle mutation.
• Whole-genome sequencing confirmed no off-target mutations in 1467 potential sites or in critical genes.

Conclusions:

• Adenoviral delivery of reprogramming factors and CRISPR/Cas9 provides a rapid and efficient method for generating gene-corrected, patient-specific iPSCs.
• This approach demonstrates safety by avoiding off-target modifications.
• The developed method holds significant potential for therapeutic applications in sickle cell disease.