Efficient gene editing of human long-term hematopoietic stem cells validated by clonal tracking

Area of Science:

• Hematology
• Gene Therapy
• Molecular Biology

Background:

• Targeted gene editing in hematopoietic stem cells (HSCs) offers potential for treating genetic diseases.
• Clinical translation is hindered by low homology-directed repair (HDR) efficiency and unclear effects on HSC clonal dynamics.
• Understanding and overcoming these barriers are crucial for advancing HSC gene therapy.

Purpose of the Study:

• To investigate the impact of gene editing on HSC clonal composition and dynamics.
• To enhance the efficiency of HDR in HSCs for therapeutic applications.
• To develop a protocol for safe and effective clinical translation of HSC gene editing.

Main Methods:

• Utilized a barcoding strategy (BAR-Seq) for clonal tracking of edited HSCs in hematochimeric mice.
• Investigated the role of p53 activation in response to gene editing and its effect on the HSC repertoire.
• Employed transient p53 inhibition and adenovirus 5 E4orf6/7 protein expression to enhance HDR efficiency.

Main Results:

• Gene editing activated p53, leading to a significant reduction in the HSC clonal repertoire.
• Transient p53 inhibition restored polyclonal graft composition.
• Combined E4orf6/7 expression and p53 inhibition achieved up to 50% HDR editing efficiency in human grafts without affecting HSC repopulation or self-renewal.

Conclusions:

• Gene editing in HSCs can be optimized by managing p53 activation and enhancing HDR efficiency.
• The developed protocol shows potential for broad clinical applicability in treating genetic diseases.
• This approach paves the way for successful clinical translation of HSC gene editing therapies.