Intragenic integration in DLC1 sustains factor VIII expression in primary human cells without insertional...

Area of Science:

• Gene therapy
• Genomics
• Cellular biology

Background:

• Precise genome modifications are crucial for safe gene-based therapies.
• The DLC1 gene is a known integration site for phiC31 integrase-mediated gene transfer.
• Understanding integration site effects is vital for therapeutic development.

Purpose of the Study:

• To evaluate the DLC1 gene as a safe and effective locus for durable transgene expression.
• To assess the impact of transgene integration into DLC1 on cellular and genomic stability.
• To explore the potential of DLC1 for site-specific integration in ex vivo cell therapy.

Main Methods:

• Integration of a coagulation factor VIII transgene into intron 7 of the DLC1 gene in human umbilical cord-lining epithelial cells.
• Analysis of DLC1 and neighboring gene expression.
• Assessment of transcriptional alterations in surrounding genes.
• In vivo tumorigenicity studies.
• High-resolution genomic DNA copy number analysis.

Main Results:

• Durable expression of factor VIII was achieved in primary human cells with DLC1 integration.
• DLC1 transgene integration did not alter DLC1 or nearby gene expression significantly.
• Minimal transcriptional changes (1.9% of genes) were observed, primarily affecting cell cycle and DNA repair pathways.
• DLC1-integrated cells showed no signs of tumor formation and normal genomic copy number.
• No features of insertional oncogenesis were detected.

Conclusions:

• The DLC1 gene serves as a suitable locus for achieving durable transgene expression.
• Site-specific integration into DLC1 offers a safe approach, mitigating risks of insertional oncogenesis.
• This finding expands therapeutic options for ex vivo cell therapies utilizing site-specific integration.