Lentiviral vector integration in the human genome induces alternative splicing and generates aberrant transcripts

Area of Science:

• Molecular Biology
• Gene Therapy
• Virology

Background:

• Retroviral vectors are used in gene therapy but can cause genotoxicity.
• Integration into host genes can lead to transcriptional and posttranscriptional deregulation.
• Interference with splicing and polyadenylation are key concerns for retroviral vector safety.

Purpose of the Study:

• To investigate the impact of retroviral vector integration on transcript splicing.
• To identify the mechanisms driving aberrant splicing events.
• To develop strategies for reducing posttranscriptional genotoxicity of lentiviral vectors.

Main Methods:

• Transduction of human cells with HIV-derived vectors containing reporter or human β-globin genes.
• Random cloning of transduced cells and determination of integration sites.
• Analysis of transcript splicing patterns in targeted genes.

Main Results:

• Aberrantly spliced, chimeric transcripts were found in over half of targeted genes across all cell types.
• Chimeric transcripts resulted from the use of constitutive and cryptic splice sites within the vector and transgene.
• Nonsense-mediated mRNA degradation limited the accumulation of most aberrant transcripts.

Conclusions:

• A subset of cryptic splice sites is responsible for the majority of aberrant splicing events.
• Recoding lentiviral vector backbones and transgenes can mitigate posttranscriptional genotoxicity.
• Understanding integration-induced splicing alterations is crucial for safer gene therapy vectors.