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Updated: Aug 8, 2026
Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models
Published on: October 3, 2019
Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector
Eugenio Montini1, Daniela Cesana, Manfred Schmidt
1San Raffaele-Telethon Institute for Gene Therapy, via Olgettina 58, 20132, Milan, Italy.
Abstract:
Insertional mutagenesis represents a major hurdle to gene therapy and necessitates sensitive preclinical genotoxicity assays. Cdkn2a-/- mice are susceptible to a broad range of cancer-triggering genetic lesions. We exploited hematopoietic stem cells from these tumor-prone mice to assess the oncogenicity of prototypical retroviral and lentiviral vectors. We transduced hematopoietic stem cells in matched clinically relevant conditions, and compared integration site selection and tumor development in transplanted mice. Retroviral vectors triggered dose-dependent acceleration of tumor onset contingent on long terminal repeat activity. Insertions at oncogenes and cell-cycle genes were enriched in early-onset tumors, indicating cooperation in tumorigenesis. In contrast, tumorigenesis was unaffected by lentiviral vectors and did not enrich for specific integrants, despite the higher integration load and robust expression of lentiviral vectors in all hematopoietic lineages. Our results validate a much-needed platform to assess vector safety and provide direct evidence that prototypical lentiviral vectors have low oncogenic potential, highlighting a major rationale for application to gene therapy.
Insights
Gene therapy faces challenges from insertional mutagenesis. Prototypical lentiviral vectors show low oncogenic potential in preclinical genotoxicity assays using Cdkn2a-/- mice, unlike retroviral vectors.
Area of Science:
- * Gene Therapy Vector Safety
- * Cancer Genomics
- * Hematopoietic Stem Cell Biology
Background:
- * Insertional mutagenesis is a significant risk in gene therapy, requiring robust genotoxicity testing.
- * Cdkn2a-/- mice are highly susceptible to genetic lesions, making them a valuable model for cancer research.
- * Assessing the oncogenic potential of viral vectors is crucial for safe gene therapy development.
Purpose of the Study:
- * To evaluate the oncogenic potential of retroviral and lentiviral vectors using a preclinical genotoxicity assay.
- * To compare integration site selection and tumor development following transduction of hematopoietic stem cells.
- * To validate a platform for assessing vector safety in gene therapy.
Main Methods:
- * Hematopoietic stem cells were isolated from Cdkn2a-/- mice.
- * Cells were transduced with prototypical retroviral and lentiviral vectors under clinically relevant conditions.
- * Transduced cells were transplanted into mice, and tumor development and integration sites were analyzed.
Main Results:
- * Retroviral vectors accelerated tumor onset in a dose-dependent manner, linked to long terminal repeat activity.
- * Early-onset tumors showed enrichment for insertions in oncogenes and cell-cycle genes, indicating cooperative oncogenesis.
- * Lentiviral vectors did not affect tumorigenesis or show enrichment for specific integration sites, despite higher integration loads.
Conclusions:
- * Prototypical lentiviral vectors demonstrate low oncogenic potential, supporting their use in gene therapy.
- * The Cdkn2a-/- mouse model provides a validated platform for preclinical assessment of gene therapy vector safety.
- * Understanding integration site profiles is critical for evaluating the risks associated with different vector types.
