Regulatory agencies require genotoxicity testing because genotoxicity may occur in gene and gene-editing therapies. Genotoxicity refers to damage to the host DNA, which can result from the integration of the vector, a therapeutic transgene or an off-target gene-editing event. The genotoxic outcome occurs when such damage inactivates a tumor suppressor gene, activates an oncogene, or triggers a pathway involved in tumorigenesis.
What is genotoxicity?
Natural adenoviruses and lentiviruses integrate into the genome at low frequency. Even when engineered not to integrate for use in gene therapies, integration can still mistakenly occur. Integration events may happen during attempts to repair double-strand DNA breaks, through homologous recombination, or due to reversion events where the viral vector regains integration-competent elements. Integration can also result from unintended contamination with wild-type viruses, replication-competent variants, or helper viruses from production. For vectors designed to integrate, the risk of oncogenesis is naturally higher.
Other impurities during production, such as residual host cell DNA, may also cause genotoxic effects. Host cell DNA can randomly integrate into the patient genome or through mechanisms like double-strand DNA break repair or homologous recombination. Also, host cell DNA may harbor viral oncogenes integrated into the production cell line that could integrate into the patient’s genome. For example, HEK293 cells contain the adenovirus serotype 5 E1 gene, while HeLa cells contain human papillomavirus E6 and E7 oncogenes.
Genotoxicity occurs when critical patient genes, such as tumor suppressor genes, oncogenes, or genes activating pathways involved in tumorigenesis, are altered in a way that drives cell division and oncogenesis.
Genotoxicity risk mitigation
In gene therapies, viral vectors are modified to prevent integration. Lentiviruses, for example, use an integrase enzyme to process the host cell’s chromosomal DNA for integration. When this enzyme is inactivated, the viral DNA remains episomal.
Adeno-associated viral vectors (rAAVs) are engineered by removing all viral genes except inverted terminal repeats (ITRs). In natural AAVs, Rep proteins are essential for viral DNA replication and integration into the host genome. rAAVs lack Rep proteins, which prevents integration into the host genome. After delivery into the target cell, the rAAV genome can self-ligate (join its ends together) or combine with other rAAV genomes to form circular DNA molecules called concatemers. These concatemers do not integrate into the host genome but instead exist as episomes, which are extrachromosomal DNA elements. Episomal viral vectors allow for long-term transgene expression without genomic integration.
Regulatory requirement for genotoxicity testing
Regulatory agencies recommend testing for integration events and integration site analysis to detect rearrangements, recombinations, or gene editing that might lead to cancer. For vectors intended to integrate into the patient genome, the frequency of rearrangements, the recombination events, and the oncogenic occurrences must be tested to ensure that the integrated vector remains stable over time.
Integration testing should align with the expected persistence of the transgene expression in non-clinical studies.
In clinical trials, monitoring the presence of integrated vector sequences, the integration sites, and the clonal expansion of cells with integration events is essential, as this could indicate an oncogenic process. Long-term follow-up is crucial, with the FDA recommending up to 15 years of post-treatment monitoring for specific integrating vectors or those targeting long-lived cells.
Regulatory agencies also require the control of host cell DNA residuals in the gene therapy product during manufacturing and before administration.
Techniques for genotoxicity testing
qPCR and dPCR are optimal tools for quantifying integration events, offering the sensitivity to detect low-frequency occurrences. Extraction protocols and primer design must be optimized to target only integrated vectors, avoiding the quantification of episomal vectors.
Next-generation sequencing (NGS) techniques provide powerful tools for detecting integration sites and other genomic alterations. Whole-genome sequencing (WGS) enables high-resolution detection of integration sites, indels, and translocations anywhere in the genome. Whole-exome sequencing (WES) focuses on coding regions, while RNA-Seq can identify unintended genomic changes that alter expression patterns.
Host cell DNA residuals are typically detected using highly sensitive and specific qPCR or dPCR assays targeting particular sequences, such as E1A or other oncogenes. We utilize a proprietary DeltaAmp assay to detect the genomic 18S ribosomal RNA gene. This gene is present in multiple copies within the genome, ensuring reliable detection of host DNA if present. The assay is sensitive to even small DNA fragments and provides additional data on DNA degradation.