Abstract
Lentivirus vectors are effective for treatment of genetic disease. However,
safety associated with vector related genotoxicity is of concern and currently available
models are not reliably predictive of safety in humans. We have developed hInGeTox
as the first human in vitro platform that uses induced pluripotent stem cells and their
hepatocyte like cell derivatives to better understand vector-host interactions that relate
vectors to their potential genotoxicity. Using lentiviral vectors carrying the eGFP
expression cassette under SFFV promoter activity, that only differ by their LTR and
SIN configuration, we characterised vector host interactions potentially implicated in
genotoxicity. To do this, lentiviral infected cells were subjected to an array of assays
and data from these was used for multi-omics analyses of vector effects on cells at early
and late harvest time points. Data on the integration sites of lentiviral vectors in cancer
genes and differential expression levels of these genes, showed that both vector
configurations are capable of activating cancer genes. Through IS tracking in bulk
infected cell populations, we also saw an increase in the viral sequence count in cancer
genes present over time which were differentially regulated. RNASeq also showed each
vector had potential to generate fusion transcripts with the human genome suggestive
of gene splicing or vector mediated readthrough from the internal SFFV promoter.
Initially, after infection, both vector configurations were associated with differential
expression of genes associated cytokine production, however, after culturing over time
there were differences in differential expression in cells infected by each LV. This was
marked in particular by the expression of genes involved in the response to DNA
damage in cells transduced by the SIN vector, suggesting effects likely to prevent
tumour development, in contrast to the expression of genes involved in methylation,
characteristic of tumour development, in cells transduced by the LTR vector. Both sets
of lentiviral infected cells were also found associated with differential expression of
MECOM and LMO2 genes known to be associated with clonal dominance, supporting
their potential genotoxicity. Alignment of transcriptomic signatures from iPSC and
HLC infected cultures with known cancer gene signatures showed the LTR vector with
a higher cancer score than the SIN vector over time in iPSC and also in HLC, which
further suggests higher genotoxic potential by the LTR configuration lentivirus.
By application of hInGeTox to cells infected with LV at the pre-clinical stage of
development, we hope that hInGeTox can act as a useful pre-clinical tool to identify
3
lentivirus-host interactions that may be considered contributory to genotoxicity to
improve safer lentiviral vector design for gene therapy.
safety associated with vector related genotoxicity is of concern and currently available
models are not reliably predictive of safety in humans. We have developed hInGeTox
as the first human in vitro platform that uses induced pluripotent stem cells and their
hepatocyte like cell derivatives to better understand vector-host interactions that relate
vectors to their potential genotoxicity. Using lentiviral vectors carrying the eGFP
expression cassette under SFFV promoter activity, that only differ by their LTR and
SIN configuration, we characterised vector host interactions potentially implicated in
genotoxicity. To do this, lentiviral infected cells were subjected to an array of assays
and data from these was used for multi-omics analyses of vector effects on cells at early
and late harvest time points. Data on the integration sites of lentiviral vectors in cancer
genes and differential expression levels of these genes, showed that both vector
configurations are capable of activating cancer genes. Through IS tracking in bulk
infected cell populations, we also saw an increase in the viral sequence count in cancer
genes present over time which were differentially regulated. RNASeq also showed each
vector had potential to generate fusion transcripts with the human genome suggestive
of gene splicing or vector mediated readthrough from the internal SFFV promoter.
Initially, after infection, both vector configurations were associated with differential
expression of genes associated cytokine production, however, after culturing over time
there were differences in differential expression in cells infected by each LV. This was
marked in particular by the expression of genes involved in the response to DNA
damage in cells transduced by the SIN vector, suggesting effects likely to prevent
tumour development, in contrast to the expression of genes involved in methylation,
characteristic of tumour development, in cells transduced by the LTR vector. Both sets
of lentiviral infected cells were also found associated with differential expression of
MECOM and LMO2 genes known to be associated with clonal dominance, supporting
their potential genotoxicity. Alignment of transcriptomic signatures from iPSC and
HLC infected cultures with known cancer gene signatures showed the LTR vector with
a higher cancer score than the SIN vector over time in iPSC and also in HLC, which
further suggests higher genotoxic potential by the LTR configuration lentivirus.
By application of hInGeTox to cells infected with LV at the pre-clinical stage of
development, we hope that hInGeTox can act as a useful pre-clinical tool to identify
3
lentivirus-host interactions that may be considered contributory to genotoxicity to
improve safer lentiviral vector design for gene therapy.
| Original language | English |
|---|---|
| Journal | Gene Therapy |
| DOIs | |
| Publication status | Published - 15 Jul 2025 |