RNA 3′ Readthrough of Oncoretrovirus and Lentivirus: Implications for Vector Safety and Efficacy

Abstract

The expression of reporter genes driven by the same human elongation factor 1␣ (EF1␣) promoter in murine leukemia virus (MLV)-and human immunodeficiency virus type 1 (HIV-1)-based vectors was studied in either transfected or virally transduced cells. The HIV-1 vectors consistently expressed 3 to 10 times higher activity than the MLV vectors at both the RNA and protein levels. The difference was not attributable to transcriptional interference, alternative enhancer/silencer, or differential EF1␣ intron splicing. Based on nuclear run-on assays, both vectors exhibited similar EF1␣ transcriptional activity. The reduced RNA levels of MLV vectors could not be explained by the decrease in RNA half-lives. Southern analysis of proviral DNA indicated that both HIV-1 and MLV vectors efficiently propagated the EF1␣ intron in the transduced cells. To decipher the discrepancy in transgene expression between MLV and HIV-1 vectors, the role of RNA 3-end processing was examined using a sensitive Cre/lox reporter assay. The results showed that MLV vectors, but not HIV-1 vectors, displayed high frequencies of readthrough of the 3 polyadenylation signal. Interestingly, the polyadenylation signal of a self-inactivating (SIN) HIV-1 vector was as leaky as that of the MLV vectors, suggesting a potential risk of oncogene activation by the lentiviral SIN vectors. Together, our results suggest that an efficient polyadenylation signal would improve both the efficacy and the safety of these vectors.

Risk assessment in skin gene therapy: viral–cellular fusion transcripts generated by proviral transcriptional read-through in keratinocytes transduced with self-inactivating lentiviral vectors

“…It is, however, consistent with an original estimation of read-through transcription from avian leukosis retroviruses at 15% of total proviral transcription. 8 In agreement with previous work on 3¢ reporter activation using transient transfection assays of SIN vector constructs, 10 we found comparable levels of read-through transcription from our K14 promoter-driven SIN g-retroviral and lentiviral proviruses, as estimated by northern blot analysis of viral-cellular fusion transcripts. Therefore, both vectors seem to carry comparable risks concerning this mode of insertional mutagenesis.…”

“…A genotoxicity mechanism that has attracted considerable interest for gene therapy biosafety is the generation of chimeric viral and cellular transcripts. 10,17,18 We have therefore focused our study on the insertional mutagenesis effects caused by inefficient 3¢ RNA processing of retroviral vectors, as the extent and characteristics of this kind of transcription from SIN vector insertions have not yet been fully determined. Our study shows readily detectable, and therefore abundant, read-through-derived viral-cellular transcripts in all K14-enhanced green gluorescent protein SIN lentiviral and g-retroviral vector-infected keratinocyte clones analyzed by northern blot (Figures 1a and b).…”

“…Our study shows readily detectable, and therefore abundant, read-through-derived viral-cellular transcripts in all K14-enhanced green gluorescent protein SIN lentiviral and g-retroviral vector-infected keratinocyte clones analyzed by northern blot (Figures 1a and b). This is a significant finding, as the extent of read-through transcription of SIN retroviral vectors had been estimated before only in transient transfection analysis of vectors carrying a 3¢ reporter cassette, 10,12 but not in the genomic scenario of integrated vector proviruses. It is, however, consistent with an original estimation of read-through transcription from avian leukosis retroviruses at 15% of total proviral transcription.…”

“…g-Retroviral (Moloney leukemia virus)-derived vectors displayed high frequency of 3¢ polyadenylation signal readthrough, as shown in transient transfection assays of retroviral vector constructs carrying a reporter expression cassette situated downstream from the vector sequence. 10,11 U3 deletion in SIN lentiviral vectors results in decreased RNA 3¢ processing and the increased generation of chimeric transcripts, 12 demonstrating that the U3 enhancer deletion, aimed at mitigating insertional mutagenesis by enhancer transactivation, might be the source of potentially troubling unintended transcription of viral-cellular fusion genes.…”

Functional transfer of CD40L gene in human B-cell precursor ALL blasts by second-generation SIN lentivectors

“…60,61 However, the SIN deletion should guarantee a greatly reduced risk of recombination with wild-type sequences in addition to reducing the probability of unwanted transcriptional activation at the insertion sites. 15,19,[62][63][64] Moreover, our vectors contain two cis-acting elements (cPPT and Wpre) which should have optimized the expression of the transgene in the transduced B-ALL cells. [8][9][10][11]24,25 Their direct role, however, cannot be implied by these data, even if our previous data suggest an improved activity of the cPPT element in B-cell lines.…”