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

Abstract

Cutaneous gene therapy can be envisioned through the use of keratinocyte stem cell clones in which retroviral genotoxic risks can be pre-assessed. While transactivation of cellular genes by the retroviral long terminal repeat enhancer has been proven in experimental and clinical settings, the formation of chimeric viral-cellular transcripts originated by the inefficient termination (read-through) of retroviral transcripts remains to be studied in depth. We now demonstrate the widespread presence of viral-cellular fusion transcripts derived from integrated proviruses in keratinocytes transduced with self-inactivating (SIN) retroviral vectors. We have detected high molecular weight RNAs in northern blot analysis of retroviral vector expression in individual cell clones. Characterization of some of these transcripts revealed that they originate from genes located at the proviral integration sites. One class of transcripts corresponds to fusions of the viral vectors with intronic sequences, terminating at cryptic polyadenylation sites located in introns. A second class comprises fusion transcripts with coding sequences of genes at the integration sites. These are generated through splicing from a cryptic, not previously described donor site in the lentiviral vectors to exons of cellular genes, and have the potential to encode unintended open reading frames, although they are downregulated by cellular mechanisms. Our data contribute to a better understanding of the impact of SIN lentiviral vector integration on cellular gene transcription, and will be helpful in improving the design of this type of vectors.

Gene therapy: too much splice can spoil the dish

“…They also found that the presence of a wild-type LTR increased the incidence of aberrant splicing, but since Mavilio and colleagues performed all of their analyses with SIN lentiviral vectors, this nuance gives no real comfort. These findings mirror the recent report of proviral transcriptional read-through transcripts in keratinocytes derived from skin stem cells transduced with SIN lentiviral vectors (15). It is thus likely that some degree of vector-induced aberrant splicing always occurs within a population of retrovirally transduced cells and at least a fraction of these cells harbor RNAs generated by 5ʹ or 3ʹ fusion of viral and cellular transcripts ( Figure 1, A and B).…”

Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations

“…Conceivably, the mRNAs produced by read-through transcription within vector sequences undergo regulation by the nonsense mRNA decay (18,19,26). As shown in the work of Almarza et al (20), the noncoding transcripts generated by read-through transcription from the internal promoter of an integrated SINLV are regulated by the nonsense mRNA decay mechanism. This would affect the abundance of specific transcripts with respect to all the potential aberrant transcripts that can be generated by all genomic integrations.…”

“…These polyadenylation sites, however, are used only in a context-dependent manner and when specific requirements are met (18,19). Moreover, viral-cellular fusion transcripts terminating at cryptic polyadenylation sites located in the host cellular genome were found in keratinocytes transduced with SIN LVs (20). Thus, a complex interplay among the presence, relative strength, position, and distance of promoters; splice site consensus sequences; and mRNA polyadenylation signals is ultimately responsible for the production of specific aberrant mRNAs (17,21).…”

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

“…Preclinical and clinical studies suggested that the insertion of retroviral splicing and polyadenylation signals within transcription units may cause posttranscriptional deregulation of gene expression with a certain frequency (3,18,21). This may include aberrant splicing, premature transcript termination, and the generation of chimeric, read-through transcripts originating from vector-borne promoters (21), a classical cause of insertional oncogenesis (22). The propensity of LVs to integrate into the body of transcribed genes increases the probability of such events compared with that of MLV-derived vectors.…”