Cell-culture assays reveal the importance of retroviral vector design for insertional genotoxicity.
Retroviral vectors with long terminal repeats (LTRs), which contain strong enhancer/promoter sequences at both ends of their genome, are widely used for stable gene transfer into hematopoietic cells. However, recent clinical data and mouse models point to insertional activation of cellular proto-oncogenes as a dose-limiting side effect of retroviral gene delivery that potentially induces leukemia. Self-inactivating (SIN) retroviral vectors do not contain the terminal repetition of the enhancer/promoter, theoretically attenuating the interaction with neighboring cellular genes. With a new assay based on in vitro expansion of primary murine hematopoietic cells and selection in limiting dilution, we showed that SIN vectors using a strong internal retroviral enhancer/promoter may also transform cells by insertional mutagenesis. Most transformed clones, including those obtained after dose escalation of SIN vectors, showed insertions upstream of the third exon of Evi1 and in reverse orientation to its transcriptional orientation. Normalizing for the vector copy number, we found the transforming capacity of SIN vectors to be significantly reduced when compared with corresponding LTR vectors. Additional modifications of SIN vectors may further increase safety. Improved cell-culture assays will likely play an important role in the evaluation of insertional mutagenesis.
Modlich
Ute
U
Department of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany.
Bohne
Jens
J
Schmidt
Manfred
M
von Kalle
Christof
C
Knöss
Sabine
S
Schambach
Axel
A
Baum
Christopher
C
D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000166:cytology / Q000821:virology; D018929:Cell Culture Techniques; D004279:DNA, Viral / Q000235:genetics; D005786:Gene Expression Regulation; D015316:Genetic Therapy / Q000009:adverse effects / Q000379:methods; D005822:Genetic Vectors; D006801:Humans; D007938:Leukemia / Q000209:etiology; D051379:Mice; D008810:Mice, Inbred C57BL; D016254:Mutagenesis, Insertional; D011519:Proto-Oncogenes; D012190:Retroviridae / Q000235:genetics*; D012449:Safety; D020079:Terminal Repeat Sequences; D014161:Transduction, Genetic
D004279:DNA, Viral