Endogenous murine leukemia virus DNA sequences in murine cell lines: Implications for gene therapy safety testing by PCR

被引:7
作者
Allan, DS [1 ]
DeKoven, A [1 ]
Wild, A [1 ]
KamelReid, S [1 ]
Dube, ID [1 ]
机构
[1] QUEENS UNIV,FAC MED,KINGSTON,ON K7L 3N6,CANADA
关键词
retrovectors; safety testing; gene therapy; PCR; murine; leukemia virus;
D O I
10.3109/10428199609054842
中图分类号
R73 [肿瘤学];
学科分类号
100214 ;
摘要
Safety testing for replication-competent retrovirus (RCR) is an important requirement in gene transfer clinical trials using retroviral vectors. A sensitive polymerase chain reaction (PCR) method is one approach to RCR detection. Only in the presence of RCR will the pol-env encoding sequences, necessary for viral replication and packaging, be amplified from proviral DNA in infected indicator cells. To avoid false-positive results in this assay it is crucial that indicator cell lines are free of endogenous retroviral sequences that could also be amplified with pol-env PCR primers. We screened candidate murine indicator cell lines and determined that while Mus dunni is free of detectable pol-env sequences, endogenous retroviral sequences do indeed exist in several cell lines and lead to false-positive results in the PCR assay for RCR. Furthermore, these endogenous retroviral sequences are expressed as RNA transcripts in NIH 3T3 and SC-1 cell lines, as determined by PCR amplification of cDNA but, nevertheless, do not give rise to replication-competent particles. We recognize the potential for murine cell lines to undergo spontaneous rearrangements of endogenous viral sequences in culture and give rise to recombinants containing newly acquired contiguous pol-env sequences. Indicator cell lines should thus be carefully selected and monitored on an ongoing basis when used in safety testing using PCR approaches for the detection of RCR.
引用
收藏
页码:375 / 381
页数:7
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