Endogenous Murine Leukemia Virus DNA Sequences in Murine Cell Lines: Implications for Gene Therapy Safety Testing by PCR

Abstract

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.

Gene Therapy for Canine alpha-L-Iduronidase Deficiency: In Utero Adoptive Transfer of Genetically Corrected Hematopoietic Progenitors Results in Engraftment but Not Amelioration of Disease

“…The adherent LTMC cells were harvested by trypsinization and aliquots of cells were used in progenitor and molecular assays of gene transfer. Culture supernatant was tested in a marker rescue assay for replicationcom petent retrovirus (Allan et al, 1996). The remaining cells were either cryopreserved or infused directly.…”

“…LCa IDSN and LN supernatants were produced by the PA317 retroviral packaging cell line (Miller and Buttimore, 1986), and M48a ID was produced by the C CRIP retroviral packaging cell line (Danos and Mulligan, 1988). To ensure the absence of replication-comp etent retrovirus, supernatants from producer cell lines were routinely assayed by a sensitive marker rescue assay (Allan et al, 1996).…”

Proteomic profiling of human embryonic stem cell-derived microvesicles reveals a risk of transfer of proteins of bovine and mouse origin

“…Many mouse strains constitutively express proteins of retroviral origin, and these proteins can be detected in CM [50,51]. Although Amit et al [52] reported that hESC maintained with mouse feeder cells are not infected with mouse viruses, it is known that under certain circumstances latent viruses can be spontaneously activated in cells that previously tested negative for infectious virus [53]. Thus we cannot completely rule out the possibility that retroviral Gag protein originating from mouse feeder cells is indicative of the production of active virus.…”

In UteroInjection ofα-L-Iduronidase-Carrying Retrovirus in Canine Mucopolysaccharidosis Type I: Infection of Multiple Tissues and Neonatal Gene Expression

“…The infection potential of hightiter clones and proviral a-ID expression were confirmed by transduction of MPSI canine kidney cells. To ensure the absence of replication-competent retrovirus, supernatants from producer cell lines were assessed by a sensitive marker rescue assay (Allan et al, 1996). Two PG-13 clones, A12 and C6, were used for all further studies.…”