Oncogenicity of DNA in vivo: Tumor induction with expression plasmids for activated H-ras and c-myc

Division of Viral Products, OVRR, CBER, FDA, Building 29A, Room 3D08, 29 Lincoln Drive, Bethesda, MD 20892, USA.
Biologicals (Impact Factor: 1.21). 06/2008; 36(3):184-97. DOI: 10.1016/j.biologicals.2007.11.003
Source: PubMed


All vaccines and other biological products contain contaminating residual DNA derived from the production cell substrate. Whether this residual cell-substrate DNA can induce tumors in vaccine recipients and thus represent a risk factor has been debated for over 50 years without resolution. As a first step in resolving this issue, we have generated expression plasmids for the activated human H-ras oncogene and for the murine c-myc proto-oncogene. Their oncogenic activity was confirmed in vitro using the focus-formation transformation assay. Two strains of adult and newborn immune-competent mice were inoculated with different amounts of either plasmid alone or with a combination of the H-ras and c-myc plasmids. Tumors developed only in mice inoculated with both plasmids and only at the highest amount of DNA (12.5 microg of each plasmid). The NIH Swiss mouse was more sensitive than the C57BL/6 mouse, and newborn animals were more sensitive than adults. Cell lines were established from the tumors. PCR and Southern hybridization analyses demonstrated that both inoculated oncogenes were present in all of the tumor-derived cell lines and that the cells in the tumors were clonal. Western analysis demonstrated that both oncoproteins were expressed in these cell lines. These results demonstrate that cellular oncogenes can induce tumors following subcutaneous inoculation. Such information provides a possible way of evaluating and estimating the theoretical oncogenic risk posed by residual cell-substrate DNA in vaccines.

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    • "The majority of published studies on rDNA infectivity and oncogenicity are from Peden et al. (2004) at the Food and Drug Administration (FDA). In a recent study on rDNA oncogenicity, they used both H-ras and c-myc to study tumor induction, and it was found that 12.5 mg each of plasmid containing H-ras and c-myc were needed to induce tumor growth in adult or newborn mice (Li et al., 2008). If this information was put into the context of the whole human genome, it translates to genomic DNA in the range of 1–10 g to have similar cell transformation effect because this test was using the specified gene only whereas in a genomic DNA preparation this gene is only a very small proportion of the whole genome. "
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    ABSTRACT: Residual DNA (rDNA) is comprised of deoxyribonucleic acid (DNA) fragments and longer length molecules originating from the host organism that may be present in samples from recombinant biological processes. Although similar in basic structural base pair units, rDNA may exist in different sizes and physical forms. Interest in measuring rDNA in recombinant products is based primarily on demonstration of effective purification during manufacturing, but also on some hypothetical concerns that, in rare cases, depending on the host expression system, some DNA sequences may be potentially infectious or oncogenic (e.g., HIV virus and the Ras oncogene, respectively). Recent studies suggest that a sequence known as long interspersed nucleotide element-1 (LINE-1), widely distributed in the mammalian genome, is active as a retrotransposon that can be transcribed to RNA, reverse-transcribed into DNA and inserts into a new site in genome. This integration process could potentially disrupt critical gene functions or induce tumorigenesis in mammals. Genomic DNA from microbial sources, on the other hand, could add to risk of immunogenicity to the target recombinant protein being expressed, due to the high CpG content and unmethylated DNA sequence. For these and other reasons, it is necessary for manufacturers to show clearance of DNA throughout production processes and to confirm low levels in the final drug substance using an appropriately specific and quantitative analytical method. The heterogeneity of potential rDNA sequences that might be makes the testing of all potential analytes challenging. The most common methodology for rDNA quantitation used currently is real-time polymerase chain reaction (RT-PCR), a robust and proven technology. Like most rDNA quantitation methods, the specificity of RT-PCR is limited by the sequences to which the primers are directed. To address this, primase-based whole genome amplification is introduced herein. This paper will review the recent advancement in rDNA quantitation and recent findings regarding potential risks of immunogenicity, infectivity, and oncogenicity of rDNA.
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    • "Š Number of doses taken to induce an oncogenic or infective event K. Peden et al. (2004), Vaccine Cell Substrate. and L. Sheng et al. (2008) "
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