+------------+---------------------------------------------------------+
| **A        | Due to the risk from emerging (*e.g*., SARS virus) and  |
| bstract:** | re-emerging (pandemic influenza virus strains and West  |
|            | Nile virus) infectious pathogens and the heightened     |
|            | risk of bioterrorism, there is an increased need for    |
|            | rapid vaccine development. Immortalized (neoplastic)    |
|            | cells are the cell substrate of choice for this effort. |
|            | However, unresolved safety concerns with the use of     |
|            | neoplastic cells for vaccine manufacture present        |
|            | challenges. For example, while there is an              |
|            | international consensus concerning the amount of        |
|            | residual cell-substrate DNA in vaccines prepared in     |
|            | cells for which there is extensive human experience     |
|            | (*e.g*., MRC-5, WI-38, VERO), no agreement has been     |
|            | reached with respect to neoplastic cell lines that are  |
|            | tumorigenic or were derived from tumors. The            |
|            | possibility that DNA from these cells could be          |
|            | oncogenic remains a concern. To address this concern,   |
|            | we are developing the reagents necessary for assays     |
|            | that can be used to evaluate the risk of oncogenic      |
|            | activity of cell-substrate DNA. Expression plasmids for |
|            | the cloned oncogenes human *H-ras* and mouse *c-myc*    |
|            | have been constructed and inoculated into mice. In a    |
|            | preliminary, single assay experiment, tumors were found |
|            | only in mice inoculated with both *H-ras* and *c-myc*   |
|            | (*H-ras* alone was insufficient). NIH Swiss mice appear |
|            | to be more susceptible than C57BL/6, and newborns       |
|            | appear to be more susceptible than adults. Cell lines   |
|            | were established from these tumors, and PCR analysis of |
|            | their DNA demonstrated that the *ras* and *myc* genes   |
|            | were present in all lines. A possible dose-response     |
|            | relationship was revealed, as a 10-fold reduction in    |
|            | the amount of DNA inoculated resulted in loss of        |
|            | oncogenicity, suggesting a two-hit mechanism for tumor  |
|            | induction. These experiments demonstrate that activated |
|            | cellular oncogenes can be oncogenic in animals. In      |
|            | contrast to current assumptions, we conclude that       |
|            | residual DNA from neoplastic cells that carry activated |
|            | oncogenes do represent some level of risk for oncogenic |
|            | activity. To assess these risks for viral vaccines      |
|            | developed in neoplastic cells, *in vivo* oncogenicity   |
|            | assays are needed. Based on our single assay data, we   |
|            | can calculate a safety factor of 10e6-10e7 for the risk |
|            | of a tumor-inducing event by a vaccine containing 10 ng |
|            | of residual neoplastic cell DNA. However, as 2-hit      |
|            | biological processes can be quite variable, it is       |
|            | essential to confirm the initial results, and any risk  |
|            | estimates based on our initial results must be          |
|            | considered to be preliminary. In this proposal, we plan |
|            | to repeat these experiments and expand them to develop  |
|            | a database that can be used to establish a sensitive    |
|            | *in vivo* assays to assess quantitatively the oncogenic |
|            | activity of cellular DNA. This will require: additional |
|            | DNA plasmids to be constructed and evaluated; the       |
|            | testing of transfection \"facilitators\" to assess      |
|            | their effect on the sensitivity of oncogene detection;  |
|            | and the testing of additional strains of mice. The      |
|            | outcome from the experiments described in this proposal |
|            | should be the establishment of a sensitive and          |
|            | quantitative *in* *vivo* assay for the detection        |
|            | oncogenic activity of DNA.                              |
+------------+---------------------------------------------------------+
| **Specific | 1\. To construct plasmids containing both the *H-ras*   |
| aims:**    | and the *c-myc* genes and use theses plasmid to compare |
|            | the kinetics of tumor induction by the individual       |
|            | *H-ras* and the *c-myc* expression plasmids with        |
|            | plasmids where the oncogenes are expressed on the same  |
|            | plasmid and to establish the dynamic range of the assay |
|            |                                                         |
|            | 2\. To evaluate the effect of DNA transfection          |
|            | \"facilitators\", both cationic lipids and other types, |
|            | on the efficiency of oncogene-induced tumor formation.  |
|            |                                                         |
|            | 3\. To establish the most sensitive animal model for    |
|            | detecting oncogenic activity of these plasmids by       |
|            | inoculation into T-cell deficient adult and newborn     |
|            | nude mice and CD3 epsilon mice, which are deficient in  |
|            | both T cells and NK cells. In the future, it may also   |
|            | be necessary to evaluate their oncogenic activity in    |
|            | mice genetically modified to be more susceptible to     |
|            | neoplastic development (\"pre-initiated\" Tg.AC mice,   |
|            | constitutively \"promoted\" K6.ODC mice, and possibly   |
|            | p53 heterozygous mice). However, due to the costs of    |
|            | these mice, these experiments are beyond the scope of   |
|            | this proposal.                                          |
|            |                                                         |
|            | 4\. To determine the tumor-forming potential of the     |
|            | cells isolated from the *myc*-*ras*--induced tumors and |
|            | to confirm the role of the *H-ras* and *c-myc* genes in |
|            | determining the tumorigenic phenotype of the            |
|            | transformed cells by blocking their expression with     |
|            | siRNAs directed to *myc* and *ras* mRNAs.               |
|            |                                                         |
|            | 5\. To assess the oncogenic activity of DNA and         |
|            | chromatin from the *ras*-*myc*--induced tumor cells,    |
|            | from other types of weakly (the Ad5 transformed 293)    |
|            | and highly (HeLa) tumorigenic cells, and from vaccine   |
|            | cell substrates (VERO), and from potential vaccine cell |
|            | substrates (MDCK).                                      |
+------------+---------------------------------------------------------+
| **Bac      | For over forty years, neoplastic cells have not been    |
| kground:** | permitted for the manufacture of vaccines, a            |
|            | proscription that was due in part to the concerns       |
|            | regarding the potential oncogenicity of their DNA.      |
|            | Because appropriate assays are not available to         |
|            | evaluate the risks associated with residual             |
|            | cell-substrate DNA, it is not possible to objectively   |
|            | assign risks to the use of such cells for vaccine       |
|            | manufacture, making it difficult to assure the public   |
|            | of the safety of vaccines produced in them. Until such  |
|            | assays are developed and validated, the levels of       |
|            | residual cell-substrate DNA from tumorigenic cells that |
|            | CBER can safely permit in vaccines cannot be            |
|            | ascertained objectively. Our work is directed towards   |
|            | establishing sensitive and quantitative *in vivo*       |
|            | assays to assess the oncogenic activity of DNA from all |
|            | types of mammalian cells. The resolution of this issue  |
|            | for the manufacture of vaccines against emerging and    |
|            | re-emerging infectious pathogens and bioterrorism       |
|            | agents should have important consequences for the       |
|            | regulation of cell substrates in general, as these      |
|            | types of substrates have been proposed for the          |
|            | production of other vaccines (*e.g.* HIV/AIDS).         |
+------------+---------------------------------------------------------+

  ---------------- ----------------------------------------------------------
  **Study          Our earlier work (OSCC funded 2001 -- 2002) was directed
  design/Plan:**   at determining whether the establishment of a quantitative
                   *in vivo* assay to assess DNA oncogenicity was possible.
                   Expression plasmids for the cloned oncogenes human *H-ras*
                   and mouse *c-myc* were constructed and inoculated into
                   different strains of mice. Tumors developed only in mice
                   inoculated with both *H-ras* and *c-myc* (*H-ras* alone
                   was insufficient), and a dose-response relationship was
                   detected (a 10-fold reduction in the amount of DNA
                   inoculated resulted in loss of oncogenicity, indicating a
                   two-hit mechanism). It was found that NIH Swiss mice were
                   more susceptible than C57BL/6, and newborns were more
                   susceptible than adults. Cell lines were established from
                   the tumors, and PCR analysis of their DNA demonstrated
                   that the *ras* and *myc* genes were present in all lines.
                   These experiments demonstrated that an *in vivo*
                   oncogenicity assay is possible. To develop such an assay,
                   several critical issues need to be addressed: 1. To
                   document whether a two-hit mechanism is operating and to
                   increase the dynamic range of the assay, plasmids
                   containing expression cassettes in different orientations
                   for both the *ras* and the *myc* genes will be
                   constructed. The activity of these double plasmids will be
                   first assessed using *in vitro* transformation assays on
                   NIH 3T3 and Rat-1 cells, and their activity and dose
                   response will be compared with the separate plasmids.
                   These experiments will document whether two-hit kinetics
                   are operating *in vitro*. The plasmid(s) with the highest
                   activity *in vitro* will be tested initially in Swiss and
                   nude mice, both newborn and adult, to evaluate the
                   sensitivity of these models and document whether the
                   double plasmids are more efficient *in vivo* and whether
                   two-hit kinetics are operating. 2. To evaluate the effect
                   of DNA transfection \"facilitators\" on the efficiency of
                   oncogene-induced tumor formation, a number of agents, both
                   cationic lipids (*e.g*., Lipofectamine, DIMRIE, Effectene)
                   and other types (PolyFect), will be tested using the
                   double expression plasmids (should they be more efficient)
                   in several strains of mice. 3. Mouse strains with various
                   immunodeficiencies will be tested. Our original study
                   included introducing *H-ras* into the skin of
                   constitutively \"promoted\" K6.ODC mice, which only
                   require an \"initiator\" for tumor induction. Due to
                   difficulties in breeding these mice, this part of the
                   original study has just begun. The mouse strains to be
                   tested in this proposal include adult and newborn nude
                   mice and CD3 epsilon mice. 4. To confirm the role of the
                   *H-ras* and *c-myc* genes in tumor formation, the
                   tumor-forming potential of the *ras-myc* tumor cells will
                   be determined after silencing Ras and Myc expression using
                   small interfering (si)RNA methodology. 5. Once the most
                   sensitive mouse strain(s) has (have) been identified, the
                   most efficient facilitator identified, and the assay has
                   been standardized, we will then be in a position to ask,
                   using a controlled assay, the critical question of whether
                   cellular DNA that contains dominant activated cellular
                   oncogenes (DNA from the *ras*-*myc*--induced tumor cell
                   lines), dominant viral oncogenes, DNA from weakly (HEK
                   293) and highly (HeLa) tumorigenic cells, and DNA from
                   vaccine cell substrates (VERO) can induce tumors *in
                   vivo*. In addition, because residual cell-substrate DNA is
                   in the form of chromatin rather than free DNA, this form
                   of cellular DNA can be inoculated as well.

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