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| **          | Number range CHAPTER The ability of plasmid DNA        |
| Abstract:** | vaccines to integrate into the genome of host cells is |
|             | a major safety concern. Studies conducted in the late  |
|             | 1990's demonstrated that "conventional" DNA vaccines   |
|             | (plasmids dissolved in saline and administered         |
|             | intramuscularly) integrated at a frequency far lower   |
|             | that the spontaneous mutation rate. Based on that      |
|             | finding, clinical trials to study the safety and       |
|             | immunogenicity of "conventional" DNA vaccines were     |
|             | initiated.                                             |
|             |                                                        |
|             | Novel strategies designed to improve DNA vaccine       |
|             | immunogenicity by substantially increasing plasmid     |
|             | uptake by host cells were recently developed.          |
|             | Preliminary findings suggest these strategies,         |
|             | specifically in vivo electroporation and liposome      |
|             | encapsulation, may have the undesirable side effect of |
|             | increasing the frequency of plasmid integration by     |
|             | several orders of magnitude. This proposal will        |
|             | address this safety issue.                             |
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| **Specific  | 1\) Determine whether electroporation and/or liposome  |
| aims:**     | encapsulation alter the biodistribution of DNA         |
|             | vaccines in vivo.                                      |
|             |                                                        |
|             | 2\) Determine whether electroporation and/or liposome  |
|             | encapsulation alter the persistence of DNA vaccines in |
|             | vivo.                                                  |
|             |                                                        |
|             | 3\) Determine whether electroporation and/or liposome  |
|             | encapsulation increase the integration frequency of    |
|             | DNA vaccines in vivo.                                  |
|             |                                                        |
|             | 4\) Develop strategies to rapidly and accurately       |
|             | monitor integration events, improving CBER's ability   |
|             | to assess whether forthcoming changes in DNA vaccine   |
|             | composition and delivery adversely impact safety.      |
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| **Ba        | Number range CHAPTERDNA vaccines encoding              |
| ckground:** | pathogen-specific antigens are capable of inducing     |
|             | protective immunity against a variety of infectious    |
|             | microorganisms. For example, considerable effort is    |
|             | currently being directed towards developing DNA        |
|             | vaccines against category A biothreat agents in order  |
|             | to reduce the threat posed by biowarfare pathogens.    |
|             |                                                        |
|             | One of the major safety concerns raised by DNA         |
|             | vaccines is their potential to integrate into the host |
|             | genome, causing mutations, genetic instability,        |
|             | translocation, or gene activation/ inactivation. The   |
|             | pathogenic consequences of DNA plasmid integration     |
|             | range from dysregulation of normal gene expression to  |
|             | tumor formation. At the recommendation of the FDA      |
|             | Vaccines Advisory Committee and the NIH Recombinant    |
|             | DNA Advisory Committee, pre-clinical safety studies    |
|             | were conducted to establish the integration frequency  |
|             | of "conventional" DNA vaccines. Based on results       |
|             | showing that the spontaneous mutation rate             |
|             | substantially exceeded the plasmid integration rate,   |
|             | clinical trials of "conventional" DNA vaccines were    |
|             | allowed to proceed.                                    |
|             |                                                        |
|             | Very recently, novel delivery strategies designed to   |
|             | increase the immunogenicity of DNA vaccines were       |
|             | developed. These strategies use in vivo                |
|             | electroporation and liposome encapsulation to increase |
|             | the uptake of antigen-encoding plasmids by 100 --      |
|             | 1,000 fold. Future strategies are likely to have even  |
|             | more profound effects on plasmid uptake. Preliminary   |
|             | evidence suggests that such delivery strategies may    |
|             | increase the frequency of plasmid integration by many  |
|             | orders of magnitude. Given the risk posed by high      |
|             | levels of integration, it is critical that CBER gain   |
|             | objective and reliable data on the biodistribution,    |
|             | persistence and integration of DNA plasmids delivered  |
|             | by these novel approaches. Unfortunately, industry has |
|             | not taken the lead in this issue, since experimental   |
|             | evidence of increased integration interferes with      |
|             | their vaccine development plans. The studies described |
|             | below will assess the effect of novel methods of DNA   |
|             | vaccine delivery on plasmid persistence and            |
|             | integration in vivo. They will also improve CBER's     |
|             | ability to provide guidance to industry on the conduct |
|             | of pre-clinical studies designed to rapidly and        |
|             | efficiently assess whether changes in DNA vaccine      |
|             | formulation or delivery increase the rate of           |
|             | integration.                                           |
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| **Study     | Number range CHAPTERThis project will examine whether  |
| des         | novel strategies of DNA vaccine delivery result in a   |
| ign/Plan:** | significant increase in the frequency of plasmid       |
|             | integration. All experiments will be performed using   |
|             | two different plasmids (representative of those        |
|             | currently in clinical trials). The biodistribution,    |
|             | persistence and integration frequency of these         |
|             | plasmids will be evaluated after i) conventional       |
|             | intramuscular delivery in saline ii) electroporation   |
|             | (novel strategy #1) and iii) liposome formulation      |
|             | (DMRIE/DOPE, novel strategy #2). Groups of mice will   |
|             | be sacrificed 2 days post plasmid administration (to   |
|             | monitor the initial uptake and distribution of the     |
|             | DNA), and at days 28 and 56 (to monitor plasmid        |
|             | persistence). Experiments will utilize 10              |
|             | mice/sex/group, and will be repeated to confirm the    |
|             | statistical significance of observed outcomes. DNA     |
|             | from the gonads, spleen, liver, draining lymph nodes,  |
|             | kidneys, bone marrow, and site of injection (muscle    |
|             | and/or skin) will be studied. Taqman RT-PCR will be    |
|             | used to detect and quantify the amount of plasmid      |
|             | present in each sample using primers internal to the   |
|             | plasmid. Our group has considerable experience in the  |
|             | delivery of DNA plasmids, and in detecting their       |
|             | localization and persistence by RT-PCR. All of the     |
|             | reagents and equipment needed to perform the           |
|             | experiments described above are currently available in |
|             | the laboratory.                                        |
|             |                                                        |
|             | The amount of integrated vs non-integrated plasmid     |
|             | present at relevant sites (those with high levels of   |
|             | persisting plasmid) will be studied at multiple time   |
|             | points to determine the rapidity with which            |
|             | integration occurs. This will entail quantifying the   |
|             | total amount of plasmid present in each target tissue, |
|             | and then determining what fraction has integrated at   |
|             | each time point. This will be accomplished by cutting  |
|             | the genomic DNA with SfiI (an 8-cutter that cleaves at |
|             | a single site in the plasmids), followed by size       |
|             | fractionation over agarose gels. Integration will be   |
|             | characterized by persistence of plasmid DNA (8 kb) in  |
|             | the high molecular weight fraction of genomic DNA      |
|             | (\>24 kb in length). Up to 5 iterations of the SfiI    |
|             | digestion/genomic DNA isolation will be performed (as  |
|             | necessary) to insure that non-integrated plasmid has   |
|             | been eliminated from the sample. These studies will    |
|             | utilize up to 20 mice/time point, since tissues will   |
|             | need to be pooled to obtain sufficient DNA for         |
|             | multiple rounds of purification.                       |
|             |                                                        |
|             | We estimate that at least 5 time points/vaccination    |
|             | strategy will need to be evaluated. Initially studies  |
|             | will focus on tissues isolated 1 week, 1 month, and 2  |
|             | months after vaccination. Results from these studies   |
|             | will guide the selection of additional time points for |
|             | analysis.                                              |
|             |                                                        |
|             | The results of these studies will clarify how each     |
|             | method of DNA vaccine delivery affects the frequency   |
|             | of plasmid integration, and will provide reliable data |
|             | on the magnitude of such effects. They will also       |
|             | establish the kinetics with which integration occurs.  |
|             | These results will substantially improve CBER's        |
|             | understanding of how novel vaccination strategies      |
|             | alter the integration frequency (and thus safety) of   |
|             | DNA vaccines. This project will also improve our       |
|             | ability to help sponsors design experiments to rapidly |
|             | determine whether future changes in vaccine design or  |
|             | delivery significantly increase the risk of            |
|             | integration.                                           |
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|             |                                                        |
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