Suezanne E. Parker

Plasmid DNA Malaria Vaccine: The Potential for Genomic Integration after Intramuscular Injection

Plasmid-based (naked DNA) genetic vaccines are now entering clinical trials to test their safety and efficacy in healthy human volunteers. A safety concern unique to this new class of vaccines is the potential risk of deleterious integration into host cell genomic DNA following direct intramuscular injection. To address this issue experimentally, a preclinical safety study was conducted in mice to determine the structural nature of plasmid DNA sequences persisting in total muscle DNA at both 30 and 60 days following a single intramuscular injection of a plasmid expressing the Plasmodium falciparum circumsporozoite protein. In a protocol described for the first time, total DNA was extracted from muscle tissue and was subsequently linearized with a restriction endonuclease to enable agarose gel size fractionation of all extrachromosomal plasmid DNAs from high molecular weight mouse genomic DNA. Using PCR assays to quantitate plasmid-specific sequences, it was found that the amount of plasmid DNA persisting in muscle tissue varied but averaged about 10 fg per microgram of genomic DNA (in the range of 1500 copies per 150,000 genomes). In two of four separate experimental injections of mouse muscle, PCR assays of genomic DNA fractions indicated that agarose gel purification removed plasmid DNA down to a level of < or =3 copies per 150,000 mouse genomes. In the two other experimental samples, 3-30 copies of plasmid DNA remained associated with purified genomic DNA. The time following injection (i.e., 30 or 60 days) was not a factor in the number of copies of plasmid associating with genomic DNA and it was not possible to conclude if such sequences were covalently linked to genomic DNA or simply adventitiously associated with the genomic DNA. However, if an assumption is made that the highest level plasmid DNA found associated with genomic DNA (i.e., 30 copies) represented covalently integrated plasmid inserts and that each insert resulted in a mutational event, the calculated rate of mutation would be 3000 times less than the spontaneous mutation rate for mammalian genomes. This level of integration, if it should occur, was not considered to pose a significant safety concern.

Plasmid DNA Malaria Vaccine: Tissue Distribution and Safety Studies in Mice and Rabbits

To evaluate the safety of a plasmid DNA vaccine, tissue distribution studies in mice and safety studies in mice and rabbits were conducted with VCL-2510, a plasmid DNA encoding the gene for the malaria circumsporozoite protein from Plasmodium falciparum (PfCSP). After intramuscular administration, VCL-2510 plasmid DNA was detected initially in all of the highly vascularized tissues, but at later time points was found primarily in the muscle at the site of injection, where it persisted for up to 8 weeks. After intravenous administration, plasmid DNA initially distributed at a relatively low frequency to all the tissues examined except the gonads and brain. However, plasmid DNA rapidly cleared, and by 4 weeks postadministration could be detected only in the lung of one of six animals evaluated. In a safety study in mice, eight repeated intramuscular injections of VCL-2510 at plasmid DNA doses of 1, 10, and 100 microg had no adverse effects on clinical chemistry or hematology, and did not result in any organ pathology or systemic toxicity. In a safety study in rabbits, six repeated intramuscular injections of VCL-2510 at plasmid DNA doses of 0.15 and 0.45 mg had no discernible effects on clinical chemistry, hematology, or histopathology. No evidence of autoimmune-mediated pathology, anti-nuclear antibodies (ANA), or antibodies to dsDNA were observed in the mouse or rabbit studies.

Safety, tolerability, and lack of antibody responses after administration of a PfCSP DNA malaria vaccine via needle or needle-free jet injection, and comparison of intramuscular and combination intramuscular/intradermal routes

Introduction of a new vaccine requires choosing a delivery system that provides safe administration and the desired level of immunogenicity. The safety, tolerability, and immunogenicity of three monthly 2.5-mg doses of a PfCSP DNA vaccine were evaluated in healthy volunteers as administered intramuscularly (IM) by needle, IM by jet injection (Biojector or IM/intradermally (ID) by jet injection. Vaccine administration was well-tolerated. Adverse events were primarily mild and limited to the site of injection (98%). Jet injections (either IM or ID) were associated with approximately twice as many adverse events per immunization as needle IM, but nevertheless were strongly and consistently preferred in opinion polls taken during the study. No volunteers had clinically significant biochemical or hematologic changes or detectable anti-dsDNA antibodies. In conclusion, the injection of Plasmodium falciparum circumsporozoite (PfCSP) DNA vaccine appeared to be safe and well-tolerated when administered by any of the three modes of delivery. However, despite improved antibody responses following both jet injection and ID delivery in animal models, no antibodies could be detected in volunteers by immunofluorescence antibody test (IFAT) or enzyme-linked immunosorbent assay (ELISA) after DNA vaccination.

Studies of direct intratumoral gene transfer using cationic lipid-complexed plasmid DNA

Cationic lipid-mediated gene transfer is a safe and effective means of delivering potent immunomodulatory cytokines directly into tumors. This approach avoids undesirable side effects, including systemic toxicities. To investigate key factors affecting intratumoral (i.t.) gene transfer, cationic lipid-DNA complexes were injected into subcutaneous human melanoma tumors in severe combined immunodeficient mice. Animals received i.t. injections of VR1103, a DNA plasmid encoding the gene for human interleukin-2 (IL-2), either alone or complexed with the cationic lipid N-(1-(2,3-dimyristyloxypropyl)-N,N-dimethyl-(2-hydroxyethyl) ammonium bromide/dioleoyl phosphatidylethanolamine (DMRIE/DOPE). Tumors were subcultured and supernatants were tested for IL-2 secretion by enzyme-linked immunosorbent assay. IL-2 secretion was consistently higher when lipid:DNA (L:D) complexes were formulated at high L:D ratios (wt/wt), and IL-2 transgene expression increased in a DNA dose-dependent manner. A comparison of naked plasmid and lipid-complexed DNA revealed that lipid complexes were more effective for i.t. gene transfer. Using an enhanced green fluorescent protein reporter plasmid and flow cytometry, i.t. transfection efficiency was 1.74% (± 1.08%). Tumor injection technique, including injection volume and location, had a limited impact on i.t. gene transfer. These results indicate that the formulation and dosage of cationic L:D complexes, but not injection technique, play a key role in determining the level of i.t. transgene expression.

The development of a bicistronic plasmid DNA vaccine for B-cell lymphoma

Tumor vaccines are a promising alternative to chemotherapy for the treatment of metastatic cancer. To be effective and safe, a therapeutic cancer vaccine should specifically target antigens expressed only on metastatic tumor cells. A vaccine directed against the unique surface immunoglobulin or idiotype expressed on non-Hodgkins B-cell lymphoma fulfills these criteria, as both primary and metastatic tumor cells express tumor specific immunoglobulins. Using the murine 38C13 B-cell lymphoma tumor as a model system, a plasmid DNA vaccine was designed to express a bicistronic mRNA encoding both the light and heavy tumor immunoglobulin (idiotype) proteins expressed on the surface of the 38C13 tumor. To increase the immunogenicity of the plasmid DNA vaccine, each of the murine variable domains (light and heavy) were fused to their respective human immunoglobulin constant domains. In addition, a eukaryotic expression cassette was constructed to effect both high-level expression of the mouse/human chimeric immunoglobulin, and to elicit a protective immune response in vivo. Unique Sfi I restriction sites were used for the rapid cloning of any tumor specific immunoglobulin idiotype domains and a series of plasmid constructs were made to test changes to the J domain and/or the human C domain to insert the Sfi I restriction sites. Such changes were found to have significant effects on both expression and immunogenicity. Vaccination of mice with prototype idiotype vaccines was found to generate a protective immune response to the 38C13 tumor. This study indicates that a novel bicistronic plasmid DNA-based vaccine can be used to develop a tumor specific vaccine against B-cell lymphoma.

DNA vaccines for cancer therapy

Vaccination with a tumour antigen-expressing plasmid DNA (pDNA) is a novel approach to human cancer immunotherapy. Initial results in preclinical rodent tumour models are promising, revealing that pDNA cancer vaccines can elicit both humoral, as well as cell-mediated immunity and, in some cases, protect against tumour growth. Compared to peptide, viral or dendritic cell vaccines, the delivery of tumour antigens using pDNA has the advantages of ease of manufacture, lack of toxicity and broad applicability to large populations. With advances in modern genomics strategies and the identification of an increasing number of tumour antigen genes, pDNA-based cancer vaccines may be used in the future to treat a wide variety of human cancers.

Local Delivery of Therapeutic Proteins by Intratumoral Injection of Plasmid DNA-Lipid Complexes.

There are several strategies by which one may deliver a plasmid DNA (pDNA) encoding a therapeutic gene to a tumor. One may transfect cells ex vivo, single cell clone, expand the clone in vitro, and reinject the cells at the tumor site. This is a labor-intensive process and is especially impractical for human tumor therapy. Another method is intramuscular (im) injection of the therapeutic pDNA to achieve circulating levels of the protein (discussed in Chapter 14 by Horton and Parker). A third method is to directly inject the therapeutic pDNA into the tumor. For accessible neoplasms, this is a simple procedure, and can be useful for delivery of a therapeutic gene, such as a cytokine gene, to the tumor site. Using this technique, one may achieve high local levels of a therapeutic protein, yet have low systemic levels, thereby reducing side effects (1,2). In addition, producing a cytokine locally may attract immune cells to the tumor site and promote an antitumor immune response (1-3). Furthermore, certain cytokines may be more effective when delivered locally, rather than systemically (Horton, unpublished results).

Systemic Delivery of Therapeutic Proteins by Intramuscular Injection of Plasmid DNA

In vivo delivery of a cytokine gene to treat a tumor has usually involved either injection of ex vivo transfected cells around the tumor site or direct intratumoral injection of a virus or plasmid DNA (pDNA) vector encoding the cytokine gene (1,2). In this manner, transfected cells in or around the tumor site may secrete cytokine locally and stimulate an antitumor immune response (3,4). Recently, a new method of cytokine gene delivery for treating tumors was described. In this method, a naked pDNA encoding a cytokine, in this case, interferon-α (IFN-α), was injected intramuscularly (im) into C57BL/6 mice bearing solid or metastatic B16F10 melanoma tumors (5). The mice treated in this manner had a striking inhibition of tumor growth.