Jan Schultz

Intradermal delivery of interleukin-12 plasmid DNA by in vivo electroporation.

Gene therapy depends on safe and efficient gene delivery. The skin is an attractive target for gene delivery because of its accessibility. Recently, in vivo electroporation has been shown to enhance expression after injection of plasmid DNA. In this study, we examined the use of electroporation to deliver plasmid DNA to cells of the skin in order to demonstrate that localized delivery can result in increased serum concentrations of a specific protein. Intradermal injection of a plasmid encoding luciferase resulted in low levels of expression. However, when injection was combined with electroporation, expression was significantly increased. When performing this procedure with a plasmid encoding interleukin-12, the induced serum concentrations of gamma-interferon were as much as 10 fold higher when electroporation was used. The results presented here demonstrate that electroporation can be used to augment the efficiency of direct injection of plasmid DNA to skin.

Passive Immunization against β-Amyloid Peptide Protects Central Nervous System (CNS) Neurons from Increased Vulnerability Associated with an Alzheimer's Disease-causing Mutation

To characterize the effects of the familial Alzheimers disease-causing Swedish mutations of amyloid precursor protein (SwAPP) on the vulnerability of central nervous system neurons, we induced epileptic seizures in transgenic mice expressing SwAPP. The transgene expression did not change the seizure threshold, but consistently more neurons degenerated in brains of SwAPP mice as compared with wild-type littermates. The degenerating neurons were stained both by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and by Gallyas silver impregnation. A susceptible population of neurons accumulated intracellular Aβ and immunoreacted with antibodies against activated caspase-3. To demonstrate that increased Aβ levels mediated the increased vulnerability, we infused antibodies against Aβ and found a significant reduction in neuronal loss that was paralleled by decreased brain levels of Aβ. Because the SwAPP mice exhibited no amyloid plaques at the age of these experiments, transgenic overproduction of Aβ in brain rendered neurons susceptible to damage much earlier than the onset of amyloid plaque formation. Our data underscore the possibility that Aβ is toxic, that it increases the vulnerability of neurons to excitotoxic events produced by seizures, and that lowering Aβ by passive immunization can protect neurons from Aβ-related toxicity.

Long-Lasting Anti-Metastatic Efficiency of Interleukin 12-Encoding Plasmid DNA

Intramuscular injection of plasmid DNA encoding both subunits of the cytokine interleukin 12 (IL-12) exhibits strong antimetastatic activity against lung metastases induced by the malignant melanoma cell line B16-F10. The protective effect of IL-12 DNA is long-lasting, since administration of tumor cells 9 days after IL-12 DNA treatment prevented metastasis formation. No effects were observed with empty plasmid controls, DNA encoding the melanoma-associated antigen pmel17/gp100, the granulocyte-macrophage colony-stimulating factor GM-CSF, B7.1, or CpG-containing oligodeoxynucleotides. IL-12 DNA is required during early phases of metastasis formation and is ineffective when administered later. Its efficiency is dose dependent. The cytotoxic T cell response contributes to the antimetastatic effect as evidenced by genetically modified CD8- or perforin knockout mice. Depletion of natural killer (NK) cells by antibodies completely abrogated the effect. In contrast, the IL-12-induced antimetastatic effect was not mediated by interferon gamma (IFN-gamma) or tumor necrosis factor alpha (TNF-alpha) as shown with IFN-gamma receptor and TNF-alpha knockout mice, respectively. Toxic side effects by IL-12 were low. Our results suggest that plasmid DNA encoding IL-12 might have potential value as gene medicine against the initiation of metastasis formation.

Passive immunization against beta-amyloid peptide protects CNS neurons from increased vulnerability associated with an Alzheimer's disease-causing mutation

To characterize the effects of the familial Alzheimers disease-causing Swedish mutations of amyloid precursor protein (SwAPP) on the vulnerability of central nervous system neurons, we induced epileptic seizures in transgenic mice expressing SwAPP. The transgene expression did not change the seizure threshold, but consistently more neurons degenerated in brains of SwAPP mice as compared with wild-type littermates. The degenerating neurons were stained both by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and by Gallyas silver impregnation. A susceptible population of neurons accumulated intracellular Aβ and immunoreacted with antibodies against activated caspase-3. To demonstrate that increased Aβ levels mediated the increased vulnerability, we infused antibodies against Aβ and found a significant reduction in neuronal loss that was paralleled by decreased brain levels of Aβ. Because the SwAPP mice exhibited no amyloid plaques at the age of these experiments, transgenic overproduction of Aβ in brain rendered neurons susceptible to damage much earlier than the onset of amyloid plaque formation. Our data underscore the possibility that Aβ is toxic, that it increases the vulnerability of neurons to excitotoxic events produced by seizures, and that lowering Aβ by passive immunization can protect neurons from Aβ-related toxicity.

Tumor regression of human and murine melanoma after intratumoral injection of IL‐12‐encoding plasmid DNA in mice

Abstract: DNA coding for murine interleukin 12 (IL‐12) prevents the formation of B16‐melanoma metastasis when administered intramuscularly. Here, the antitumor effect of IL‐12‐encoding DNA on established mouse B16 melanoma and human melanoma tumors was investigated in vivo using two animal models: B16 melanoma in C57B/6 mice and human melanoma in nude mice. In B16 melanoma, intratumoral injections of IL‐12‐encoding DNA resulted in highly significant growth retardation when compared with mice injected with control vector. In the case of the human melanoma model, treatment with DNA coding for IL‐12 induced regression of tumors in all cases, with complete disappearance of the tumor in two out of five animals. DNA treatment did not induce systemic side‐effects. In the animals injected with control vector the human melanoma tumors grew expansively. The therapeutic effect of the DNA injection was mediated in part by natural killer (NK) cells as shown by NK‐depletion experiments. An antivascular effect of IL‐12 treatment was evident in histological examination with endothelial thickening and abrupt changes in vessel diameters. These results suggest that intratumoral plasmid DNA coding for IL‐12 holds some promise as a new therapeutic tool for accessible melanoma lesions and should be tested in clinical trial.

Update on Antiviral DNA Vaccine Research (1998–2000)

DNA vaccines can induce protective cellular and humoral immune responses and have therefore been used during the last decade to develop vaccines against a variety of different pathogens. Because current antiviral vaccines predominantly generate humoral immunity, DNA immunization may be especially useful to provide long-term protection against viral diseases that also require cellular immunity (e.g. HIV). A significant number of articles published in the field of DNA vaccines are dealing with viral diseases, reflecting the need for better and alternative vaccination strategies against viruses. The success of DNA immunization depends on a variety of parameters (e.g. type of antigen, method of application and usage of adjuvants). Therefore, different strategies have been explored to modulate the induced immune response with respect to the requirements necessary to protect against a specific pathogen (e.g. induction of mucosal or cell-mediated immunity). The following article provides an update on different aspects of antiviral DNA vaccine research that have previously been reviewed by others.

Induction of long-lasting cytokine effect by injection of IL-12 encoding plasmid DNA

We have recently demonstrated that DNA coding for both subunits of the murine IL-12 heterodimer exhibits a strong antimetastatic effect against B16-melanoma in C57BL/6 mice and after intratumoral injection tumor regression. Here we show that the antimetastatic effect can be detected when the DNA is injected intramuscularly 30 days before tumor cell challenge. A long-term IL-12 expression was measured for up to 50 days in the serum with a peak at day 20 amounting to about 10 ng/mL in C57BL/6 mice. CpG oligodeoxynucleotides also induce IL-12 expression, however, only for a few hours. IL-12 DNA administration induces long-lasting systemic IFN-γ production, whereas IL-4 and TNF-α levels remained undetectable. NK cell–depleted mice showed a strong but reduced expression of murine IL-12. Expression of DNA encoding human instead of murine IL-12 resulted in a significantly lower and transient expression, indicating that not plasmid-derived IL-12 production alone but the immune system of the host contributes to the long-lasting antimetastatic effect. It may be attributable to an autocrine feedback mechanism maintaining murine IL-12 expression, whereby several cell populations including NK cells are involved. Cancer Gene Therapy (2000) 7, 1557–1565

DNA-based vaccine against La Crosse virus: protective immune response mediated by neutralizing antibodies and CD4+ T cells.

La Crosse virus (LACV)-mediated encephalitis is the most frequently reported arboviral disease in the United States, but to date no vaccine against this virus is available. We have established a new animal model, genetically targeted mice lacking a functional interferon type I receptor (IFNAR-1). These mice show an age-independent susceptibility to LACV and develop an acute encephalitis within 6 days of infection, thereby allowing the evaluation of vaccines against LACV. Taking advantage of this knockout mouse model, we have assessed the feasibility of DNA vaccination against this viral disease. Plasmid DNAs, encoding either the virus surface glycoproteins G1 and G2 or the internal nucleocapsid protein N, were used to immunize IFNAR-1-deficient mice. Mice vaccinated with DNA encoding the glycoproteins G1 and G2 produced neutralizing antibodies and exhibited a high degree of protection against challenge with high doses of LACV. Depletion of CD4+ T cells in mice vaccinated with DNA encoding G1/G2 reduced their capacity to control the infection. Virus titration and immunohistological analysis revealed that the protected mice showed no evidence of LACV particles in the brain. This indicates that the vaccine-induced immune response efficiently blocked viral spreading from the primary replication site to the brain. In contrast, immunization with DNA encoding protein N yielded only a partial protective effect that can be attributed to the cellular immune response. Taken together, this study shows that DNA vaccines can be designed to efficiently induce a protective immune response based on neutralizing antibodies and CD4+ T cells.

IP‐10‐encoding plasmid DNA therapy exhibits anti‐tumor and anti‐metastatic efficiency

Abstract:  We report here that the interferon‐induced protein of 10 kDa (IP‐10 or CXCL10) elicits strong anti‐tumor and anti‐metastatic responses in mice when administered by plasmid DNA. Intratumoral but not intramuscular IP‐10 DNA inoculation resulted in reduced tumor formation of malignant melanoma (B16F10) and Lewis lung carcinoma (LL/2) in C57BL/6 mice. In addition, plasmid DNA‐encoding IP‐10 substantially reduced the establishment of metastases when injected systemically by the intramuscular route. In contrast to the primary tumor model, the anti‐metastatic effect of DNA‐encoding IP‐10 was primarily mediated by NK cells. Compared to DNA‐encoding interleukin‐12 (IL‐12), therapy with DNA‐encoding IP‐10 exhibits lower efficacy against primary melanoma tumors but equivalent efficacy against primary Lewis lung tumors and against B16F10 lung metastasis formation. Co‐administration of DNA‐encoding IP‐10 and IL‐12 enhanced the anti‐tumor activity of IL‐12 in the lung metastasis model but had little effect in the local treatment of established subcutaneous tumors. Interestingly, treatment of nude mice lacking T lymphocytes with DNA‐encoding IP‐10 or IL‐12 still resulted in a pronounced reduction of tumor growth or metastasis formation.

DNA vaccination against La Crosse virus.

For the development of effective conventional vaccines or DNA vaccines against viruses, the availability of suitable animal models is an essential prerequisite. For many recently emerging zoonotic viruses, suitable animal models are still missing. We have established a novel small animal model for DNA vaccines using mice lacking a functional interferon-α/β receptor (IFNAR-1). IFNAR-1-deficient mice are highly susceptible to many different viruses despite their ability to mount a normal humoral and cellular immune response. Taking advantage of this animal model, we show that mice can be completely protected from lethal challenge with a single injection of plasmid DNA encoding the viral envelope proteins G1 and G2. By contrast, vaccination with a plasmid encoding the internal nucleocapsid protein N had little effect. In an effort to enhance the protective immune response to N we assessed the efficacy of vaccination with plasmid DNA encoding N in combination with a plasmid encoding the cytokine IL-12 as adjuvant. IL-12 enhanced the survival of mice following viral challenge, but the effect was independent of N indicating the involvement of components of the innate immune system such as NK cells.