Design and Structural Requirements of the Potent and Safe TLR-9 Agonistic Immunomodulator MGN1703.

Single-stranded oligodeoxynucleotides (ODN), containing nonmethylated cytosine-guanine motifs (CpG ODN), are recognized by the innate immune system as "danger signals." CpG ODN are efficacious immunomodulators but require phosphorothioate (PT) or other backbone modifications for metabolic stability, which cause toxicities in mice and primates. We therefore designed a covalently closed DNA molecule (dSLIM(®)) where two single-stranded loops containing CG motifs are connected through a double-stranded stem in the absence of any nonnatural DNA component. The most promising immunomodulator, MGN1703, comprises two loops of 30 nucleotides containing three CG motifs each, and a connecting stem stem of 28 base pairs. MGN1703 stimulates cytokine secretion [interferon (IFN)-α, IFN-γ, interleukin (IL)-12, IL-6, and IL-2] and activates immune cells by increased expression of CD80, CD40, human leukocyte antigen (HLA)-DR and ICAM-1. Efficacy of immunomodulation strictly depends on the descriptive dumbbell shape and size of the molecule. Variations in stem length and loop size lead to reduced potency of the respective members of the dSLIM(®) class. In a representative mouse model, toxicities from injections of high amounts of a CpG ODN-PT and of MGN1703 were evaluated. The CpG ODN-PT group showed severe organ damage, whereas no such or other pathologies were found in the MGN1703 group. Oncological clinical trials of MGN1703 already confirmed our design.

Minimal size MIDGE vectors improve transgene expression in vivo.

Viral and plasmid vectors may cause immunological side-effects resulting from the expression of therapeutically unwanted genes and from CpG motifs contained in their sequence. A new vector type for minimalistic, immunological-defined gene expression (MIDGE) may overcome these problems. MIDGE is a minimal size gene transfer unit consisting of the expression cassette, including promotor, gene and RNA-stabilizing sequences, flanked by two short hairpin oligonucleotide sequences. DNA not encoding the desired gene is reduced to a minimum. To compare transfection efficiencies in vivo hydrodynamics-based, systemic transfection was performed in BALB/c mice with MIDGE vectors and corresponding plasmids. The transfection efficiencies of the MIDGE vectors as measured by luciferase expression were significantly higher in liver (2.5-fold), lung (3.5-fold), kidneys (3.9-fold) and heart (17-fold) as compared to plasmids. The mean numbers of MIDGE vector molecules per cell as measured by quantitative PCR were also significantly higher. These advantages suggest the preferential use of this new vector type for clinical gene therapy studies.

Protection of mice against Philadelphia chromosome-positive acute lymphoblastic leukemia by cell-based vaccination using nonviral, minimalistic expression vectors and immunomodulatory oligonucleotides.

PURPOSE: Childhood Philadelphia chromosome positive (Ph(+)) acute lymphoblastic leukemia (ALL) has a poor prognosis. Because leukemia cell burden is reduced but not eradicated by polychemotherapy, improved treatment strategies should enhance those immune mechanisms responsible for the maintenance of complete remission. The aim of this study was to evaluate the protection of mice challenged with the syngeneic Ph(+) ALL cell line BM185 using genetically modified leukemia cell vaccines and immunomodulating oligonucleotides. EXPERIMENTAL DESIGN: Because retroviral vectors are ineffective at transducing nondividing primary cells from human hematopoietic malignancies, we first evaluated nonviral techniques (electroporation and ballistic transfer) using minimalistic immunogenically defined gene expression vectors to generate B7.1 or granulocyte macrophage colony-stimulating factor (GM-CSF)-expressing BM185 cells. Subsequently, protective vaccination experiments with these cells were performed in a leukemia challenge mouse model. RESULTS: Electroporation yielded a high transfection rate (82.6% for B7.1) with moderate GM-CSF secretion/1 x 10(6) cells (228 pg), whereas ballistic transfer led to a lower transfection rate (30.9%) with high GM-CSF secretion (614 pg). Secondly, we immunized mice with B7.1/interleukin 2- or B7.1/GM-CSF-expressing BM185 cell vaccines. We observed a better protection of mice that received the B7.1/GM-CSF vaccine compared with these receiving the B7.1/interleukin 2 vaccine. Protection was additionally enhanced by application of a double stem-loop immunomodulating oligonucleotide containing CpG motifs. CONCLUSION: Our data indicate that immunization with B7.1/GM-CSF-expressing cell vaccines generated by electroporation and application of double stem-loop immunomodulating oligonucleotide protected mice against a murine Ph(+) ALL challenge. Ultimately, this approach may also lead to clinical benefit in patients with Ph(+) ALL.

Minimizing side effects of ballistic gene transfer into the murine corneal epithelium.

BACKGROUND: The beneficial effect of modulating an allospecific immune response by ballistic IL-4 and CTLA4 gene transfer to deliver minimalistic immunologically defined gene expression (MIDGE) vectors into the corneal epithelium was demonstrated in corneal transplantation. However, side effects reduced graft survival in control animals after ballistic transfer without DNA. METHODS: An adapter was constructed for the gene gun apparatus to enlarge and keep constant the distance between the gun and the cornea. Mice were treated by ballistic transfer of luciferase- or IL-10 -encoding MIDGE vectors using gold particles different in quantity, size and size uniformity. Levels of protein expression were determined. Treated corneas were observed under the scanning electron microscope and immunohistologically. Three groups of Balb/c (H-2d) mice received a C3H (H-2 k) corneal graft and two of them had gold particles delivered into the corneal epithelium by gene gun. RESULTS: Using the gene gun and the distance piece, scanning electron microscopy did not reveal morphological differences of the corneal surface compared with untreated corneas on day 2 and 5. Sagittal histological sections of the central cornea did not show an invasion of macrophages 24 h after treatment. The expression of luciferase and IL-10 was not reduced when a smaller amount of gold (0.1 mg instead of 0.5 mg) was employed. Ballistic gold treatment did not reduce graft survival. CONCLUSION: Ballistic gene transfer into the corneal epithelium allows high cytokine expression in the cornea without measurable side effects if an apparatus is used that is adapted for this specific purpose.