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.

[Gene therapy in corneal diseases]. / Gentherapie bei Hornhauterkrankungen.

Gene therapy raised euphoric expectations in the past that have yet to be met and have even been lowered due to the absence of concrete clinical successes and the occurrence of some tragic incidents. In spite of the great future potential of gene therapy, numerous pilot studies in this field will probably be discontinued for a long time. However, despite these failures, diseases will continue to be the aim of gene transfer studies with experimental protocols using only temporary gene expression or those restricted to individual organs and thus requiring only a low dose of the vehicle (vector). The eye is exceptionally well suited as a potential target organ because of its good and selective accessibility, low volume and the resultant low number of gene ferries required as well as its special immunological status. The prognosis of corneal grafting can be improved and profit from the methods catalogue of gene transfer protocols. Moreover, the cornea can be used for ex vivo gene transfer before grafting. Ophthalmology could thus occupy a pioneer position in clinical gene transfer. A survey of the literature describes the current state of experimental improvement in corneal grafting, the effect on scarring, neovascularization, and herpetic corneal infection. The essential problem of gene therapy is the unsatisfactory gene transfer technique. Difficulties and current improvements are discussed.

Ballistic CTLA4 and IL-4 gene transfer into the lower lid prolongs orthotopic corneal graft survival in mice.

PURPOSE: To explore outflow from the eye and to determine and modulate the influence of lymphatic drainage on corneal graft survival in mice. METHODS: Tracer experiments were conducted in BALB/c mice using the (99m)Tc colloidal albumin Nanocoll. Count rates were determined in the eyes, submandibular lymph nodes, spleen, liver and blood 24 h after subconjunctival, intracorneal, intracameral (anterior chamber), intravenous and subcutaneous lower-lid or upper-lid injections ( n=6 each). Four groups of BALB/c mice ( n=8) received corneal transplants from C3H mice; two of them were treated ballistically with vector CTLA4+IL-4 onto the leg or the lower lid, one group was untreated and the other control group was treated with an empty minimalistic, immunologically defined, gene expression (MIDGE) vector. RESULTS: Radioactivity was detected in the liver, spleen and ipsilateral submandibular lymph node after intracameral injection as follows: 91.9%, 6.6% and 1.2% respectively. Radioactivity uptake of the ipsilateral submandibular lymph node was also low after intravenous injection (0.1%) but high after intracorneal (33.8%), lower-lid (62.0%) and subconjunctival (71.2%) injection. Vector CTLA4+IL-4 treatment of the lower lid but not of the leg prolonged graft survival ( P=0.004). CONCLUSION: These tracer studies confirmed for the first time identical lymphatic drainage from the cornea and the lower lid. Logically, lymphatic drainage could be manipulated and graft survival improved by gene transfer to the lower lid.

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.