PURPOSE: To assess the safety and feasibility of direct vitrectomy-sparing subretinal injection for gene delivery in a large animal model. METHODS: The experimental Liběchov minipigs were used for subretinal delivery of a plasmid DNA vector (pS/MAR-CMV-copGFP) with cytomegalovirus (CMV) promoter, green fluorescent protein (GFP) reporter (copGFP) and a scaffold/matrix attachment region (S/MAR) sequence. The eyes were randomized to subretinal injection of the vector following pars plana vitrectomy (control group) or a direct injection without prior vitrectomy surgery (experimental group). Intra- and post-operative observations up to 30 days after surgery were compared. RESULTS: Six eyes of three mini-pigs underwent surgery for delivery into the subretinal space. Two eyes in the control group were operated with a classical approach (lens-sparing vitrectomy and posterior hyaloid detachment). The other four eyes in the experimental group were injected directly with a subretinal cannula without vitrectomy surgery. No adverse events, such as endophthalmitis, retinal detachment and intraocular pressure elevation were observed post-operatively. The eyes in the experimental group had both shorter surgical time and recovery while achieving the same surgical goal. CONCLUSIONS: This pilot study demonstrates that successful subretinal delivery of gene therapy vectors is achievable using a direct injection without prior vitrectomy surgery.
- MeSH
- Genetic Therapy * methods MeSH
- Genetic Vectors * administration & dosage MeSH
- Injections, Intraocular MeSH
- Swine, Miniature * MeSH
- Disease Models, Animal MeSH
- Pilot Projects MeSH
- Plasmids administration & dosage MeSH
- Swine MeSH
- Retina MeSH
- Feasibility Studies * MeSH
- Gene Transfer Techniques * MeSH
- Vitrectomy * methods MeSH
- Green Fluorescent Proteins genetics MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
In DNA vaccination, CD4(+) T-cell help can be enhanced by fusion of a gene encoding an immunization protein with a foreign gene or its part providing T(h) epitopes. To study the effect of helper epitope localization in a protein molecule, the influence of the vicinity of the helper epitope, and the impact of chimeric protein cellular localization, we fused the helper epitope p30 from tetanus toxin (TT, aa 947-967) with the N- or C-terminus of the mutated E7 oncoprotein (E7GGG) of human papillomavirus type 16, enlarged the p30 epitope with the flanking residues containing potential protease-sensitive sites and altered the cellular localization of the fusion constructs by signal sequences. The p30 epitope enhanced the E7-specific response, but only in constructs without added signal sequences. After localization of the fusion proteins into the endoplasmic reticulum and endo/lysosomal compartment, the TT-specific T(h)2 response was increased. The synthetic Pan DR epitope (PADRE) induced a stronger E7-specific response than the p30 epitope and its stimulatory effect was not limited to nuclear/cytoplasmic localization of the E7 antigen. These results suggest that in the optimization of immune responses by adding helper epitopes to DNA vaccines delivered by the gene gun, the cellular localization of the antigen needs to be taken into account.
- MeSH
- Biolistics methods MeSH
- NIH 3T3 Cells MeSH
- Cytokines metabolism MeSH
- Vaccines, DNA administration & dosage pharmacology MeSH
- Endoplasmic Reticulum immunology metabolism MeSH
- HEK293 Cells MeSH
- Humans MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Papillomavirus E7 Proteins genetics metabolism pharmacology MeSH
- Peptide Fragments genetics pharmacology MeSH
- Plasmids administration & dosage MeSH
- Recombinant Fusion Proteins metabolism pharmacology MeSH
- Tetanus Toxin genetics pharmacology MeSH
- Malaria Vaccines pharmacology MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
To test the hypothesis that neonatal GLP-1 exposure may program myosin heavy chain (MyHC) composition in adult skeletal muscle, two-day-old rats were transfected intramuscularly with vacant vector plasmid (VP), or recombinant plasmid expressing secretory GLP-1 at the doses of 60 µg (LG) and 120 µg (HG), respectively. Expression of GLP-1 mRNA was detected in muscles of both LG and HG rats 7 days after transfection, with more abundant GLP-1 transcript seen in LG rats. In accordance with the GLP-1 expression, LG rats demonstrated more significant responses to neonatal GLP-1 exposure. Small yet significant growth retardation was observed in LG rats, which is accompanied with significantly reduced serum insulin concentration at 8 weeks of age compared to VP rats. The responses of skeletal muscle were dependent on muscle type. Significant increase of PGC-1? and GLUT4 mRNA expression was detected in soleus of LG rats, whereas a MyHC type switch from ?B to ? was seen in gastrocnemius. These results indicate that neonatal exposure of healthy pups to ectopic GLP-1 causes growth retardation with decreased serum insulin as well as muscle type-dependent modifications in MyHC type composition and metabolic gene expression in adult rats.
- MeSH
- Down-Regulation MeSH
- Electroporation MeSH
- Financing, Organized MeSH
- Glucagon-Like Peptide 1 biosynthesis genetics MeSH
- Injections, Intramuscular MeSH
- Insulin blood MeSH
- Muscle, Skeletal metabolism growth & development MeSH
- Blood Glucose metabolism MeSH
- Rats MeSH
- RNA, Messenger metabolism MeSH
- Animals, Newborn MeSH
- Pancreas metabolism growth & development MeSH
- Plasmids administration & dosage MeSH
- Growth Disorders genetics metabolism MeSH
- Rats, Wistar MeSH
- Glucose Transporter Type 4 genetics MeSH
- Protein Isoforms MeSH
- RNA-Binding Proteins genetics MeSH
- Body Weight MeSH
- Myosin Heavy Chains genetics metabolism MeSH
- Transfection MeSH
- Transcription Factors genetics MeSH
- Age Factors MeSH
- Organ Size MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Male MeSH
- Female MeSH
- Animals MeSH
AIM: To compare pH and conductivity used in the determination of growth in reconstituted skim milk (RSM), to determine whether the presence of one or two plasmids in Lactococcus lactis had any influence on growth, and whether AbiS improved bacteriophages resistance of L. lactis.
- MeSH
- Colony-Forming Units Assay MeSH
- Bacterial Proteins genetics MeSH
- Transformation, Bacterial MeSH
- Bacteriophages physiology MeSH
- Electric Conductivity MeSH
- Fermentation MeSH
- Financing, Organized MeSH
- Hydrogen-Ion Concentration MeSH
- Lactococcus lactis genetics growth & development MeSH
- Milk microbiology MeSH
- Plasmids administration & dosage MeSH
- Food Microbiology MeSH
- Cheese MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Comparative Study MeSH
