RESUMEN
The cellular transport process of DNA is hampered by cell membrane barriers, and hence, a delivery vehicle is essential for realizing the potential benefits of gene therapy to combat a variety of genetic diseases. Virus-based vehicles are effective, although immunogenicity, toxicity and cancer formation are among the major limitations of this approach. Cationic polymers, such as polyethyleneimine are capable of condensing DNA to nanoparticles and facilitate gene delivery. Lack of biodegradation of polymeric gene delivery vehicles poses significant toxicity because of the accumulation of polymers in the tissue. Many attempts have been made to develop biodegradable polymers for gene delivery by modifying existing polymers and/or using natural biodegradable polymers. This review summarizes mechanistic aspects of gene delivery and the development of biodegradable polymers for gene delivery.
Asunto(s)
Quitosano/metabolismo , Técnicas de Transferencia de Gen/clasificación , Nanopartículas/metabolismo , Polietileneimina/metabolismo , Polilisina/metabolismo , Animales , Transporte Biológico , Quitosano/química , Dextranos/química , Dextranos/metabolismo , Endosomas/metabolismo , Terapia Genética/métodos , Glucanos/química , Glucanos/metabolismo , Humanos , Ácido Hialurónico/química , Ácido Hialurónico/metabolismo , Hidrólisis , Lisosomas/metabolismo , Nanopartículas/química , Polietileneimina/química , Polilisina/químicaRESUMEN
OBJECTIVES: We explored for the first time the possibility to deliver a reporter gene (Green Fluorescence Protein) to human primary periodontal ligament (PDL) cells and human gingival fibroblasts (HGF) using shuttle vectors derived from adeno-associated virus (AAV). Since AAV transduction rates on other human primary fibroblasts have been previously shown to depend on the particular cell lineage and on the employed viral serotype, we determined the most effective AAV variant for periodontal cells comparing different vector types. METHODS: AAV serotypes 1-5 encoding GFP in single stranded (ss) and self-complementary (sc) vector genome conformations were used to infect primary HGF and PDL cells. Two days post-infection, the percentage of GFP expressing cells was determined by flow cytometry. RESULTS: Highest transduction rates for both cell types were achieved with self-complementary vectors derived from AAV-2, resulting in GFP expression in up to 86% of PDL cells and 50% of HGF. Transgene expression could be observed by optical microscopy for 2 months after infection. Lower but detectable rates were obtained with serotypes 1, 3 and 5. CONCLUSIONS: The efficacy demonstrated here and the safety and versatility of AAV technology indicated in previous studies clearly suggest the potential of AAV vectors as tools for gene transfer to periodontal tissues.
Asunto(s)
Dependovirus/genética , Fibroblastos/citología , Técnicas de Transferencia de Gen/clasificación , Vectores Genéticos/genética , Encía/citología , Ligamento Periodontal/citología , Adolescente , Adulto , Células Cultivadas , ADN de Cadena Simple/genética , Dependovirus/clasificación , Células Epiteliales/citología , Citometría de Flujo , Regulación de la Expresión Génica/genética , Genes Reporteros/genética , Vectores Genéticos/clasificación , Genoma Viral/genética , Proteínas Fluorescentes Verdes/genética , Humanos , Plásmidos/genética , Serotipificación , Factores de Tiempo , Transducción Genética , Transgenes/genética , Adulto JovenRESUMEN
Gene and RNA interference therapies are promising cures for intractable renal failure. However, low delivery efficiency of the therapeutic nucleic acid into the nucleus of the target cell is a significant obstacle in the clinical application of nonviral gene therapy. Various mechanical techniques (hydrodynamic injection, electroporation and ultrasound-microbubble) and topically applied preparations (HVJ liposome and cationic liposome/polymer), which introduce transgenes into specific renal compartments depending on the administration route, have been reported. Additional improvements in renal application of nonviral gene vectors must address the important issue of how to control intracellular trafficking. Therefore, novel vectors based on the 'programmed packaging' concept are desirable in which all functional devices are integrated into a single system so that each function occurs at the appropriate time and correct place. In parallel with development of the carrier, quantitative evaluation of intracellular trafficking is essential to determine the efficacy of the modified devices in the cellular environment. In particular, comparison of the intracellular trafficking of the engineered devices with that of viruses (i.e. adenovirus) is useful in identifying the rate-limiting intracellular processes of the vectors during development.