RESUMEN
Cell surface targeting of recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to modify AAV's natural tropism. As modification of the capsid surface is likely to affect the mechanism of vector internalization and consequently the vector's intracellular fate, we investigated early steps in cell transduction of rAAV capsid insertion mutants. Mutants displaying peptides with neutral overall charge at position 587 transduced cells independently of AAV2's primary receptor heparan sulfate proteoglycan (HSPG), whereas mutants carrying positively charged insertions were capable of HSPG binding with affinities correlating with their net positive charge. Whereas rAAV2 is internalized via an HSPG- and clathrin-dependent pathway, HSPG-binding mutants used a clathrin- and caveolin-independent mechanism. Surprisingly, although this pathway was as efficient in mediating vector entry as the one used by rAAV2, successful cell transduction was hampered at a post-entry step, presumably caused by inefficient endosomal escape. In contrast, HSPG-independent, clathrin-dependent internalization used by non-HSPG-binding mutants correlated with efficient nuclear delivery of vector genomes and robust transgene expression. These findings indicate that cell surface targeting strategies should direct uptake of rAAV targeting vectors to clathrin-mediated endocytosis, the naturally evolved entry route of AAV, to promote successful intracellular processing and re-targeting of rAAV's tropism.
Asunto(s)
Clatrina/genética , Dependovirus/genética , Endocitosis/genética , Terapia Genética , Proteoglicanos de Heparán Sulfato/metabolismo , Proteínas de la Cápside/genética , Clatrina/metabolismo , Vectores Genéticos/administración & dosificación , Células HEK293 , Células HeLa , Células Hep G2 , Proteoglicanos de Heparán Sulfato/genética , Humanos , Mutagénesis Insercional/genética , Mutación , Transducción Genética , Internalización del VirusRESUMEN
Clinical application of viral vectors is often hampered by the lack of selectivity of viral particles for the targeted tissue. This drawback decreases the efficiency of gene delivery and raises safety concerns. We successfully established a novel in vitro evolution protocol to engineer adeno-associated virus vectors with increased selectivity for designated target cells. Subjecting a peptide-display library of AAV capsids to negative selection cycles on human primary fibroblasts and to positive selection cycles on a human melanoma cell line, we isolated several variants with up to 3.7-fold increased specificity for malignant cells in comparison to fibroblasts and other cell types. These mutants can be used to achieve high levels of gene transfer to target cells reducing undesired transduction of neighbouring tissues.
Asunto(s)
Dependovirus/genética , Marcación de Gen , Vectores Genéticos/genética , Línea Celular Tumoral , Evolución Molecular , Humanos , Receptores de Superficie Celular/genéticaRESUMEN
Targeted delivery of biological agents to atherosclerotic plaques may provide a novel treatment and/or useful tool for imaging of atherosclerosis in vivo. However, there are no known viral vectors that possess the desired tropism. Two plaque-targeting peptides, CAPGPSKSC (CAP) and CNHRYMQMC (CNH) were inserted into the capsid of adeno-associated virus 2 (AAV2) to assess vector retargeting. AAV2-CNH produced significantly higher levels of transduction than unmodified AAV2 in human, murine and rat endothelial cells, whereas transduction of nontarget HeLa cells was unaltered. Transduction studies and surface plasmon resonance suggest that AAV2-CNH uses membrane type 1 matrix metalloproteinase as a surface receptor. AAV2-CAP only produced higher levels of transduction in rat endothelial cells, possibly because the virus was found to be affected by proteasomal degradation. In vivo substantially higher levels of both peptide-modified AAV2 vectors was detected in the brachiocephalic artery (site of advanced atherosclerotic plaques) and aorta, whereas reduced levels were detected in all other organs examined. These results suggest that in the AAV2 platform the peptides are exposed on the capsid surface in a way that enables efficient receptor binding and so creates effective atherosclerotic plaque targeted vectors.
Asunto(s)
Aterosclerosis/terapia , Dependovirus/genética , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Animales , Apolipoproteínas E/genética , Línea Celular , Dependovirus/metabolismo , Marcación de Gen , Ingeniería Genética , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Heparina/metabolismo , Humanos , Metaloproteinasa 1 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/genética , Unión Proteica , Ratas , Resonancia por Plasmón de Superficie , Transducción Genética/métodos , TransgenesRESUMEN
The high prevalence of human serum antibodies against adeno-associated virus type 2 (AAV) vectors represents a potential limitation for in vivo applications. Consequently, the development of AAV vectors able to escape antibody binding and neutralization is of importance. To identify capsid domains which contain major immunogenic epitopes, six AAV capsid mutants carrying peptide insertions in surface exposed loop regions (I-261, I-381, I-447, I-534, I-573, I-587) were analyzed. Two of these mutants, I-534 and I-573, showed an up to 70% reduced affinity for AAV antibodies as compared to wild-type AAV in the majority of serum samples. In addition, AAV mutant I-587 but not wild-type AAV efficiently transduced cells despite the presence of neutralizing antisera. Taken together, the results show that major neutralizing effects of human AAV antisera might be overcome by the use of AAV capsid mutants.
Asunto(s)
Anticuerpos Antivirales/inmunología , Cápside/inmunología , Dependovirus/genética , Vectores Genéticos/genética , Afinidad de Anticuerpos/genética , Afinidad de Anticuerpos/inmunología , Dependovirus/inmunología , Vectores Genéticos/inmunología , Células HeLa/inmunología , Humanos , Sueros Inmunes/inmunología , Mutagénesis Insercional/métodos , Mutación/genética , Mutación/inmunología , Transducción Genética/métodosRESUMEN
Adeno-associated virus (AAV) is a promising vector for human somatic gene therapy. However, its broad host range is a disadvantage for in vivo gene therapy, because it does not allow the selective tissue- or organ-restricted transduction required to enhance the safety and efficiency of the gene transfer. Therefore, increasing efforts are being made to target AAV-2-based vectors to specific receptors. The studies summarized in this review show that it is possible to target AAV-2 to a specific cell. So far, the most promising approach is the genetic modification of the viral capsid. However, the currently available AAV-2 targeting vectors need to be improved with regard to the elimination of the wild-type AAV-2 tropism and the improvement of infectious titers. The creation of highly efficient AAV-2 targeting vectors will also require a better understanding of the transmembrane and intracellular processing of this virus.