RESUMEN
Pancreatic islet transplantation is a promising treatment for type 1 diabetes mellitus offering improved glycaemic control by restoring insulin production. Improved human pancreatic islet isolation has led to higher islet transplantation success. However, as many as 50% of islets are lost after transplantation due to immune responses and cellular injury, gene therapy presents a novel strategy to protect pancreatic islets for improved survival post-transplantation. To date, most of the vectors used in clinical trials and gene therapy studies have been derived from mammalian viruses such as adeno-associated or retrovirus. However, baculovirus BacMam vectors provide an attractive and safe alternative. Here, a novel BacMam was constructed containing a frameshift mutation within fp25, which results in virus stocks with higher infectious titres. This improved in vitro transduction when compared to control BacMams. Additionally, incorporating a truncated vesicular stomatitis virus G protein increased transduction efficacy and production of EGFP and BCL2 in human kidney (HK-2) and pancreatic islet ß cells (EndoC ßH3). Lastly, we have shown that our optimized BacMam vector can deliver and express egfp in intact pancreatic islet cells from human cadaveric donors. These results confirm that BacMam vectors are a viable choice for providing delivery of transgenes to pancreatic islet cells.
Asunto(s)
Baculoviridae/genética , Diabetes Mellitus Tipo 1/terapia , Terapia Genética/instrumentación , Células Secretoras de Insulina/virología , Transducción Genética , Baculoviridae/fisiología , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/genética , Vectores Genéticos/fisiología , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Islotes Pancreáticos/virologíaRESUMEN
The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is able to transduce a wide range of mammalian cells and shows preferential uptake in some, particularly liver and kidney cells. This suggests that the virus may be useful for delivery of protective genes for ameliorating the effects of ischaemia reperfusion injury (IRI) in solid organs during transplantation procedures. In this chapter we discuss the advantages of the baculovirus over other virus vectors for gene delivery in organ transplantation and describe some of the protective genes which may be used to ameliorate the effects of IRI. We then describe a method for concentrating baculovirus for use in an ex vivo transduction model. Data are also provided for the effects of virus transduction in vitro on the innate and adaptive immune response. We conclude with a discussion on the future considerations for using baculovirus for delivery and expression of protective genes in organ transplantation.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos , Nucleopoliedrovirus/genética , Trasplante de Órganos , Células Cultivadas , Regulación Viral de la Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Daño por Reperfusión/genética , Daño por Reperfusión/terapiaRESUMEN
Concerns over the safety of conventional viral vectors have limited the translation of gene transfer from an exciting experimental procedure to a successful clinical therapy in transplantation. Baculoviruses are insect viruses, but have the ability to enter mammalian cells and deliver potential therapeutic molecules with no evidence of viral replication. This study provides evidence of the ability of recombinant baculovirus to enter mammalian kidneys and livers during cold preservation. Six kidneys and six liver lobules retrieved from large pigs were perfused with University of Wisconsin (UW) solution containing a baculovirus tagged with green fluorescent protein and preserved for 8 h. In addition, six kidneys were perfused with UW containing a baculovirus expressing red fluorescent protein and preserved for 24 h. Green fluorescent virus particles were detected within transduced kidneys and livers after 8 h standard cold storage and red fluorescent protein mRNA was detected in kidneys after 24 h of cold preservation. There were no significant differences in tissue architecture, cell morphology or ATP content between experimental organs and their controls. Ex vivo transduction of organs with recombinant baculovirus during conventional cold preservation was demonstrated with no evidence of additional injury or reduction in cell viability.