A novel system for the production of fully deleted adenovirus vectors that does not require helper adenovirus

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In September 1999, the perceptions of the use of adenoviral (Ad) vectors for gene therapy were altered when a patient exposed via the hepatic artery to a high dose of adenoviral vector succumbed to the toxicity related to vector administration. Appropriately, concerns were raised about continued use of the Ad vector system and, importantly, there were increased efforts to more fully understand the toxicity. Today it is recognized that there is no ideal vector system, and that while Ad vectors are not suitable for all applications, the significant advantages over other vector systems including efficient transduction of a variety of cell types, both quiescent and dividing, make it optimal for certain applications. These include protocols where high levels of short-term expression are sufficient to provide a therapeutic benefit. Potential target applications include therapeutic angiogenesis, administration into immune-privileged sites such as the CNS, or treatments where the adjuvant effect of adenovirus can be of benefit such as cancer vaccines. Broader applicability of Ad vectors will require resolution of toxicity issues. This review will therefore focus on studies conducted over the last 2 years that have advanced our understanding of the toxicity associated with Ad vectors, studies that have employed methods to reduce toxicity and improvements in Ad vectors themselves that will reduce toxicity by one of several mechanisms. These mechanisms include retargeting vector to the tissue of interest, minimizing or eliminating viral gene expression that is thought to result in loss of transduced cells, or by methods that seek to reduce the vector dose required for therapeutic benefit. An area where there remains significant room for improvement is when readministration of vector is required because transgene expression has decreased to background levels.

Section: Helper-dependent Fully Deleted Ad Vectors Offer Great Potenmentioning

confidence: 99%

Gene therapy progress and prospects: adenoviral vectors

George¹

2003

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“…The baculovirus genome is large, thus conferring a huge cloning capacity of up to 38 kb. 39 Moreover, recombinant baculovirus construction is easy and can be achieved in 7-10 days, and baculovirus can be propagated to high titers easily by infecting its natural host insect cells. 18 One general disadvantage associated with baculovirus is that the virus confers only transient expression.…”

Section: Discussionmentioning

confidence: 99%

Baculovirus as a new gene delivery vector for stem cell engineering and bone tissue engineering

et al. 2007

Baculovirus has emerged as a novel vector for in vitro and in vivo gene delivery due to its low cytotoxicity and nonreplication nature in mammalian cells, but the applications of baculovirus in the genetic modification of human mesenchymal stem cells (hMSCs) and tissue engineering are yet to be reported. In this study, we genetically engineered hMSCs with a baculovirus (Bac-CB) expressing bone morphogenetic protein-2 (BMP-2). Bac-CB transduction of hMSCs at a multiplicity of infection of 40 triggered effective differentiation of hMSCs into osteoblasts. Supertransduction at day 6 after initial transduction enhanced the BMP-2 expression and further accelerated the in vitro osteogenesis, as confirmed by alkaline phosphatase assay, Alizarin red staining and reverse transcription-polymerase chain reaction analysis of osteoblastic genes. Implantation of the supertransduced cells at ectopic sites in the nude mice resulted in efficient cell differentiation into osteoblasts at week 2 and induced progressive mineralization and partial bone formation at week 6, as confirmed by hematoxylin and eosin, immunohistochemical and Alizarin red staining. These data collectively demonstrated, for the first time, the potential of baculovirus in hMSCs engineering and implicated its use in bone tissue engineering.

“…21 These attributes minimize the potential side effects and ease the safety concerns. Furthermore, the large baculovirus genome confers a huge cloning capacity of up to 38 kb 22 and baculovirus can be propagated to high titers easily by infecting its natural host insect cells. 10 These advantages prompted us to exploit the potential of baculovirus-mediated gene transfer to modulate the differentiation status of chondrocytes and enhance ECM production.…”

Section: Discussionmentioning

confidence: 99%

Combination of baculovirus-expressed BMP-2 and rotating-shaft bioreactor culture synergistically enhances cartilage formation

et al. 2007

Baculovirus is an emerging gene delivery vector, thanks to a number of unique advantages. Herein, we genetically modified the rabbit articular chondrocytes with a recombinant baculovirus (Bac-CB) encoding bone morphogenetic protein-2 (BMP-2), which conferred high level BMP-2 expression and triggered the re-differentiation of dedifferentiated third passage (P3) chondrocytes in the monolayer culture. The transduced and mock-transduced P3 cells were seeded into porous scaffolds and cultured in either the dishes or the rotating-shaft bioreactor (RSB), a novel bioreactor imparting a dynamic, two-phase culture environment. Neither mocktransduced constructs in the RSB culture nor the Bac-CBtransduced constructs in the static culture grew into uniform cartilaginous tissues. Only the Bac-CB-transduced constructs cultured in the RSB for 3 weeks resulted in cartilaginous tissues with hyaline appearance, uniform cell distribution, cartilage-specific gene expression and considerably enhanced cartilage-specific extracellular matrix deposition, as determined by histological staining, reverse transcription-PCR analyses and biochemical assays. This is the first study demonstrating that combination of baculovirusmediated growth factor expression and RSB culture synergistically enhanced in vitro creation of cartilaginous tissues from dedifferentiated chondrocytes. Since baculovirus transduction is generally considered safe, this approach represents a viable alternative to stimulate the formation of engineered cartilage in a more cost-effective way than the growth factor supplementation.