A new adenoviral helper–dependent vector results in long-term therapeutic levels of human coagulation factor IX at low doses in vivo

Ehrhardt, Anja; Kay, Mark A.

doi:10.1182/blood.v99.11.3923

Cited by 125 publications

(154 citation statements)

References 45 publications

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“…However, there are examples of first-generation vectors that provide prolonged expression. 40 In addition, partially deleted vectors have resulted in persistent expression. 41 It is therefore unclear how much of the viral genome must be deleted to achieve the desired length of expression.…”

Section: Advanced Generations Of Ad Vectors Provide Increased Persistmentioning

confidence: 99%

Gene therapy progress and prospects: adenoviral vectors

George¹

2003

Gene Ther

261

163

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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.

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Section: Advanced Generations Of Ad Vectors Provide Increased Persistmentioning

confidence: 99%

Gene therapy progress and prospects: adenoviral vectors

George¹

2003

Gene Ther

261

163

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show abstract

“…11,14 -16 In addition, the elimination of all the adenoviral genes leaves room to allocate large expression cassettes, and therefore gutless adenoviruses are also designed as high-capacity adenoviral vectors. 11,14 -16 For correction of metabolic and/or hereditary diseases, different authors have used gutless vectors with nonregulable promoters showing long-term expression of transgenes such as ␣1-antitrypsin, 17,18 leptin, 19 apolipoprotein E, 20 and factor VIII and IX [21][22][23][24] at therapeutic levels without apparent hepatic toxicity.…”

mentioning

confidence: 99%

Prolonged and inducible transgene expression in the liver using gutless adenovirus: A potential therapy for liver cancer

et al. 2004

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“…The system for the construction of HCAdV genomes is based on the plasmid pAdFTC carrying a HCAdV genome devoid of all viral coding sequences and the shuttle plasmid pHM5 [9][10][11][12] . Any gene of interest of up to 14 kilo bases (kb) can be cloned into the shuttle vector pHM5 in which the multiple cloning site is flanked by recognition/cleaving sites of the homing endonucleases PI-SceI and I-CeuI.…”

Section: Introductionmentioning

confidence: 99%

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

Ehrke‐Schulz

Zhang

Schiwon

et al. 2016

JoVE

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High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks and shortcuts developed over the past years working with this HCAdV vector system are presented. Video LinkThe video component of this article can be found at

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A new adenoviral helper–dependent vector results in long-term therapeutic levels of human coagulation factor IX at low doses in vivo

Cited by 125 publications

References 45 publications

Gene therapy progress and prospects: adenoviral vectors

Gene therapy progress and prospects: adenoviral vectors

Prolonged and inducible transgene expression in the liver using gutless adenovirus: A potential therapy for liver cancer

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

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