Helper‐Dependent Adenoviral Vectors

Abstract

The helper-dependent adenovirus (HDAd) is a recently developed adenovirus-based vector with an improved safety profile and long-term transgene expression. In this unit, a basic procedure for HDAd production using the Cre-loxP system is presented. Amplification and large-scale production of the vector can be done in both adherent and suspension cell culture systems. Included are protocols for Southern blot analysis to monitor vector amplification, slot blot assay to determine the infectious titer of the purified HDAd, and real-time PCR to detect helper virus contamination in the preparation.

Gene Therapy for Diabetes: Metabolic Effects of Helper-dependent Adenoviral Exendin 4 Expression in a Diet-induced Obesity Mouse Model

“…For HDAd construction, the Ex4 cassette was ligated with pLPBL1 downstream of the elongation factor 1α (BOS) promoter and upstream of the rabbit β-globin polyadenylation signal 18. The fragment containing the promoter, Ex4 cassette, and polyadenylation signal was inserted into pΔ28 (ref.…”

“…The fragment containing the promoter, Ex4 cassette, and polyadenylation signal was inserted into pΔ28 (ref. 18). …”

“…HDAd was produced in the 293Cre66 cell line in the Adenoviral Core Laboratory at Baylor College of Medicine as described 18. After seven passages, final HDAd-Ex4 and the control HDAd-0 preparations were purified from empty capsid by cesium chloride gradient, and HDAd DNA was analyzed by Southern blotting to confirm <1% helper virus contamination.…”

Efficient synthesis of stably adenylated DNA and RNA adapters for microRNA capture using T4 RNA ligase 1

“…This method is only feasible if pre-adenylated adapters can be synthesized in large quantities and with high purity. Adapter pre-adenylation for microRNA detection was first performed by chemical synthesis, in which AMP was activated and adenosine 5′-phosphorimidazolidate was coupled to the 5′-phosphorylated oligonucleotide 18 19 . This method is complex and requires significant chemistry expertise as well as a final purification step.…”

Virus-mediated Genetic Surgery: Homologous Recombination With a Little “Helper” From My Friends

“…The HDAV system is effectively a “gutted” adenovirus from which all viral genes have been deleted, retaining only those viral sequences required for direct packaging of the DNA vector into the virus capsid. 24 The HDAV vector is packaged by cotransfection into a producer mammalian cell line with a “helper” virus genome that contains all of the viral genes necessary for vector replication, capsid production, and vector packaging. 25 An advantage of the recombinant HDAV vector is that it can accommodate between 25–35 kb of DNA, as opposed to, for example, the ~4.7 kb limitation imposed on the adeno-associated virus (AAV) system.…”