Research Article: Genetic strategy to decrease complement activation with adenoviral therapies

Date Published: April 26, 2019

Publisher: Public Library of Science

Author(s): Christopher M. Gentile, Anton V. Borovjagin, Jillian R. Richter, Aditi H. Jani, Hongju Wu, Kurt R. Zinn, Jason M. Warram, Ilya Ulasov.

http://doi.org/10.1371/journal.pone.0215226

Abstract

A major obstacle to using recombinant adenoviral vectors in gene therapy is the natural ability of human adenovirus to activate the classical and alternate complement pathways. These innate immune responses contribute to hepatic adenoviral uptake following systemic delivery and enhance the humoral immune responses associated with adenoviral infection.

A recombinant Ad5 vector was genetically modified to display a peptide sequence (“rH17d’”), a known inhibitor of the classical complement pathway. The replication-defective vectors Ad5.HVR2-rH17d’ and Ad5.HVR5-rH17d’ were constructed by engineering the rH17d’ peptide into the hypervariable region (HVR)-2 or HVR5 of their major capsid protein hexon. Control Ad5 vectors were created by incorporation of a 6-histidine (His6)-insert in either HVR2 or HVR5 (Ad5.HVR2-His6 and Ad5.HVR5-His6, respectively). All vectors encoded CMV promoter-controlled firefly luciferase (Luc). The four vectors were evaluated in TIB76 mouse liver cells and immunocompetent mice to compare infectivity and liver sequestration, respectively.

In vitro studies demonstrated that preincubation of all the Ad5 vectors with fresh serum significantly increased their gene transfer relative to preincubation with PBS except Ad5.HVR5-rH17d’, whose infectivity of liver cells showed no serum-mediated enhancement. In line with that, mice injected with Ad5.HVR2-rH17d’ or Ad5.HVR5-rH17d’ showed significantly lower luciferase expression levels in the liver as compared to the respective control vectors, whereas efficiency of tumor transduction by rH17d’ and His6 vectors following their intratumoral injection was similar.

Displaying a complement-inhibiting peptide on the Ad5 capsid surface by genetic modification of the hexon protein could be a suitable strategy for reducing Ad5 liver tropism (Ad5 sequestration by liver), which may be applicable to other gene therapy vectors with natural liver tropism.

Partial Text

Activation of innate immunity and induction of inflammation are major obstacles for the use of adenoviral vectors for human gene therapy.[1] Systemic intravascular delivery of human adenovirus type 5(Ad5) rapidly induces a dose-dependent toxic response that is transcription-independent and mediated by activation of the complement system,[2,3] production of proinflammatory cytokines/chemokines,[4] and blood-based coagulation factors.[5] These innate mechanisms lead not only to potentially deadly host responses such as liver damage and coagulopathy, but also to sequestration of gene delivery vectors from circulation, preventing their transduction of target tissues. Modification of the Ad hexon to evade the natural innate immune response to Ad5 is a promising strategy for improving the safety and clinical utility of Ad5-based gene therapies.[6]

SDS-PAGE analysis (Fig 1) shows characteristic banding pattern for adenoviral proteins. The top band (~119 kDa) represents the hexon protein, whose size is consistent between all modified and unmodified Ad vectors since neither the rH17d’ or His6 epitope insertion causes a visible change in the hexon protein size. Gelcode gel staining also demonstrates consistency between gel-loaded amounts of all the viruses and their physical titers, estimated at different times, since loading of the same number of viral particles (VP; 1010 VP) of each viral preparation resulted in similar intensities of the major protein bands on the gel.

Activation of the complement pathway is a potentially adverse response to adenovirus-based gene therapy. Strategies to down-regulate the complement pathway could minimize the undesired consequences that compromise the safety and efficacy of gene delivery vectors. In this study, a genetically-modified adenovirus that displayed on its capsid surface numerous copies of a peptide, known to inhibit the classical complement pathway, was evaluated for its ability to reduce unwanted liver transduction.

 

Source:

http://doi.org/10.1371/journal.pone.0215226

 

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