Rapid Cloning of Novel Rhesus Adenoviral Vaccine Vectors

Abbink, Peter; Kirilova, Marinela; Boyd, Michael; Mercado, Noe B.; Li, Zhenfeng; Nityanandam, Ramya; Nanayakkara, Ovini; Peterson, Rebecca; Larocca, Rafael A.; Aïd, Malika; Tartaglia, Lawrence J.; Mutetwa, Tinaye; Blass, Eryn; Jetton, David; Maxfield, Lori F.; Borducchi, Erica N.; Badamchi-Zadeh, Alexander; Handley, Scott A.; Zhao, Guoyan; Virgin, Herbert W.; Havenga, Menzo; Barouch, Dan H.

doi:10.1128/jvi.01924-17

Cited by 27 publications

(30 citation statements)

References 42 publications

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“…Some recent publications have described the construction of adenoviral vectors through direct DNA assembly of several PCR products. [29][30][31] The strategy of direct assembly is brief, practical, and easy to understand. For the strategy followed in this study, it takes some effort to find the proper restriction sites and to program the whole procedure before construction begins.…”

Section: Discussionmentioning

confidence: 99%

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Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems

Liu¹,

Zhang

et al. 2019

Human Gene Therapy

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Existing adenoviral vector systems have two drawbacks. It is labor-intensive and time-consuming to load a transgene in these systems, and transgene-harboring vectors are dead ends: they cannot be reused to construct a vector carrying another transgene or achieving new characteristics. To conquer these shortcomings, single plasmid-based adenoviral vector systems were constructed where a unique PmeI site was located at the position for insertion of the exogenous gene. The polymerase chain reaction (PCR) amplified transgene could be cloned into PmeI-linearized starting plasmids using one step of Gibson assembly to generate target adenoviral plasmids, which were then ready for virus rescue. This procedure was termed restriction assembly. To expand the application of these systems, two ClaI sites were created upstream and downstream of the fiber gene to generate an upgraded starting plasmid pKAd5f11pABR-EPG. The modified fiber gene, amplified by overlap extension PCR, could be used to substitute the original fiber in pKAd5f11pABR-EPG to generate an adenoviral plasmid with a new fiber by restriction assembly. On the other hand, pKAd5f11pABR-EPG was also a starting adenoviral plasmid for expressing other transgenes.In conclusion, easy-to-use and upgradable adenoviral vector systems are introduced here, which offer extensive versatility and can serve as a basic platform and functional component library for the synthetic biology of adenoviral vectors.

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Section: Discussionmentioning

confidence: 99%

“…35 Adenoviral vector has been synthesized by assembling several parts of the virus genome. [29][30][31] However, these parts are physical modules. Here, two functional modules (one for transgene cloning and the other one for fiber modification) and the strategy of restriction assembly for vector construction are provided.…”

Section: Discussionmentioning

confidence: 99%

Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems

Liu¹,

Zhang

et al. 2019

Human Gene Therapy

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“…Here, the adenovirus genome is divided into for instance seven DNA blocks, which can be PCR‐amplified and cloned into high‐copy plasmids. The PCR‐amplified fragments, containing 20–60 base pairs (bp) overlapping regions at each end, can be in vitro assembled using the Gibson assembly methodology or a slightly modified approach called the Adenobuilder . For both methods, the overlapping regions are included in the PCR fragment via PCR primers, followed by treatment with a 5′‐3′ exonuclease to chew back the 5′ ends and subsequent DNA polymerase treatment to close the gaps.…”

Section: Techniques For Getting Access To the Natural Diversity Of Humentioning

confidence: 99%

“…For both methods, the overlapping regions are included in the PCR fragment via PCR primers, followed by treatment with a 5′‐3′ exonuclease to chew back the 5′ ends and subsequent DNA polymerase treatment to close the gaps. Finally, fragments are in vitro assembled in a seamless manner resulting in a complete Ad genome , which can be directly transduced into Ad producer cells such as HEK293 cells or a large portion of the Ad genome can be cloned into a plasmid such as a Cosmid . These methods were exemplified for the HCAdV‐C5 genome and for Ads derived from rhesus monkeys .…”

Section: Techniques For Getting Access To the Natural Diversity Of Humentioning

confidence: 99%

“…Finally, fragments are in vitro assembled in a seamless manner resulting in a complete Ad genome , which can be directly transduced into Ad producer cells such as HEK293 cells or a large portion of the Ad genome can be cloned into a plasmid such as a Cosmid . These methods were exemplified for the HCAdV‐C5 genome and for Ads derived from rhesus monkeys . Note, that it is important to point out, that PCR can be error‐prone to mutation incorporation dependent on the polymerase used.…”

Section: Techniques For Getting Access To the Natural Diversity Of Humentioning

confidence: 99%

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State‐of‐the‐art human adenovirus vectorology for therapeutic approaches

et al. 2019

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Human adenoviruses (Ads) have long been studied in the basic virology field and are exploited as vectors for gene therapy, vaccination, and oncolytic therapy. Ads are usually mild pathogens, but they can cause severe infections and symptoms in immunocompromised individuals. Ads show a large natural diversity and a broad spectrum of hosts. However, replication-competent and replication-deficient Ad vectors with therapeutic applications have been built mainly starting from human Ad type 5, because generating vectors from other human and animal Ads has proven challenging. This review provides an updated overview of vectors that are not derived from human Ad type 5. We discuss genetic engineering techniques for getting access to the natural diversity of human Ads and for vectorization of alternative Ad types. A catalogue of currently available vectorized human Ads and translational applications thereof is also compiled. We conclude with a perspective on Ad vectorology that looks into the future of Ad vectors in translational medicine.

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Fiber modifications enable fowl adenovirus 4 vectors to transduce human cells

Zhang

Guo

Yin

et al. 2021

The Journal of Gene Medicine

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Background: Pre-existing immunities hamper the application of human adenovirus (HAdV) vectors in gene therapy or vaccine development. Fowl adenovirus (FAdV)based vector might represent an alternative. Methods: An intermediate plasmid containing FAdV-4 fiber genes, pMD-FAV4Fs, was separated from FAdV-4 adenoviral plasmid pKFAV4GFP. An overlap extension polymerase chain reaction (PCR) was employed for fiber modification in pMD-FAV4Fs, and the modified fibers were restored to generate new adenoviral plasmids through restriction-assembly. FAdV-4 vectors were rescued and amplified in chicken LMH cells. Fluorescence microscopy and flow cytometry were used to evaluate the gene transfer efficiency. The amount of viruses binding to cells was determined by a real-time PCR. A plaque-forming assay and one-step growth curve were used to evaluate virus growth. Results: Four sites in the CD-, DE-, HI-and IJ-loop of fiber1 knob could tolerate the insertion of exogenous peptide. The insertion of RGD4C peptide in the fiber1 knob significantly promoted FAdV-4 transduction to human adherent cells such as 293, A549 and HEp-2, and the insertion to the IJ-loop demonstrated the best performance. The replacement of the fiber2 knob of FAdV-4 with that of HAdV-35 improved the gene transfer to human suspension cells such as Jurkat, K562 and U937. Fiber-modified FAdV-4 vectors could transduce approximately 80% human cells at an acceptable multiplicity of infection. Enhanced gene transfer mainly resulted from increased virus binding. Fiber modifications did not significantly influence the growth of recombinant FAdV-4 in packaging cells.Conclusions: As a proof of principle, it was feasible to enhance gene transduction of FAdV-4 vectors to human cells by modifying the fibers.

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Rapid Cloning of Novel Rhesus Adenoviral Vaccine Vectors

Cited by 27 publications

References 42 publications

Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems

Single Plasmid-Based, Upgradable, and Backward-Compatible Adenoviral Vector Systems

State‐of‐the‐art human adenovirus vectorology for therapeutic approaches

Fiber modifications enable fowl adenovirus 4 vectors to transduce human cells

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