Evolving Horizons: Adenovirus Vectors’ Timeless Influence on Cancer, Gene Therapy and Vaccines

Trivedi, Prasad D.; Byrne, Barry J.; Corti, Manuela

doi:10.3390/v15122378

Cited by 7 publications

(4 citation statements)

References 205 publications

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“…Despite the compact size of the designed system was compatible with adenoviral vectors, this study investigated the use of S/MAR minicircle plasmids for the expression of the genes incorporated into the system. We aimed to address concerns associated with viral vectors, such as limited capacity, potential immune responses, manufacturing complexities, and risk of insertional mutagenesis, by using MC vectors 16,17 . These episomal plasmids facilitated stable gene expression without the use of viral vectors, and we tested gene expression with these plasmids within 30 days.…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, due to the drawbacks associated with viral vectors, including limited cargo capacity, immune responses, manufacturing complexity, risk of insertional mutagenesis, and potential for off-target effects, alternative methods for gene expression in mammalian cells have emerged. 16,17 One such method is the use of minicircle (MC) plasmids for gene therapy, which enables long-term transgene episomal expression both in vitro and in vivo . This advantage stems from the absence of bacterial sequences, typically characterized by CpG dinucleotides, which are known triggers for vector-silencing mechanisms.…”

Section: Introductionmentioning

confidence: 99%

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Enzymatic Assembly for CRISPR Split-Cas9 System: The Emergence of a Sortase-based Split-Cas9 Technology

Helalat,

Kristinsdóttir,

Petersen

et al. 2024

Preprint

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CRISPR Cas9 has been widely used in scientific research and medical investigations as a pioneering technology. However, challenges such as the large size of the Cas9 sequence and potential off target effects have impeded its widespread adoption. In response, various alternatives, such as split Cas9 technology, have emerged. Split Cas9 systems allow the large Cas9 sequence to be divided into two segments to aid in the delivery of the enzyme. Nevertheless, challenges persist in achieving precise control over the timing and location of Cas9 reassembly and activity to minimize off target effects. This study presents an enzymatic based split Cas9 system, introducing a new approach utilizing the Sortase enzyme for the reconstitution of the full Cas9 protein. The developed method eliminates the need for chemical or physical induction and allows for precise genome editing in specific cells through the utilization of various specific promoters or targeted drug delivery. Experimental validation of the enzymatic system was conducted in E. coli, HEK cells, and Jurkat cells, demonstrating successful assembly and activity of the assembled Cas9 enzyme. In addition, this study explored the incorporation of nuclear localization signals, the evaluation of inducible promoters, and the delivery of the systems components in mRNA or protein form. Furthermore, we investigated the potential of SMAR minicircle technology instead of viral vectors within the system. Overall, we highlighted the feasibility and utility of the Sortase based split Cas9 system to enhance control and efficiency compared to traditional CRISPR Cas9 approaches. Additionally, this study revealed the potential of using the Sortase enzyme for posttranslational modifications and protein assembly in human cells.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enzymatic Assembly for CRISPR Split-Cas9 System: The Emergence of a Sortase-based Split-Cas9 Technology

Helalat,

Kristinsdóttir,

Petersen

et al. 2024

Preprint

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“…Adenoviridae is classified into six genera, among which Mastadenovirus and Aviadenovirus infect mammalian hosts and birds, respectively [1]. Adenoviral vectors have been widely used in biological research, gene therapy, and vaccine development [2][3][4][5]. A cell tropism is one of the key characteristics to be considered with priority in adenoviral vector utilization.…”

Section: Introductionmentioning

confidence: 99%

Redirect Tropism of Fowl Adenovirus 4 Vector by Modifying Fiber2 with Variable Domain of Heavy-Chain Antibody

Wang,

Zou,

Guo

et al. 2024

Genes

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The variable domain of a heavy-chain antibody (VHH) has the potential to be used to redirect the cell tropism of adenoviral vectors. Here, we attempted to establish platforms to simplify the screening of VHHs for their specific targeting function when being incorporated into the fiber of adenovirus. Both fowl adenovirus 4 (FAdV-4) and simian adenovirus 1 (SAdV-1) have two types of fiber, one of which is dispensable for virus propagation and is a proper site for VHH display. An intermediate plasmid, pMD-FAV4Fs, was constructed as the start plasmid for FAdV-4 fiber2 modification. Foldon from phage T4 fibritin, a trigger for trimerization, was employed to bridge the tail/shaft domain of fiber2 and VHHs against human CD16A, a key membrane marker of natural killer (NK) cells. Through one step of restriction-assembly, the modified fiber2 was transferred to the adenoviral plasmid, which was linearized and transfected to packaging cells. Five FAdV-4 viruses carrying the GFP gene were finally rescued and amplified, with three VHHs being displayed. One recombinant virus, FAdV4FC21-EG, could hardly transduce human 293 or Jurkat cells. In contrast, when it was used at a multiplicity of infection of 1000 viral particles per cell, the transduction efficiency reached 51% or 34% for 293 or Jurkat cells expressing exogenous CD16A. Such a strategy of fiber modification was transplanted to the SAdV-1 vector to construct SAdV1FC28H-EG, which moderately transduced primary human NK cells while the parental virus transduced none. Collectively, we reformed the strategy of integrating VHH to fiber and established novel platforms for screening VHHs to construct adenoviral vectors with a specific tropism.

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“…Adenoviruses are a group of non-enveloped, icosahedral viruses with a genome of linear, double-stranded DNA of 26-48 kb in length [1]. Adenoviral vectors have been widely used in basic biomedical research, gene therapy, and vaccine development due to their properties of manipulable genome, high production, physicochemical stability, and efficient transient transduction [2][3][4][5]. However, the application of common adenoviral vectors is hampered by the pre-existing immunity in humans [6,7].…”

Section: Introductionmentioning

confidence: 99%

User-Friendly Replication-Competent MAdV-1 Vector System with a Cloning Capacity of 3.3 Kilobases

Zhang,

Guo,

Hou

et al. 2024

Viruses

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Mouse adenoviruses (MAdV) play important roles in studying host–adenovirus interaction. However, easy-to-use reverse genetics systems are still lacking for MAdV. An infectious plasmid pKRMAV1 was constructed by ligating genomic DNA of wild-type MAdV-1 with a PCR product containing a plasmid backbone through Gibson assembly. A fragment was excised from pKRMAV1 by restriction digestion and used to generate intermediate plasmid pKMAV1-ER, which contained E3, fiber, E4, and E1 regions of MAdV-1. CMV promoter-controlled GFP expression cassette was inserted downstream of the pIX gene in pKMAV1-ER and then transferred to pKRMAV1 to generate adenoviral plasmid pKMAV1-IXCG. Replacement of transgene could be conveniently carried out between dual BstZ17I sites in pKMAV1-IXCG by restriction-assembly, and a series of adenoviral plasmids were generated. Recombinant viruses were rescued after transfecting linearized adenoviral plasmids to mouse NIH/3T3 cells. MAdV-1 viruses carrying GFP or firefly luciferase genes were characterized in gene transduction, plaque-forming, and replication in vitro or in vivo by observing the expression of reporter genes. The results indicated that replication-competent vectors presented relevant properties of wild-type MAdV-1 very well. By constructing viruses bearing exogenous fragments with increasing size, it was found that MAdV-1 could tolerate an insertion up to 3.3 kb. Collectively, a replication-competent MAdV-1 vector system was established, which simplified procedures for the change of transgene or modification of E1, fiber, E3, or E4 genes.

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Evolving Horizons: Adenovirus Vectors’ Timeless Influence on Cancer, Gene Therapy and Vaccines

Cited by 7 publications

References 205 publications

Enzymatic Assembly for CRISPR Split-Cas9 System: The Emergence of a Sortase-based Split-Cas9 Technology

Enzymatic Assembly for CRISPR Split-Cas9 System: The Emergence of a Sortase-based Split-Cas9 Technology

Redirect Tropism of Fowl Adenovirus 4 Vector by Modifying Fiber2 with Variable Domain of Heavy-Chain Antibody

User-Friendly Replication-Competent MAdV-1 Vector System with a Cloning Capacity of 3.3 Kilobases

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