Development of Optimized AAV Serotype Vectors for High-Efficiency Transduction at Further Reduced Doses

Chen, Ling; Li, Baozheng; Ma, Wenqin; Srivastava, Arun

doi:10.1089/hgtb.2016.054

Cited by 18 publications

(12 citation statements)

References 33 publications

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“…An alternative approach to identify HBoV capsids with improved properties that complements screening of natural HBoV variants is rational engineering of the viral capsid. To this end, the modification of surface-exposed tyrosine residues in the viral capsid is especially promising, as these residues play important roles in assembly, ubiquitination, and degradation as well as in transcription and transduction of parvoviruses (29)(30)(31)(32)(33)(34)(35). Accordingly, we mutated six different tyrosine residues in the VP1 capsid protein to phenylalanine that we had originally predicted by structural modeling to be located on the HBoV1 capsid surface (Fig.…”

Section: Resultsmentioning

confidence: 99%

Impact of Natural or Synthetic Singletons in the Capsid of Human Bocavirus 1 on Particle Infectivity and Immunoreactivity

Fakhiri

Linse

Mietzsch

et al. 2020

J Virol

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Human bocavirus 1 (HBoV1) is a parvovirus that gathers increasing attention due to its pleiotropic role as a pathogen and emerging vector for human gene therapy. Curiously, albeit a large variety of HBoV1 capsid variants has been isolated from human samples, only one has been studied as a gene transfer vector to date. Here, we analyzed a cohort of HBoV1-positive samples and managed to PCR amplify and sequence 29 distinct HBoV1 capsid variants. These differed from the originally reported HBoV1 reference strain in 32 nucleotides or four amino acids, including a frequent change of threonine to serine at position 590. Interestingly, this T590S mutation was associated with lower viral loads in infected patients. Analysis of the time course of infection in two patients for up to 15 weeks revealed a gradual accumulation of T590S, concurrent with drops in viral loads. Surprisingly, in a recombinant vector context, T590S was beneficial and significantly increased titers compared to that of T590 variants but had no major impact on their transduction ability or immunoreactivity. Additional targeted mutations in the HBoV1 capsid identified several residues that are critical for transduction, capsid assembly, or DNA packaging. Our new findings on the phylogeny, infectivity, and immunoreactivity of HBoV1 capsid variants improve our understanding of bocaviral biology and suggest strategies to enhance HBoV1 gene transfer vectors. IMPORTANCE The family of Parvoviridae comprises a wide variety of members that exhibit a unique biology and that are concurrently highly interesting as a scaffold for the development of human gene therapy vectors. A most notable example is human bocavirus 1 (HBoV1), which we and others have recently harnessed to cross-package and deliver recombinant genomes derived from another parvovirus, the adeno-associated virus (AAV). Here, we expanded the repertoire of known HBoV1 variants by cloning 29 distinct HBoV1 capsid sequences from primary human samples and by analyzing their properties as AAV/HBoV1 gene transfer vectors. This led to our discovery of a mutational hot spot at HBoV1 capsid position 590 that accumulated in two patients during natural infection and that lowers viral loads but increases vector yields. Thereby, our study expands our current understanding of HBoV1 biology in infected human subjects and concomitantly provides avenues to improve AAV/HBoV1 gene transfer vectors.

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Section: Resultsmentioning

confidence: 99%

Impact of Natural or Synthetic Singletons in the Capsid of Human Bocavirus 1 on Particle Infectivity and Immunoreactivity

Fakhiri

Linse

Mietzsch

et al. 2020

J Virol

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“…The results were analyzed using one-way ANOVA followed by Student's t-test [ 19 , 20 ]. An inspection level was considered to indicate a significant difference when p < 0.05 [ 21 , 22 ].…”

Section: Methodsmentioning

confidence: 99%

Extraction of Amana edulis Induces Liver Cancer Apoptosis

Fan

Hou

Guo

et al. 2018

Evidence-Based Complementary and Alternative Medicine

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HCC is one of the fastest-rising causes of cancer-related death. Novel therapeutic approaches for treatment are warranted. The goal of this study is to find effective components from Chinese herbal medicines, which is an important alternative source of anticancer medicine. To this end, six different herbs were selected from various traditional literatures. Soxhlet extractor was used to distill the strong polar and weak polar components of each herb. The inhibitive effect of each component was determined using liver cancer cells BEL7404. From total of 12 extractions, it was found that the combined crude lysate of Amana edulis from water and ethanol system had the best efficacy. At the concentration of 0.1 mg/mL, this component has the highest inhibition rate up to 70%. To investigate the underlying molecular reasons, we observed that the component can significantly induce the liver cancer cells apoptosis and retard the cell reproduction at G2/M stage. Verification experiments showed that this component also has apparent inhibitive effects on other liver cancer cells, such as HepG2 and Huh7. On the other hand, it has less effectiveness on another cell line HepaRG, which retains many characteristics of primary human hepatocytes. The results suggested that there might be highly efficient antihepatoma ingredient in the water and ethanol extraction of Amana edulis. The pure substances remain to be isolated and further research on their targets is required.

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“…One of the promising strategies to overcome this issue was to mutagenize the surface-exposed tyrosine residues on the AAV capsid in order to avoid AAV degradation by the host cell proteasome machinery and to improve AAV intracellular trafficking to the nucleus, which can lead to high transduction efficiency at lower vector doses 107 . The combined use of capsid-modified and genome-modified next-generation AAV vectors has allowed higher transduction efficiency and transgene expression at further reduced doses 108 . To date, the AAV-CRISPR gene-editing system was tested in non-human animals only, but the first in vivo gene therapy using CRISPR/Cas9 technology will be carried out in human clinical trials soon.…”

Section: Crispr/cas9-based Human Gene and Cell Therapiesmentioning

confidence: 99%

In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease

2017

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Adeno-associated virus (AAV) has shown promising therapeutic efficacy with a good safety profile in a wide range of animal models and human clinical trials. With the advent of clustered regulatory interspaced short palindromic repeat (CRISPR)-based genome-editing technologies, AAV provides one of the most suitable viral vectors to package, deliver, and express CRISPR components for targeted gene editing. Recent discoveries of smaller Cas9 orthologues have enabled the packaging of Cas9 nuclease and its chimeric guide RNA into a single AAV delivery vehicle for robust in vivo genome editing. Here, we discuss how the combined use of small Cas9 orthologues, tissue-specific minimal promoters, AAV serotypes, and different routes of administration has advanced the development of efficient and precise in vivo genome editing and comprehensively review the various AAV-CRISPR systems that have been effectively used in animals. We then discuss the clinical implications and potential strategies to overcome off-target effects, immunogenicity, and toxicity associated with CRISPR components and AAV delivery vehicles. Finally, we discuss ongoing non-viral-based ex vivo gene therapy clinical trials to underscore the current challenges and future prospects of CRISPR/Cas9 delivery for human therapeutics.

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Development of Optimized AAV Serotype Vectors for High-Efficiency Transduction at Further Reduced Doses

Cited by 18 publications

References 33 publications

Impact of Natural or Synthetic Singletons in the Capsid of Human Bocavirus 1 on Particle Infectivity and Immunoreactivity

Impact of Natural or Synthetic Singletons in the Capsid of Human Bocavirus 1 on Particle Infectivity and Immunoreactivity

Extraction of Amana edulis Induces Liver Cancer Apoptosis

In vivo genome editing in animals using AAV-CRISPR system: applications to translational research of human disease

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