Adeno-Associated Virus (AAV) Gene Delivery: Dissecting Molecular Interactions upon Cell Entry

Large, Edward; Silveria, Mark; Zane, Grant M.; Weerakoon, Onellah; Chapman, Michael S.

doi:10.3390/v13071336

Cited by 34 publications

(18 citation statements)

References 204 publications

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“…Since the discovery of adeno-associated viruses (AAVs) in 1965 and up through the creation of the first AAV-mediated gene therapies to treat lipoprotein lipase deficiency (Glybera in 2012) and to treat patients with Leber congenital amaurosis (Luxturna in 2017), numerous studies of AAV biology have contributed to the development of new gene delivery strategies to facilitate clinical success (Wang, Tai, & Gao, 2019). Furthermore, the beneficial features of AAV vectors, including low immunogenicity, nonpathogenicity, and effective, stable, and long-term transgene expression (Large, Silveria, Zane, Weerakoon, & Chapman, 2021), have broadened their applications in cell culture and animal model research. Additionally, AAV tropism, which is dependent on serotype, allows tissue-specific and conditional cell-type-specific transduction (Nectow & Nestler, 2020).…”

Section: Introductionmentioning

confidence: 99%

Single‐Cell Quantification of Triple‐AAV Vector Genomes Coexpressed in Neurons

2022

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Adeno-associated viruses (AAVs) are one of the most widely used types of viral vectors for research and gene therapy. AAV vectors are safe, have a low immunogenic profile, and provide efficient and long-term transgene expression in a variety of tissues and organs targeted by a specific serotype. Despite these unique features, therapeutic applications, as well as basic research studies, of AAVs have been limited by their packaging capacity of less than 5 kb. Multiple strategies have been explored to deliver large genes. One strategy is to split large transgenes into two or three fragments and package them into separate AAV capsids, generating dual or triple AAV vectors. Combining the fragments potentially allows reconstitution of an mRNA transcript containing the complete sequence of transgene in the same cell. The success of AAVs as vectors for the delivery of large or multiple genes depends directly on the efficiency of co-transduction. Here, we describe a method to measure the efficacy of codelivery, quantifying the number of AAV vectors per cell. We detail how to calculate the average number of incoming AAV genomes in neurons, given the distribution of cell fluorescence across in vitro and in vivo experimental models. To validate the method, we simulated a triple AAV strategy using three fluorescent-protein-encoding genes. We provide a general protocol for constructing plasmids and producing and purifying AAV vectors. We also include a protocol for triple AAV vector co-transduction in primary neuronal cultures and mouse brain. The method can be applied to multiple organs and tissues for the treatment of disorders caused by mutations in multiple or large genes. These protocols will be useful for researchers working to develop and improve new gene delivery technologies.

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Section: Introductionmentioning

confidence: 99%

Single‐Cell Quantification of Triple‐AAV Vector Genomes Coexpressed in Neurons

2022

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“…AAV2 uses heparan sulfate proteoglycan (HSPG) as a primary attachment receptor [ 49 ]. The negatively charged polysaccharide chain of heparan sulfate, which interacts with distinct residues of the capsid (R484, R487, K532, R585, and R588) and which is expressed on hepatocytes, is discussed as a host factor determining the liver tropism of AAV2 [ 50 , 51 , 52 ]. The HSPG density and glycosylation pattern on liver cancer cells are altered [ 53 ] and may be involved in enhanced intracellular processing as HSPG is suspected to not only serve as an attachment receptor but also to affect transduction processes.…”

Section: Discussionmentioning

confidence: 99%

Hepatocellular Carcinoma Is a Natural Target for Adeno-Associated Virus (AAV) 2 Vectors

Meumann

Schmithals

Elenschneider

et al. 2022

Cancers

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Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current—or develop novel—strategies for treating HCC.

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“…AAV9 has a preference for glycans ending in a terminal galactose. Necessary co-receptors have also been reported: the hepatocyte growth factor receptor and human fibroblast growth factor receptor-1 for AAV2; platelet-derived growth factor receptor for AAV5; integrin α5β1, LamR, AAVR and GPR108 for a variety of serotypes [18]. Following internalization by the receptor-mediated endocytosis, the AAV is transported toward the nucleus, where uncoating occurs.…”

Section: Aav Biologymentioning

confidence: 99%

PCR-Based Analytical Methods for Quantification and Quality Control of Recombinant Adeno-Associated Viral Vector Preparations

Shmidt

Егорова

2021

Pharmaceuticals

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Recombinant adeno-associated viral vectors (rAAV) represent a gene therapy tool of ever-increasing importance. Their utilization as a delivery vehicle for gene replacement, silencing and editing, among other purposes, demonstrate considerable versatility. Emerging vector utilization in various experimental, preclinical and clinical applications establishes the necessity of producing and characterizing a wide variety of rAAV preparations. Critically important characteristics concerning quality control are rAAV titer quantification and the detection of impurities. Differences in rAAV constructs necessitate the development of highly standardized quantification assays to make direct comparisons of different preparations in terms of assembly or purification efficiency, as well as experimental or therapeutic dosages. The development of universal methods for impurities quantification is rather complicated, since variable production platforms are utilized for rAAV assembly. However, general agreements also should be achieved to address this issue. The majority of methods for rAAV quantification and quality control are based on PCR techniques. Despite the progress made, increasing evidence concerning high variability in titration assays indicates poor standardization of the methods undertaken to date. This review summarizes successes in the field of rAAV quality control and emphasizes ongoing challenges in PCR applications for rAAV characterization. General considerations regarding possible solutions are also provided.

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Adeno-Associated Virus (AAV) Gene Delivery: Dissecting Molecular Interactions upon Cell Entry

Cited by 34 publications

References 204 publications

Single‐Cell Quantification of Triple‐AAV Vector Genomes Coexpressed in Neurons

Single‐Cell Quantification of Triple‐AAV Vector Genomes Coexpressed in Neurons

Hepatocellular Carcinoma Is a Natural Target for Adeno-Associated Virus (AAV) 2 Vectors

PCR-Based Analytical Methods for Quantification and Quality Control of Recombinant Adeno-Associated Viral Vector Preparations

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