MicroRNA‐based recombinant AAV vector assembly improves efficiency of suicide gene transfer in a murine model of lymphoma

Khan, Noureen; Cheemadan, Sabna; Saxena, Harshit; Bammidi, Sridhar; Jayandharan, Giridhara R.

doi:10.1002/cam4.2935

Cited by 5 publications

(6 citation statements)

References 66 publications

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“…Recombinant adeno-associated viruses (rAAV) are a class of viral vectors that are being investigated intensively in the development of gene therapies [ 14 , 15 ]. In order to develop efficient rAAV therapies, produced through controlled and economical manufacturing processes, multiple challenges need to be addressed, from capsid design through identification of optimal process and formulation conditions to comprehensive quality control of drug substance and drug product [ 16 , 17 ].…”

Section: Introductionmentioning

confidence: 99%

Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques

Cole¹,

Fernandes²,

Hussain³

et al. 2021

Pharmaceutics

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Viruses are increasingly used as vectors for delivery of genetic material for gene therapy and vaccine applications. Recombinant adeno-associated viruses (rAAVs) are a class of viral vector that is being investigated intensively in the development of gene therapies. To develop efficient rAAV therapies produced through controlled and economical manufacturing processes, multiple challenges need to be addressed starting from viral capsid design through identification of optimal process and formulation conditions to comprehensive quality control. Addressing these challenges requires fit-for-purpose analytics for extensive characterization of rAAV samples including measurements of capsid or particle titer, percentage of full rAAV particles, particle size, aggregate formation, thermal stability, genome release, and capsid charge, all of which may impact critical quality attributes of the final product. Importantly, there is a need for rapid analytical solutions not relying on the use of dedicated reagents and costly reference standards. In this study, we evaluate the capabilities of dynamic light scattering, multiangle dynamic light scattering, and SEC–MALS for analyses of rAAV5 samples in a broad range of viral concentrations (titers) at different levels of genome loading, sample heterogeneity, and sample conditions. The study shows that DLS and MADLS® can be used to determine the size of full and empty rAAV5 (27 ± 0.3 and 33 ± 0.4 nm, respectively). A linear range for rAAV5 size and titer determination with MADLS was established to be 4.4 × 1011–8.7 × 1013 cp/mL for the nominally full rAAV5 samples and 3.4 × 1011–7 × 1013 cp/mL for the nominally empty rAAV5 samples with 3–8% and 10–37% CV for the full and empty rAAV5 samples, respectively. The structural stability and viral load release were also inferred from a combination of DLS, SEC–MALS, and DSC. The structural characteristics of the rAAV5 start to change from 40 °C onward, with increasing aggregation observed. With this study, we explored and demonstrated the applicability and value of orthogonal and complementary label-free technologies for enhanced serotype-independent characterization of key properties and stability profiles of rAAV5 samples.

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Section: Introductionmentioning

confidence: 99%

Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques

Cole¹,

Fernandes²,

Hussain³

et al. 2021

Pharmaceutics

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“…Negative regulation of MAPK activity has been reported during AAV production (Chung et al, 2023). In addition, dysregulation of MAKP signaling using miR-431 and miR-636 has been reported to improve AAV2 assembly and potency by > 3-fold (Khan et al, 2020). These findings underscore the intricate interplay between cell cycle regulation and signaling pathways during AAV production and may offer strategies to enhance AAV vector yields.…”

Section: Discussionmentioning

confidence: 99%

Transcriptomics-informed pharmacology identifies epigenetic and cell cycle regulators as enhancers of AAV production

Tworig,

Grafton,

Hörer

et al. 2024

Preprint

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Recombinant adeno-associated virus (rAAV) is a widely used viral vector for gene therapy. Despite its clinical efficacy, the manufacturing of rAAV faces challenges in productivity and quality, leading to limited availability. To address the growing demand, next-generation process development should be informed by a mechanistic understanding of the cellular response to rAAV. In this study, we performed transcriptomic analysis of 5 cell lines with variable capacities for rAAV production. Using an intersectional approach, we assessed the transcriptional response to rAAV production and compared transcriptional profiles between high and baseline producers to identify possible targets for enhancing production. Modulation of cell cycle and nucleosome components suggested a reduction of proliferative capacity and a shift toward DNA replication to support rAAV production. During rAAV production, we observed upregulation of several core functions including transcription, stress response, and Golgi and endoplasmic reticulum organization. Conversely, inhibitors of DNA-binding proteins and mitochondrial components were consistently downregulated during rAAV production. We next performed a drug connectivity analysis of these results and identified 5 classes of drugs predicted to enhance rAAV production. Validation studies confirmed the efficacy of HDAC and microtubule inhibitors. Our data uncover novel and previously identified pathways that may enhance rAAV productivity, potentially enabling a path to engineer improved processes and cell lines for higher yields and better quality rAAV production.Graphical abstract

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“…This miRNA was identified based on its ability to bind to the AAV genome (ITRs) and also dysregulate mitogen-activated protein kinase (MAPK) signaling during vector production. AAV6 vectors packaged in this way demonstrated increased transduction efficiency in vitro and expression of an inducible caspase 9 suicide gene [ 94 ]. Treatment with a dimerizer drug resulted in increased cell killing in a murine T-cell lymphoma (EL-4) model following intratumoral application [ 94 ].…”

Section: Use Of Aav Vectors For Cancer Gene Therapy In Preclinicalmentioning

confidence: 99%

“…AAV6 vectors packaged in this way demonstrated increased transduction efficiency in vitro and expression of an inducible caspase 9 suicide gene [94]. Treatment with a dimerizer drug resulted in increased cell killing in a murine T-cell lymphoma (EL-4) model following intratumoral application [94].…”

Section: Cytotoxic Killing Of Tumor Cells: Suicide Gene Transfermentioning

confidence: 99%

Towards Clinical Implementation of Adeno-Associated Virus (AAV) Vectors for Cancer Gene Therapy: Current Status and Future Perspectives

Hacker

Bentler

Kaniowska

et al. 2020

Cancers

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Adeno-associated virus (AAV) vectors have gained tremendous attention as in vivo delivery systems in gene therapy for inherited monogenetic diseases. First market approvals, excellent safety data, availability of large-scale production protocols, and the possibility to tailor the vector towards optimized and cell-type specific gene transfer offers to move from (ultra) rare to common diseases. Cancer, a major health burden for which novel therapeutic options are urgently needed, represents such a target. We here provide an up-to-date overview of the strategies which are currently developed for the use of AAV vectors in cancer gene therapy and discuss the perspectives for the future translation of these pre-clinical approaches into the clinic.

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MicroRNA‐based recombinant AAV vector assembly improves efficiency of suicide gene transfer in a murine model of lymphoma

Cited by 5 publications

References 66 publications

Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques

Characterization of Recombinant Adeno-Associated Viruses (rAAVs) for Gene Therapy Using Orthogonal Techniques

Transcriptomics-informed pharmacology identifies epigenetic and cell cycle regulators as enhancers of AAV production

Towards Clinical Implementation of Adeno-Associated Virus (AAV) Vectors for Cancer Gene Therapy: Current Status and Future Perspectives

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