Universal Real-Time PCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2-Derived Inverted Terminal Repeat Sequences

Aurnhammer, Christine; Haase, Maren; Muether, Nadine; Hausl, Martin A; Rauschhuber, Christina; Huber, Ingrid; Nitschko, Hans; Busch, Ulrich; Sing, Andreas; Ehrhardt, Anja; Baiker, Armin

doi:10.1089/hum.2011.034

Cited by 70 publications

(76 citation statements)

References 30 publications

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“…AAV vectors were purified using the AAVpro Purification Kit All Serotypes (6666; Takara Bio Inc.). Virus titers were then determined by qPCR using the primer pair AAV2 ITR 38 , Luna Universal® qPCR Master Mix (M3003S; New England Biolabs, Ipswitch, MA), and the LightCycler® qPCR 2.0 system (DX400; Roche, Basel, Switzerland).…”

Section: Methodsmentioning

confidence: 99%

Visualization and molecular characterization of whole-brain vascular networks with capillary resolution

et al. 2020

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Structural elucidation and molecular scrutiny of cerebral vasculature is crucial for understanding the functions and diseases of the brain. Here, we introduce SeeNet, a method for near-complete three-dimensional visualization of cerebral vascular networks with high signalto-noise ratios compatible with molecular phenotyping. SeeNet employs perfusion of a multifunctional crosslinker, vascular casting by temperature-controlled polymerization of hybrid hydrogels, and a bile salt-based tissue-clearing technique optimized for observation of vascular connectivity. SeeNet is capable of whole-brain visualization of molecularly characterized cerebral vasculatures at the single-microvessel level. Moreover, SeeNet reveals a hitherto unidentified vascular pathway bridging cerebral and hippocampal vessels, thus serving as a potential tool to evaluate the connectivity of cerebral vasculature.

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Section: Methodsmentioning

confidence: 99%

Visualization and molecular characterization of whole-brain vascular networks with capillary resolution

et al. 2020

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“…AAV6 was produced in HEK293 cells and purified using the iodixanol density gradient ultracentrifugation method as previously reported. 29 AAV6 was titered using qPCR with probes corresponding to the inverse terminal repeat region 30 and applied to cells at a MOI of 5E4 vector genomes per cell.…”

Section: Experimental Design To Probe the Transcriptional Response Tomentioning

confidence: 99%

Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells

et al. 2018

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Genome-editing technologies are currently being translated to the clinic. However, cellular effects of the editing machinery have yet to be fully elucidated. Here, we performed global microarray-based gene expression measurements on human CD34 hematopoietic stem and progenitor cells that underwent editing. We probed effects of the entire editing process as well as each component individually, including electroporation, Cas9 (mRNA or protein) with chemically modified sgRNA, and AAV6 transduction. We identified differentially expressed genes relative to control treatments, which displayed enrichment for particular biological processes. All editing machinery components elicited immune, stress, and apoptotic responses. Cas9 mRNA invoked the greatest amount of transcriptional change, eliciting a distinct viral response and global transcriptional downregulation, particularly of metabolic and cell cycle processes. Electroporation also induced significant transcriptional change, with notable downregulation of metabolic processes. Surprisingly, AAV6 evoked no detectable viral response. We also found Cas9/sgRNA ribonucleoprotein treatment to be well tolerated, in spite of eliciting a DNA damage signature. Overall, this data establishes a benchmark for cellular tolerance of CRISPR/Cas9-AAV6-based genome editing, ensuring that the clinical protocol is as safe and efficient as possible.

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“…The purified viruses were concentrated using Amicon 10 kDa concentrator (Millipore) and stored in low retention tubes at −80°C. Packaged AAV vectors were quantified by a real‐time PCR‐based method described earlier . The physical particle titers of vectors were estimated from an average of six replicate samples and expressed as vector genomes (vg)/mL.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2020

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Recent success in clinical trials with recombinant Adeno‐associated virus (AAV)‐based gene therapy has redirected efforts in optimizing AAV assembly and production, to improve its potency. We reasoned that inclusion of a small RNA during vector assembly, which specifically alters the phosphorylation status of the packaging cells may be beneficial. We thus employed microRNAs (miR‐431, miR‐636) identified by their ability to bind AAV genome and also dysregulate Mitogen‐activated protein kinase (MAPK) signaling during vector production, by a global transcriptome study in producer cells. A modified vector assembly protocol incorporating a plasmid encoding these microRNAs was developed. AAV2 vectors packaged in the presence of microRNA demonstrated an improved gene transfer potency by 3.7‐fold, in vitro. Furthermore, AAV6 serotype vectors encoding an inducible caspase 9 suicide gene, packaged in the presence of miR‐636, showed a significant tumor regression (~2.2‐fold, P < .01) in a syngeneic murine model of T‐cell lymphoma. Taken together, we have demonstrated a simple but effective microRNA‐based approach to improve the assembly and potency of suicide gene therapy with AAV vectors.

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Universal Real-Time PCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2-Derived Inverted Terminal Repeat Sequences

Cited by 70 publications

References 30 publications

Visualization and molecular characterization of whole-brain vascular networks with capillary resolution

Visualization and molecular characterization of whole-brain vascular networks with capillary resolution

Global Transcriptional Response to CRISPR/Cas9-AAV6-Based Genome Editing in CD34+ Hematopoietic Stem and Progenitor Cells

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

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