Massively parallel in vivo Perturb-seq screening.

Self-complementary AAV Vectors; Advances and Applications

Biochemical, Pathological, and Skeletal Improvement of Mucopolysaccharidosis VI After Gene Transfer to Liver but Not to Muscle

Cationic Lipid Formulations Alter the In Vivo Tropism of AAV2/9 Vector in Lung

Adeno-associated virus (AAV) vector preparations are often contaminated with variable amounts of replication-competent AAV (rcAAV), which may influence the behavior of these vectors both in cultured cells and in animals. A packaging plasmid/vector plasmid system containing no significant homology and lacking the wild-type AAV p5 promoter was constructed to eliminate the production of wild-type AAV by recombination. Still, rcAAV was detected in vector produced by cotransfection of these plasmids at large scale. Sequence analysis revealed that nonhomologous recombination was responsible for the generation of these novel rcAAVs. A new AAV packaging plasmid carrying separate rep and cap expression cassettes in opposite transcriptional orientations was constructed. AAV vector preparations produced by using this packaging construct did not contain rcAAV.

Adeno-associated Virus-mediated, Mifepristone-regulated Transgene Expression in the Brain

Genetic manipulation in vivo is a critical method for mechanistically understanding gene function in disease and physiological processes. To facilitate this, embryonic transgenesis in popular animal models like mice has been developed. Compared to the longer, expensive methods of transgenesis, viral vectors, such as adeno-associated virus (AAV), have grown increasingly in popularity due to their relatively low cost and ease of production, translating to an overall greater versatility as a biological tool. In this article, we describe protocols for AAV production and purification for efficient transduction in vivo. Importantly, our method differs from others in application of a streamlined, more cost-effective approach. From this method, as many as 2 × 1013 genome-containing viral particles (vp), or 200 units, can be produced within 3 to 4 weeks, with a minimal cost of $1800 to $2000 for supplies and reagents and <15 hr of personnel time per week. A unit here is defined as 1 × 1011 vp, our standard dose of AAV per animal, injected via tail vein. Therefore, our method provides production and purification of AAV in quantities capable of transducing up to 200 animals. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: AAV production Basic Protocol 2: AAV purification.

Despite decades of research in adeno-associated virus (AAV) and the role of adenovirus in production, the interplay of AAV and adenovirus is not fully understood. Specific regions of the adenoviral genome containing E1, E2a, E4 open reading frame (ORF), and VA RNA have been demonstrated as necessary for AAV production; however, incorporating these regions into either a producer cell line or subcloning into an Ad helper plasmid may lead to inclusion of neighboring adenoviral sequence or ORFs with unknown function. Because AAV is frequently used in gene therapies, removing excessive adenovirus sequences improves the Ad helper plasmid size and manufacturability, and may lead to safer vectors for patients. Furthermore, deepening our understanding of the helper virus genes required for recombinant AAV (rAAV) production has the potential to increase yields and manufacturability of rAAV for clinical and commercial applications. One region continuously included in various Ad helper plasmid iterations is the adenoviral E2a promoter region that appears to be necessary for E2a expression. Due to the compact nature of viral genomes, the E2a promoter region overlaps with the Hexon Assembly/100K protein and the L4 region. The L4 region, which contains the coding sequences for 22K and 33K proteins, had not been thought to be necessary for AAV production. Through molecular techniques, this study demonstrates that the adenoviral 22K protein is essential for rAAV production in HEK293 cells by triple transfection and that the 33K protein synergistically increases rAAV yield.

Rep/Cap gene amplification and high-yield production of AAV in an A549 cell line expressing Rep/Cap.

Adeno-Associated Virus Vectors in Clinical Trials

97. Stability and Inactivation of AAV Serotype 1 Vectors

304. Gene Therapy of Malignant Brain Tumor by a Novel Double-Stranded Adeno-Associated Viral (AAV) Vector Carrying the Decorin Gene

Adeno-associated virus (AAV) is a versatile gene vector that is widely used in mammalian research. In basic studies and large-scale AAV production, genetic testing is ubiquitous and routine polymerase chain reaction (PCR)-based tests limit the efficiency due to the labor-intensive and time-consuming requirements of thermal cycling. This study introduces an assay based on recombinase-aided amplification combined with lateral flow (LF-RAA), which can quickly and accurately detect the AAV genome, thus improving the efficiency of AAV research and production. This application is the first use of an RAA approach to AAV genome detection. In this point-of-care testing (POCT) detection platform, the RAA reaction and LF readout are integrated into a user-friendly microfluidic chip that can be applied without advanced technical training. The LF-RAA chip provides high sensitivity, with a limit of detection of 10 copies/μL, and generates results quickly, and it only needs to be incubated for 10 min at a constant temperature, that is, 39 °C. Results are visualized on the LF Dipstick, and detection results are reliable, validated with 100% accuracy in 47 laboratory-produced recombination adeno-associated virus (rAAV) samples carrying target genes from several different viruses. The LF-RAA assay is applicable in AAV research and production processes requiring genome identification.

Genome-wide activation screens to increase adeno-associated virus production

Recombinant Adeno-associated Virus Vectors Efficiently Transduce Foreign Gene Into Bovine Aortic Endothelial Cells: Comparison With Adenovirus Vectors

Because our in silico analysis with a human transcription factor database demonstrated the presence of several binding sites for NF-κB, a central regulator of cellular immune and inflammatory responses, in the adeno-associated virus (AAV) genome, we investigated whether AAV uses NF-κB during its life cycle. We used small molecule modulators of NF-κB in HeLa cells transduced with recombinant AAV vectors. VP16, an NF-κB activator, augmented AAV vector-mediated transgene expression up to 25-fold. Of the two NF-κB inhibitors, Bay11, which blocks both the canonical and the alternative NF-κB pathways, totally ablated transgene expression, whereas pyrrolidone dithiocarbamate, which interferes with the classical NF-κB pathway, had no effect. Western blot analyses confirmed the abundance of the nuclear p52 protein component of the alternative NF-κB pathway in the presence of VP16, which was ablated by Bay11, suggesting that AAV transduction activates the alternative NF-κB pathway. In vivo, hepatic AAV gene transfer activated the canonical NF-κB pathway within 2 h, resulting in expression of proinflammatory cytokines and chemokines (likely reflecting the sensing of viral particles by antigen-presenting cells), whereas the alternative pathway was activated by 9 h. Bay11 effectively blocked activation of both pathways without interfering with long-term transgene expression while eliminating proinflammatory cytokine expression. These studies suggest that transient immunosuppression with NF-κB inhibitors before transduction with AAV vectors should lead to a dampened immune response, which has significant implications in the optimal use of AAV vectors in human gene therapy.