Buffer Valency Engineering Enables High-concentration and Shelf-stable DNA Transfection Particles for Viral Vector Production

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.

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.

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

515. Overlapping Adeno-Associated Viral (AAV) Vector Mediated Gene Transfer Is Dependent on Viral Serotype and the Transgene Sequence in Skeletal Muscle

The adeno-associated virus (AAV) holds great potential as a gene delivery vector for emerging gene therapies. However, AAV's production is currently limited by factors such as low viral titers, inefficient capsid packaging, and producer cell toxicity. We sought to address the issue of low titers through deep mutational scanning of the AAV rep gene, which is involved in AAV replication, transcription regulation, and packaging. After generating a library of all single codon substitutions at approximately 300 sites in the gene, we characterized the mutational effects on packaged virus particles in a high-throughput replication competition assay in Expi293F cells suspension culture. The resulting values of the enrichment (a metric of packaging efficiency of native cargo) correlated moderately with those of a previous study in HEK293 adherent cells. However, difference in the range and distribution of values between the two studies as well as experimental variability between replicas in both data sets complicated assessment of cell line differences in mutational effects. We determined the ability of select mutants to alter the packaging of a synthetic DNA cargo into the AAV capsid but did not observe statistically significant improvements in either Expi293F or HEK293 cells. Our study adds to the growing evidence that beneficial effects of mutations in rep are highly dependent on the nature of the packaged DNA.

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.

Human cytomegalovirus completely helps adeno-associated virus replication

Advances in genomics have identified thousands of risk genes impacting human health and diseases, but the functions of these genes and their mechanistic contribution to disease are often unclear. Moving beyond identification to actionable biological pathways requires dissecting risk gene function and cell type-specific action in intact tissues. This gap can in part be addressed by in vivo Perturb-seq, a method that combines state-of-the-art gene editing tools for programmable perturbation of genes with high-content, high-resolution single-cell genomic assays as phenotypic readouts. Here we describe a detailed protocol to perform massively parallel in vivo Perturb-seq using several versatile adeno-associated virus (AAV) vectors and provide guidance for conducting successful downstream analyses. Expertise in mouse work, AAV production and single-cell genomics is required. We discuss key parameters for designing in vivo Perturb-seq experiments across diverse biological questions and contexts. We further detail the step-by-step procedure, from designing a perturbation library to producing and administering AAV, highlighting where quality control checks can offer critical go-no-go points for this time- and cost-expensive method. Finally, we discuss data analysis options and available software. In vivo Perturb-seq has the potential to greatly accelerate functional genomics studies in mammalian systems, and this protocol will help others adopt it to answer a broad array of biological questions. From guide RNA design to tissue collection and data collection, this protocol is expected to take 9-15 weeks to complete, followed by data analysis.

Adeno-associated virus (AAV) -mediated gene therapy is a promising strategy to treat liver-based monogenic diseases. However, two major obstacles limit its success: first, vector dilution in actively dividing cells, such as hepatocytes in neonates/children, due to the non-integrating nature of the vector; second, development of an immune response against the transgene and/or viral vector. Crigler-Najjar Syndrome Type I is a rare monogenic disease with neonatal onset, caused by mutations in the liver-specific UGT1 gene, with toxic accumulation of unconjugated bilirubin in plasma, tissues and brain. To establish an effective and long lasting cure, we applied AAV-mediated liver gene therapy to a relevant mouse model of the disease. Repeated gene transfer to adults by AAV-serotype switching, upon neonatal administration, resulted in lifelong correction of total bilirubin (TB) levels in both genders. In contrast, vector loss over time was observed after a single neonatal administration. Adult administration resulted in lifelong TB levels correction in male, but not female Ugt1-/- mice. Our findings demonstrate that neonatal AAV-mediated gene transfer to the liver supports a second transfer of the therapeutic vector, by preventing the induction of an immune response and supporting the possibility to improve AAV-therapeutic efficacy by repeated administration.

Adeno-associated virus (AAV)-mediated gene therapy has made significant progress in the last few decades. Nevertheless, challenges imposed by the immune system remain. The very high doses of AAV vectors used for some disorders have resulted in serious adverse events (SAEs) or even deaths, demonstrating that AAV vector doses that can safely be injected into patients are limited and for some indications below the therapeutic dose. Currently used immunosuppressive drugs have not prevented the SAEs, indicating that it may be prudent to treat patients with repeated transfer of moderate doses rather than a single injection of high doses of AAV vectors. The former approach has been avoided as AAV vectors elicit neutralizing antibodies that prevent successful reapplication of serologically crossreactive vectors. Immunosuppressive regimens that block B cell responses to AAV vectors or treatments that remove AAV neutralizing antibodies thus need to be developed to allow for a shift from toxic single-dose injections of AAV vectors to repeated treatments with more moderate and safe doses. Preventing or blocking antibody responses would also allow for redosing of patients with declining transgene product expression, or for effective AAV-mediated gene transfer into patients with the pre-existing neutralizing antibodies.

Recently, adeno-associated virus (AAV)-mediated gene therapies have attracted clinical interest for treating neurodegenerative diseases including spinal muscular atrophy (SMA), Canavan disease (CD), Parkinson’s disease (PD), and Friedreich’s ataxia (FA). The influx of clinical findings led to the first approved gene therapy for neurodegenerative disorders in 2019 and highlighted new safety concerns for patients. Large doses of systemically administered AAV stimulate host immune responses, resulting in anti-capsid and anti-transgene immunity with implications for transgene expression, treatment longevity, and patient safety. Delivering lower doses directly to the central nervous system (CNS) is a promising alternative, resulting in higher transgene expression with decreased immune responses. However, neuroinflammatory responses after CNS-targeted delivery of AAV are a critical concern. Reported signs of AAV-associated neuroinflammation in preclinical studies include dorsal root ganglion (DRG) and spinal cord pathology with mononuclear cell infiltration. In this review, we discuss ways to manage neuroinflammation, including choice of AAV capsid serotypes, CNS-targeting routes of delivery, genetic modifications to the vector and/or transgene, and adding immunosuppressive strategies to clinical protocols. As additional gene therapies for neurodegenerative diseases enter clinics, tracking biomarkers of neuroinflammation will be important for understanding the impact immune reactions can have on treatment safety and efficacy.

Gene therapy for colon cancer by adeno-associated viral vector-mediated transfer of survivin Cys84Ala mutant

Sequence and symmetry requirements within the internal palindromic sequences of the adeno-associated virus terminal repeat

306. Production of Safe and Efficient AAV Vectors Using Minicircle DNA