Multivariate Process Optimization for Fixed-Bed Bioreactor-Based AAV Production Improves Total Batch Yield.

Oracle or false prophet? Can we predict AAV efficacy based on preexisting antibody titers?

Tissue ischemia is a common occurrence in many disease processes including chronic wounds, stroke, solid tumors, and myocardial infarction. The application of gene delivery for healing of wounds has demonstrated increasing therapeutic promises in animal models. Adenoviral vectors have been successfully used for gene delivery to the ischemic wound. However, these vectors typically demonstrate short, transient transgene expression while eliciting significant cytotoxic immune response. Adeno-associated viral vectors (AAV) do not have those limitations; however, scant information is available about their transfection efficiency under low-oxygen tension. The goal of this study was to compare AAV vector with adenoviral vector in terms of relative efficiency of gene delivery and cytotoxic immune response in ischemic wounds. Reporter constructs Ad5-LacZ and AAV-LacZ (108 pfu/wound) were injected onto the dermis of rabbit ear prior to creation of ischemic wounds. Wounds were harvested at postoperative day 10. Frozen sections of the wounds were fixed in cold acetone and stained with an in situ β-gal staining kit. Intense expression of β-gal was observed with both vectors; however, transduction rates with AAV vector was approximately 10-fold lower than Adenovirus. Unlike Adenovirus, no noticeable inflammatory cell infiltration was observed with AAV injection. Even when the dosage of AAV was increased to 109 pfu/wound inflammatory cell infiltration remained negligible. Thus our data indicates that both AAV and adenoviral vectors are suitable to use in gene-therapy experiments in ischemic tissues. The particular advantage of AAV is the ability to transfect with higher doses while at lower dose maximal transfection rate seems to be more with Adenovirus.

228. AAV Mediated Hypoxia-Inducible VEGF Expression and Neovascular Formation in Ischemic Pig Myocardium

Self-complementary AAV Vectors; Advances and Applications

Hepatic AAV Gene Transfer and the Immune System: Friends or Foes?

Generation of Novel AAV Variants by Directed Evolution for Improved CFTR Delivery to Human Ciliated Airway Epithelium

Efficient Intrathymic Gene Transfer Following In Situ Administration of a rAAV Serotype 8 Vector in Mice and Nonhuman Primates

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

Adeno-associated viral vectors (AAVs) have become popular for gene therapy, given their many advantages, including their reduced inflammatory profile compared with that of other viruses. However, even in areas of immune privilege such as the eye, AAV vectors are capable of eliciting host-cell responses. To investigate the effects of such responses on several ocular cell types, we tested multiple AAV genome structures and capsid types using subretinal injections in mice. Assays of morphology, inflammation, and physiology were performed. Pathological effects on photoreceptors and the retinal pigment epithelium (RPE) were observed. Müller glia and microglia were activated, and the proinflammatory cytokines TNF-α and IL-1β were up-regulated. There was a strong correlation between cis-regulatory sequences and toxicity. AAVs with any one of three broadly active promoters, or an RPE-specific promoter, were toxic, while AAVs with four different photoreceptor-specific promoters were not toxic at the highest doses tested. There was little correlation between toxicity and transgene, capsid type, preparation method, or cellular contaminants within a preparation. The toxic effect was dose-dependent, with the RPE being more sensitive than photoreceptors. Our results suggest that ocular AAV toxicity is associated with certain AAV cis-regulatory sequences and/or their activity and that retinal damage occurs due to responses by the RPE and/or microglia. By applying multiple, sensitive assays of toxicity, AAV vectors can be designed so that they can be used safely at high dose, potentially providing greater therapeutic efficacy.

723. AAV Mediated Cancer Targeting: Systemic Trafficking to Tumor Is More Important Than Vector Tumor Cell Interaction

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Young scientists grant MNS9/2021 Polish National Science Centre PRELUDIUM grant 2019/33/N/NZ1/03066 Introduction Although gene therapy has become a feasible alternative to regular treatment in numerous disorders, efficient targeting of human heart still seems out of reach. So far, adeno-associated viral vectors (AAVs) have been the most promising transgene carriers in in vivo applications. Transduction with AAVs relies on serotype-specific binding to the glycan residues on the cell surface. While AAV serotype 9 (AAV9) - binding galactose - was successfully used for delivery of therapeutic genes to cardiac muscle in murine models, its effect was underwhelming in large animals. Switching the serotype to AAV1 or AAV6 – binding sialic acids – enabled transduction of porcine hearts, and cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) in 2D in vitro culture. But, as evidenced in clinical trials where less than 1% of heart cells contained delivered transgene, such strategy was not suitable for in vivo application in humans. Purpose Taking into account differences in glycosylation patterns between humans and other mammals, we aimed to investigate the role of surface glycans in AAV6 and AAV9 transduction mechanism in 2D and 3D cultures of human cardiomyocytes. Methods We generated hiPSC- derived cardiomyocytes (hiPSC-CMs) and epicardial fibroblasts (hiPSC-CFs). Cells were cultured in regular 2D conditions or 3D co-cultures (as spheroids), since direct cells interaction may influence the availability of terminal sugar residues. Surface glycans were stained with lectins and imaged using confocal microscope. Results While in 2D conditions AAV6 transduces hiPSC-CMs very efficiently, it is significantly less potent in 3D culture. Conversely, AAV9 maintains most of its functionality in 3D model. Our data collected from spheroids, indicate that cells in such 3D cultures undergo significant changes in terminal glycosylation pattern. While the level of galactose (AAV9 receptor) increased from day 1 to day 7, the signal from sialic acids attached to galactose (AAV6 receptor) decreased. This effect was accompanied by the upregulation of sialidase-3 expression, that regulates availability of galactose residues on the cell surface. As a result, transduction efficiency was improved in spheroids exposed to AAV9 on day 7 of 3D culture, in comparison to those transduced on day 1. Inhibition of sialidases activity with NADNA reduced the level of terminal galactose in both, hiPSC-CMs and hiPSC-CFs, and was associated with more efficient transduction of cardiomyocytes with AAV6, and less efficient transduction with AAV9. Additionally, we observed that galactosylation of collagen can be another factor that influences cell-vector interaction in 2D and 3D culture. Conclusions Our results demonstrate that the availability of terminal sugar residues is a key factor regulating transduction of hiPSC-CMs with AAV vectors, and underline the need for development of appropriate models for testing of AAV functionality in preclinical studies.

Site-specific Modification of AAV Vector Particles With Biophysical Probes and Targeting Ligands Using Biotin Ligase

152. Retinal Tropism of Exosome-Associated AAV Vector Via Intravitreal Delivery

Efficient Whole-body Transduction with Trans-splicing Adeno-associated Viral Vectors

Adeno-associated viral vectors (AAV) are a leading delivery system for gene therapy in animal models and humans. With several FDA-approved AAV gene therapies on the market, issues related to vector manufacturing have become increasingly important. In this study, we focused on potentially toxic DNA contaminants that can arise from AAV proviral plasmids, the raw materials required for manufacturing recombinant AAV in eukaryotic cells. Typical AAV proviral plasmids are circular DNAs containing a therapeutic gene cassette flanked by natural AAV inverted terminal repeat (ITR) sequences, and a plasmid backbone carrying prokaryotic sequences required for plasmid replication and selection in bacteria. While the majority of AAV particles package the intended therapeutic payload, some capsids instead package the bacterial sequences located on the proviral plasmid backbone. Since ITR sequences also have promoter activity, potentially toxic bacterial open reading frames can be produced in vivo, thereby representing a safety risk. In this study, we describe a new AAV proviral plasmid for vector manufacturing that (1) significantly decreases cross-packaged bacterial sequences; (2) increases correctly packaged AAV payloads; and (3) blunts ITR-driven transcription of cross-packaged material to avoid expressing potentially toxic bacterial sequences. This system may help improve the safety of AAV vector products.