Like humans, companion dogs develop cancers spontaneously in the context of increased age, genetic variations, or environmental exposures, making them valuable as a preclinical model. Here, we assessed the safety and preliminary effectiveness of a multivalent adeno-associated virus (AAV) cancer vaccine in spontaneous canine oral melanoma. Eight companion dogs were vaccinated with equal doses of three different optimized AAV vectors encoding the melanoma others glycoprotein 100 (GP100), tyrosinase (Tyr), and tyrosinase-related protein 1 (TRP-1). Five dogs received a dose of 3 × 1012 viral genomes (vg)/dog, and three others received 9 × 1012 vg/dog. Post-vaccination monitoring established a lack of adverse effects and confirmed the presence of antigen-specific IFN-γ-producing T cells in the blood of most dogs. Although the variable survival times make it difficult to evaluate the effectiveness of the optimized AAV cancer vaccine at this point, three of the eight dogs were disease-free by the end of study, and two dogs survived 804 and 953 days after vaccination, and one is currently alive. Thus, the optimized AAV cancer vaccine showed promise in a spontaneous cancer animal model that closely recapitulates human disease development.

Choosing an optimal combination of adeno-associated virus (AAV) capsid and promoter is key to achieving precise, efficient, and safe gene delivery. However, the limited packaging capacity of AAV can make incorporating large transgenes with strong promoters challenging. To address this, we screened several promoter designs derived from portions of the 5' untranslated regions (UTRs) of the human ERCC6 and ERCC8 DNA repair genes to generate a novel 126-base pair (bp) mini-promoter (CP040). We evaluated the expression capabilities of CP040 and its components in vitro and demonstrated that intravenous (i.v.) administration of AAVDJ vector harboring enhanced green fluorescent protein (eGFP) driven by the CP040 promoter (AAVDJ-CP040-eGFP) in neonatal mice resulted in strong central nervous system (CNS) expression 6 weeks later. CP040 expression was compared with the chicken beta actin (CBA), human elongation factor 1 alpha (EF1α), and human synapsin 1 (hSYN) promoters following intracerebroventricular (ICV) and intrathecal (IT) AAVDJ administrations. The CP040 mini-promoter yielded comparable expression levels to CBA and hSYN promoters and higher levels than EF1α, with lower off-target expression in liver and kidneys. These initial characterizations support CP040 as a promising candidate to enhance AAV-mediated gene therapy targeting the CNS when cargo sequences cannot fit within AAV size constraints.

Background: Irrespective of the rapid development of AAV-based gene therapy, the production of clinical-grade vectors has a bottleneck resulting from product-related impurities such as empty and partially filled capsids, which lack a functional recombinant genome. Methods: In the current study, we applied the sequential affinity chromatography (AC)- and anion-exchange chromatography (AEX)-based method for purification of AAV9 vector harboring single-stranded genome encoding the fusion of firefly luciferase (fLuc)-yellow fluorescent protein (YFP) under chicken beta actin (CBA) promoter. We assessed the efficiency of two different pre-packed cross-linked sepharose and one monolithic AEX columns following AC purification to separate fully encapsulated with recombinant DNA AAV vectors from byproducts. Results: We showed the possibility to achieve approximately 20–80% recovery and over 90% calculated DNA-containing/empty capsid ratio depending on column and buffers composition. Additionally, we confirmed the infectivity of AAV by in vitro luciferase assay regardless of recovery method from different AEX columns. Conclusions: Our purification data indicate the effectiveness of dual chromatography method to obtain rAAV9 vectors with DNA-containing capsid content over 90%.

Recombinant adeno-associated virus (rAAV) is one of the most widely used viral vectors for gene therapy. It is used in very high doses for the treatment of many diseases, making large-scale production for clinical applications challenging. We have established a synthetic biology-based platform to construct stable production cell lines, which can be induced to produce rAAV2. In this study, we extended our cell line construction pipelines for rAAV2 to rAAV8, a serotype whose tropism makes it attractive for gene delivery in multiple tissues. The Genome Module, encoding the rAAV2 genome, and Replication Modules, containing Rep68, DBP and E4orf6 coding sequences, originally used for rAAV2 were retained, but the Packaging Module was modified to replace the AAV2 intron-less cap gene (VP123) with that of AAV8. These three genetic modules were integrated into HEK293 genome to generate four rAAV8 producer cell lines VH1-4, which all produced rAAV8 upon induction. Their productivity was similar to the initial rAAV2 producer cell lines GX2/6 constructed using the same pipeline, but was much lower than conventional triple plasmid transfection. We identified Cap protein production and capsid formation as a potential limiting factor, just as we observed in GX2/6. By integrating more copies of AAV8 VP123 into VH3 clone, the encapsidated rAAV8 titer increased 20-fold to a level comparable to triple transfection. By tuning induction conditions to modulate capsid production, the full particle content could be elevated. This study demonstrated that our rAAV producer cell line development platform is robust and applicable to different AAV serotypes.

We previously showed therapeutic advantages of using a capsid-modified and encoded antigen-optimized AAV-based cancer vaccine to initiate strong antigen-specific immune responses and increase survival in a syngeneic mouse model of melanoma. In this study, we further explore AAV vaccine dose reduction and possible mechanisms of the immune response. Immunization with extracellular vesicle (EV)-associated AAV6-S663V encoded ovalbumin (OVA) or tyrosinaserelated protein 1 (TRP-1) induced significantly higher levels of antigen-specific CD8+ T cells compared with standard AAV in mice. Importantly, a higher number of specific CD8+ T cells was achieved with EV-AAV several logs lower than optAAV-based doses. EV-optAAV-OVA was used in a dose 100 times lower, and EV-optTRP-1 10 times lower than optOVA and optTRP-1 correspondingly. Our data suggest that significant dose reduction for optimized AAV-based vaccines is possible without sacrificing efficiency. In addition, we studied the role of conventional type 1 dendritic cells (cDC1) in optimized AAV-based immunization using a C57BL/6-Irf8em1Kmm (Irf8+32-/-) mouse model lacking cDC1. Interestingly, we found that cDC1 are not essential for conveying effector T cell responses to AAV-encoded tumor antigens.

Highly efficient adeno associated viruses (AAVs) targeting the central nervous system (CNS) are needed to deliver safe and effective therapies for inherited neurological disorders. The goal of this study was to compare the organ-specific transduction efficiencies of two AAV capsids across three different delivery routes. We compared AAV9-CBA-fLucYFP to AAV-DJ-CBA-fLucYFP using the following delivery routes in mice: intracerebroventricular (ICV) 1x1012 vg/kg, intrathecal (IT) 1x1012 vg/kg, and intravenous (IV) 1x1013 vg/kg body weight. Our evaluations revealed that following ICV and IT administrations, AAV-DJ demonstrated significantly increased vector genome (vg) uptake throughout the CNS as compared to AAV9. Through the IV route, AAV9 demonstrated significantly increased vg uptake in the CNS. However, significantly fewer vgs were detected in the off-target organs (kidney and liver) following administration of AAV-DJ using the IT and IV delivery routes as compared to AAV9. Distributions of vgs correlate well with transgene transcript levels, luciferase enzyme activities, and immunofluorescence detection of YFP. Overall, between the two vectors, AAV-DJ resulted in better targeting and expression in CNS tissues paired with de-targeting and reduced expression in liver and kidneys. Our findings support further examination of AAV-DJ as a gene therapy capsid for the treatment of neurological disorders.

The production of clinical-grade recombinant adeno-associated viral (AAV) vectors for gene therapy trials remains a major hurdle in the further advancement of the gene therapy field. During the past decades, AAV research has been predominantly focused on the development of new capsid modifications, vector-associated immunogenicity, and the scale-up vector production. However, limited studies have examined the possibility to manipulate non-structural components of AAV such as the Rep genes. Historically, naturally isolated, or recombinant library-derived AAV capsids have been produced using the AAV serotype 2 Rep gene to package ITR2-flanked vector genomes. In the current study, we mutated four variable amino acids in the conservative part of the binding domain in AAV serotype 6 Rep to generate a Rep2/6 hybrid gene. This newly generated Rep2/6 hybrid had improved packaging ability over wild-type Rep6. AAV vectors produced with Rep2/6 exhibited similar in vivo activity as standard AAV6 vectors. Furthermore, we show that this Rep2/6 hybrid also improves full/empty capsid ratios, suggesting that Rep bioengineering can be used to improve the ratio of fully encapsulated AAV vectors during upstream manufacturing processes.

We previously described therapeutic opportunities provided by capsid- and expression cassette-optimized adeno-associated virus serotype 6 (AAV6) vectors to suppress tumor growth in both solid and metastatic mouse models by using artificial ovalbumin (OVA) immunogen. In the current study, we further elucidated the mechanism of function of a novel AAV-based vaccine loaded with the melanoma tumor-associated antigens premelanosome protein gp100, tyrosinase (Tyr), tyrosinase-related protein 1 (TRP1), and dopachrome tautomerase (TRP2). We showed that the AAV6-based vaccine creates cellular and humoral antigen-specific responses, while antigen expression at the site of vaccine injection was temporal, and the clearance of antigen coincided with T cell infiltration. Our data revealed the superior protective immune response of optimized AAV6-TRP1 compared with other self-antigens in a disease-free mouse model. We further assessed the ability of AAV6-TRP1 to protect animals from metastatic spread in the lungs and to extend animal survival by inhibiting solid tumor growth. Flow cytometry-based analysis indicated significant infiltration of CD8+ T cells and natural killer (NK) cells in the tumor site, as well as changes in the polarization of intratumoral macrophages. Altogether, our data strongly support the use of optimized AAV vectors for cancer vaccine development.

The accurate assessment of AAV-specific pre-existing humoral immunity due to natural viral infection is critical for the efficient use of clinical gene therapy. The method described in the present study applies equivalent infection conditions to each AAV serotype (AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAVAnc80L65). In the current study, we validated the assay by assessing AAV-neutralizing antibody titers in a limited cohort of random human donors and well-established preclinical large animal models, including dogs and non-human primates (NHPs). We achieved a rapid and accurate evaluation of neutralizing titers for each individual subject that can be used for clinical enrollment based on specific AAV serotypes and individualized selection of the most suitable AAV serotype for each specific patient.