Adeno-associated viral vectors (AAVs) are a leading delivery system for gene therapy in animal models and humans. With several Food and Drug Administration-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.

UNASSIGNED: Recent studies reported that the hepatic expression of AQP8 and AQP9 was downregulated in bile duct-ligated (BDL) rats and that overexpression of human AQP1 in the rat liver attenuated cholestasis. However, the hepatic expression of AQP10 and its regulatory mechanism in human cholestasis remain unclear.
UNASSIGNED: Serum and liver samples were collected from 34 patients with obstructive cholestasis and from 12 control patients. Eight-week-old male C57BL/6J mice were intravenously injected with an adeno-associated virus 8 (AAV8) encoding human AQP10 driven by a hepatocyte-specific Alb promotor (AAV8-Alb promotor-hAQP10) for functional studies. Constructs of the AQP10 promoter and PLC/PRF/5-ASBT cell lines were used for regulatory mechanism studies.
UNASSIGNED: AQP10 was significantly downregulated in patients with obstructive cholestasis and negatively associated with the serum levels of total bile acid (TBA). The hepatocyte-specific overexpression of hAQP10 significantly attenuated the cholestatic liver injury and intrahepatic bile acids (BA) accumulation in BDL mice. Conjugated BAs, such as TCA and inflammatory factor TNFα, significantly repressed AQP10 expression. Furthermore, NFκB p65/p50 directly bound to the AQP10 promotor and decreased its activity in PLC/RPF/5-ASBT cells and in the livers of patients with obstructive cholestasis. However, these changes were diminished by BAY 11-7082 (a specific inhibitor of NFκB signaling).
UNASSIGNED: We are the first to report that AQP10 was significantly decreased in patients with obstructive cholestasis. AQP10 overexpression significantly attenuated cholestatic liver injury in BDL mice. Therefore, overexpression of hAQP10 in the liver may be a valuable strategy for cholestasis intervention.

Progressive familial intrahepatic cholestasis (PFIC) is a liver disease that occurs during childhood and requires liver transplantation. ABCB4 is localized along the canalicular membranes of hepatocytes, transports phosphatidylcholine into bile, and its mutation causes PFIC3. Abcb4 gene-deficient mice established as animal models of PFIC3 exhibit cholestasis-induced liver injury. However, their phenotypes are often milder than those of human PFIC3, partly because of the existence of large amounts of less toxic hydrophilic bile acids synthesized by the rodent-specific enzymes Cyp2c70 and Cyp2a12. Mice with double deletions of Cyp2c70/Cyp2a12 (CYPDKO mice) have a human-like hydrophobic bile acid composition. PFIC-related gene mutations were induced in CYPDKO mice to determine whether these triple-gene-deficient mice are a better model for PFIC. To establish a PFIC3 mouse model using CYPDKO mice, we induced abcb4 gene deletion in vivo using adeno-associated viruses expressing SaCas9 under the control of a liver-specific promoter and abcb4-target gRNAs. Compared to Abcb4-deficient wild-type mice, Abcb4-deficient CYPDKO mice showed more pronounced liver injury along with an elevation of inflammatory and fibrotic markers. The proliferation of intrahepatic bile ductal cells and hematopoietic cell infiltration were also observed. CYPDKO/abcb4-deficient mice show a predominance of taurine-conjugated chenodeoxycholic acid and lithocholic acid in the liver. In addition, phospholipid levels in the gallbladder bile were barely detectable. Mice with human-like bile acids exhibit severe cholestatic liver injury when Abcb4 is knocked down using genome editing technology. This mouse model is useful for studying human cholestatic diseases and developing new treatments.

Recombinant adeno-associated virus (rAAV) is the most widely used viral vector for in vivo human gene therapy. To ensure safety and efficacy of gene therapy products, a comprehensive analytical profile of the rAAVs is needed, which provides crucial information for therapeutic development and manufacturing. Besides information on rAAV quantities and possible contaminating DNA and protein species, assessing rAAV quality is of utmost importance. In vitro biopotency and methods to determine the full/empty ratio of rAAV capsids are commonly applied, but methods to assess the integrity of the viral genome are still rarely used. Here we describe an orthogonal approach to characterize rAAV quality. Two biologically different rAAV9s from different stages of the bioprocess, generated each with two different transfection reagents, were investigated. In vitro biopotency tests in all cases demonstrated that rAAV9s generated with transfection reagent FectoVIR® possessed a higher biological activity. Mass-based analytical methods, such as sedimentation velocity analytical ultracentrifugation (AUC) and mass photometry, showed a high share of full capsids (>80 %) at late process stages but did not detect any differences in the rAAV9s from the different transfection reagents. Multiplex dPCR and Nanopore long-read sequencing both demonstrated that, also in late-stage process samples, sample heterogeneity was relatively high with a rather small share of full-length transgenes of ∼10-40 %. Intriguingly, both methods detected a higher share of complete transgenes in rAAV9 generated with transfection reagent FectoVIR® instead of Polyethylenimine (PEI), and thereby explain the differences already observed in the biopotency assays. This study therefore emphasizes the necessity to utilize multiple, orthogonal methods to gain a better understanding of recombinantly manufactured AAVs.

Adeno-associated viruses (AAVs) are gaining traction as delivery vehicles for gene therapy although the molecular understanding of AAV-transgene release is still limited. Typically, the process of viral uncoating is investigated (in vitro) through thermal stress, revealing capsid disintegration at elevated temperatures. To assess the (in)stability of different empty and filled AAV preparations, we used the light-scattering-based interferometric microscopy technique of mass photometry that, on a single-particle basis, determines the molecular weight of AAVs. By introducing a heat-stable DNA plasmid as an internal standard, we quantitatively probed the impact of heat on AAVs. Generally, empty AAVs exhibited greater heat resistance than genome-filled particles. Our data also indicate that upon DNA release, the capsids do not transform into empty AAVs, but seem to aggregate or disintegrate. Strikingly, some AAVs exhibited an intermediate state with disrupted capsids but preserved bound genome, a feature that experimentally only emerged following incubation with a nuclease. Our data demonstrate that the thermal uncoating process is highly AAV specific (i.e., can be influenced by serotype, genome, host system). We argue that nuclease treatment in combination with MP can be used as an additional analytical tool for assessing structural integrity of recombinant and/or clinical AAV vectors.

Adeno-associated viral vector (AAV) gene therapy provides a promising platform for treatment of monogenic inherited disorders. Clinical studies have demonstrated long-term expression with reduction in bleeding using this approach for the treatment of hemophilia. Despite these advances, there are unknowns surrounding the natural history of recombinant AAV (rAAV) vectors and the cellular mechanisms mediating vector persistence. These unknowns underpin questions regarding long-term efficacy and safety. The predominant mechanism via which AAV is proposed to persist is in circular double-stranded extrachromosomal DNA structures (episomes) within the nucleus. Studies of wild-type (WT-AAV) and rAAV have demonstrated that AAV also persists via integration into a host cell\'s DNA. It is important to determine whether these integration events can mediate expression or could result in any long-term safety concerns. WT-AAV infection affects a large proportion of the general population, which is thought to have no long-term sequelae. Recent studies have highlighted that this WT-AAV has been detected in cases of acute hepatitis in children and in a minority of cases of hepatocellular carcinoma. Integration following treatment using rAAV has also been reported in preclinical and clinical studies. There have been variable reports on the potential implications of integration for rAAV vectors with data in some murine studies demonstrating recurrent integration with development of hepatocellular carcinoma. These findings have not been seen in other pre-clinical or clinical studies. In this review, we will summarize current understanding of the natural history of AAV (wild-type and recombinant) with a focus on genomic integration and the cellular implications.

Osteoarthritis (OA) pain is often associated with the expression of tumor necrosis factor alpha (TNF-α), suggesting that TNF-α is one of the main contributing factors that cause inflammation, pain, and OA pathology. Thus, inhibition of TNF-α could potentially improve OA symptoms and slow disease progression. Anti-TNF-α treatments with antibodies, however, require multiple treatments and cannot entirely block TNF-α. TNF-α-induced protein 8-like 2 (TIPE2) was found to regulate the immune system\'s homeostasis and inflammation through different mechanisms from anti-TNF-α therapies. With a single treatment of adeno-associated virus (AAV)-TIPE2 gene delivery in the accelerated aging Zmpste24-/- (Z24-/-) mouse model, we found differences in Safranin O staining intensity within the articular cartilage (AC) region of the knee between TIPE2-treated mice and control mice. The glycosaminoglycan content (orange-red) was degraded in the Z24-/- cartilage while shown to be restored in the TIPE2-treated Z24-/- cartilage. We also observed that chondrocytes in Z24-/- mice exhibited a variety of senescent-associated phenotypes. Treatment with TIPE2 decreased TNF-α-positive cells, β-galactosidase (β-gal) activity, and p16 expression seen in Z24-/- mice. Our study demonstrated that AAV-TIPE2 gene delivery effectively blocked TNF-α-induced inflammation and senescence, resulting in the prevention or delay of knee OA in our accelerated aging Z24-/- mouse model.

Influenza remains a worldwide public health threat. Although seasonal influenza vaccines are currently the best means of preventing severe disease, the standard-of-care vaccines require frequent updating due to antigenic drift and can have low efficacy, particularly in vulnerable populations. Here, we demonstrate that a single administration of a recombinant adenovirus-associated virus (rAAV) vector expressing a computationally optimized broadly reactive antigen (COBRA)-derived influenza H1 hemagglutinin (HA) induces strongly neutralizing and broadly protective antibodies in naïve mice and ferrets with pre-existing influenza immunity. Following a lethal viral challenge, the rAAV-COBRA vaccine allowed for significantly reduced viral loads in the upper and lower respiratory tracts and complete protection from morbidity and mortality that lasted for at least 5 months post-vaccination. We observed no signs of antibody waning during this study. CpG motif enrichment of the antigen can act as an internal adjuvant to further enhance the immune responses to allow for lower vaccine dosages with the induction of unique interferon-producing CD4+ and CD8+ T cells specific to HA head and stem peptide sequences. Our studies highlight the utility of rAAV as an effective platform to improve seasonal influenza vaccines.
OBJECTIVE: Developing an improved seasonal influenza vaccine remains an ambitious goal of researchers and clinicians alike. With influenza routinely causing severe epidemics with the potential to rise to pandemic levels, it is critical to create an effective, broadly protective, and durable vaccine to improve public health worldwide. As a potential solution, we created a rAAV viral vector expressing a COBRA-optimized influenza hemagglutinin antigen with modestly enriched CpG motifs to evoke a robust and long-lasting immune response after a single intramuscular dose without needing boosts or adjuvants. Importantly, the rAAV vaccine boosted antibody breadth to future strains in ferrets with pre-existing influenza immunity. Together, our data support further investigation into the utility of viral vectors as a potential avenue to improve our seasonal influenza vaccines.

The adeno-associated virus (AAV) vector is one of the most advanced platforms for gene therapy because of its low immunogenicity and non-pathogenicity. The concentrations of both AAV vector empty particles, which do not contain DNA and do not show any efficacy, and AAV vector full particles (FPs), which contain DNA, are important quality attributes. In this study, a dual fluorescence-linked immunosorbent assay (dFLISA), which uses two fluorescent dyes to quantify capsid and genome titers in a single analysis, was established. In dFLISA, capture of AAV particles, detection of capsid proteins, and release and detection of the viral genome are performed in the same well. We demonstrated that the capsid and genomic titers determined by dFLISA were comparable with those of analytical ultracentrifugation. The FP ratios determined by dFLISA were in good agreement with the expected values. In addition, we showed that dFLISA can quantify the genomic and capsid titers of crude samples. dFLISA can be easily modified for measuring other AAV vector serotypes and AAV vectors with different genome lengths. These features make dFLISA a valuable tool for the future development of AAV-based gene therapies.

Neurodegenerative disorders, such as Parkinson\'s disease (PD) and Alzheimer\'s disease (AD), have a global prevalence and profoundly impact both motor and cognitive functions. Although adeno-associated virus (AAV)-based gene therapy has shown promise, its application for treating central nervous system (CNS) diseases faces several challenges, including effective delivery of AAV vectors across the blood-brain barrier, determining optimal dosages, and achieving targeted distribution. To address these challenges, we have developed a fusion delivery therapeutic cargo called AAV-aMTD-Parkin, which combines a hydrophobic cell-penetrating peptide sequence with the DNA sequences of AAV and Parkin. By employing this fusion delivery platform at lower dosages compared to zolgensma, we have achieved significant enhancements in cell and tissue permeability, while reducing the occurrence of common pathological protein aggregates. Consequently, motor and cognitive functions were restored in animal models of PD and AD. With its dual functionality in addressing PD and AD, AAV-aMTD-Parkin holds immense potential as a novel class of therapeutic biologics for prevalent CNS diseases.