AAV Vectors: The Rubik's Cube of Human Gene Therapy.

Background: Despite several studies on the effect of adeno-associated virus (AAV)-based therapeutics on osteoarthritis (OA), information on the transduction efficiency and applicable profiles of different AAV serotypes to chondrocytes in hard cartilage tissue is still limited. Moreover, the recent discovery of additional AAV serotypes makes it necessary to screen for more suitable AAV serotypes for specific tissues. Here, we compared the transduction efficiencies of 14 conventional AAV serotypes in human chondrocytes, mouse OA models, and human cartilage explants obtained from OA patients. Methods: To compare the transduction efficiency of individual AAV serotypes, green fluorescent protein (GFP) expression was detected by fluorescence microscopy or western blotting. Likewise, to compare the transduction efficiencies of individual AAV serotypes in cartilage tissues, GFP expression was determined using fluorescence microscopy or immunohistochemistry, and GFP-positive cells were counted. Results: Only AAV2, 5, 6, and 6.2 exhibited substantial transduction efficiencies in both normal and OA chondrocytes. All AAV serotypes except AAV6 and rh43 could effectively transduce human bone marrow mesenchymal stem cells. In human and mouse OA cartilage tissues, AAV2, AAV5, AAV6.2, AAV8, and AAV rh39 showed excellent tissue specificity based on transduction efficiency. These results indicate the differences in transduction efficiencies of AAV serotypes between cellular and tissue models. Conclusions: Our findings indicate that AAV2 and AAV6.2 may be the best choices for AAV-mediated gene delivery into intra-articular cartilage tissue. These AAV vectors hold the potential to be of use in clinical applications to prevent OA progression if appropriate therapeutic genes are inserted into the vector.

/pdf/cellular-and-tissue-selectivity-of-aav-serotypes-for-gene-350x91i1rv.pdf

Cellular and Tissue Selectivity of AAV Serotypes for Gene Delivery to Chondrocytes and Cartilage.

Viral gene transfer, known as transduction, is a powerful research tool for studying the biology of chondrocytes in novel ways and also a technology enabling the use of gene therapy for regenerating cartilage and treating diseases that affect cartilage, such as osteoarthritis. Adenovirus, retrovirus, lentivirus, and adeno-associated virus (AAV) are most commonly used to transduce chondrocytes. Although AAV is able to transduce chondrocytes in situ by intra-articular injection, chondrocytes are most commonly transduced in monolayer culture using the four vectors mentioned above. Protocols for achieving this are described, along with a discussion of the variables that can influence transduction efficiency. 

Viral Gene Delivery in Chondrocytes.

Abstract α‐Solanine has been shown to exhibit anti‐inflammatory and anti‐tumour properties; however, its efficacy in treating osteoarthritis (OA) remains ambiguous. The study aimed to evaluate the therapeutic effects of α‐solanine on OA development in a mouse OA model. The OA mice were subjected to varying concentrations of α‐solanine, and various assessments were implemented to assess OA progression. We found that α‐solanine significantly reduced osteophyte formation, subchondral sclerosis and OARSI score. And it decreased proteoglycan loss and calcification in articular cartilage. Specifically, α‐solanine inhibited extracellular matrix degradation by downregulating collagen 10, matrix metalloproteinase 3 and 13, and upregulating collagen 2. Importantly, α‐solanine reversed chondrocyte pyroptosis phenotype in articular cartilage of OA mice by inhibiting the elevated expressions of Caspase‐1, Gsdmd and IL‐1β, while also mitigating aberrant angiogenesis and sensory innervation in subchondral bone. Mechanistically, α‐solanine notably hindered the early stages of OA progression by reducing I‐κB phosphorylation and nuclear translocation of p65, thereby inactivating NF‐κB signalling. Our findings demonstrate the capability of α‐solanine to disrupt chondrocyte pyroptosis and sensory innervation, thereby improving osteoarthritic pathological progress by inhibiting NF‐κB signalling. These results suggest that α‐solanine could serve as a promising therapeutic agent for OA treatment.

α‐Solanine attenuates chondrocyte pyroptosis to improve osteoarthritis via suppressing NF‐κB pathway



Osteoarthritis (OA) is a highly debilitating, degenerative pathology of cartilaginous
joints affecting over 500 million people worldwide. The global economic burden of OA
is estimated at $260-519 billion and growing, driven by aging global population and increasing
rates of obesity. To date, only the multi-injection chondroanabolic treatment regimen of Fibroblast
Growth Factor 18 (FGF18) has demonstrated clinically meaningful disease-modifying efficacy in
placebo-controlled human trials. Our work focuses on the development of a novel single injection
disease-modifying gene therapy, based on FGF18’s chondroanabolic activity.



OA was induced in Sprague-Dawley rats using destabilization of the medial meniscus
(DMM) (3 weeks), followed by intra-articular treatment with 3 dose levels of AAV2-FGF18, rh-
FGF18 protein, and PBS. Durability, redosability, and biodistribution were measured by quantifying
nLuc reporter bioluminescence. Transcriptomic analysis was performed by RNA-seq on cultured
human chondrocytes and rat knee joints. Morphological analysis was performed on knee
joints stained with Safranin O/Fast Green and anti-PRG antibody.



Dose-dependent reductions in cartilage defect size were observed in the AAV2-FGF18-
treated joints relative to the vehicle control. Total defect width was reduced by up to 76% and cartilage
thickness in the thinnest zone was increased by up to 106%. Morphologically, the vehicle-
treated joints exhibited pronounced degeneration, ranging from severe cartilage erosion and
bone void formation, to subchondral bone remodeling and near-complete subchondral bone collapse.
In contrast, AAV2-FGF18-treated joints appeared more anatomically normal, with only regional
glycosaminoglycan loss and marginal cartilage erosion. While effective at reducing cartilage
lesions, treatment with rhFGF18 injections resulted in significant joint swelling (19% increase
in diameter), as well as a decrease in PRG4 staining uniformity and intensity. In contrast to
early-timepoint in vitro RNA-seq analysis, which showed a high degree of concordance between
protein- and gene therapy-treated chondrocytes, in vivo transcriptomic analysis, revealed few gene
expression changes following protein treatment. On the other hand, the gene therapy treatment exhibited
a high degree of durability and localization over the study period, upregulating several
chondroanabolic genes while downregulating OA- and fibrocartilage-associated markers.



FGF18 gene therapy treatment of OA joints can provide benefits to both cartilage
and subchondral bone, with a high degree of localization and durability.


Single Injection AAV2-FGF18 Gene Therapy Reduces Cartilage Loss
and Subchondral Bone Damage in a Mechanically Induced Model of
Osteoarthritis

Osteoarthritis (OA) is the most common arthropathy, characterized by progressive degeneration of the articular cartilage. Currently, there are no disease-modifying approaches for OA treatment. Adeno-associated virus (AAV)-mediated gene therapy has recently become a potential treatment for OA due to its exceptional characteristics; however, the tropism and transduction efficiency of different AAV serotypes to articular joints and the safety profile of AAV applications are still unknown. The present study aims to screen an ideal AAV serotype to efficiently transfer genes to arthritic cartilage. AAV vectors of different serotypes expressing eGFP protein were injected into the knee joint cavities of mice, with all joint tissues collected 30 days after AAV injection. The transduction efficiency of AAVs was quantified by assessing the fluorescent intensities of eGFP in the cartilage of knee joints. Structural and morphological changes were analyzed by toluidine blue staining. Changes to ECM metabolism and pyroptosis of chondrocytes were determined by immunohistochemical staining. Fluorescence analysis of eGFP showed that eGFP was expressed in the cartilage of knee joints injected with each AAV vector. Quantification of eGFP intensity indicated that AAV2, 7 and 8 had the highest transduction efficiencies. Both toluidine blue staining and Mankin score showed that AAV6 aggravated cartilage degeneration. The analysis of key molecules in ECM metabolism suggested that AAV5 and 7 significantly reduced collagen type II, while AAV9 increased ADAMTS-4 but decreased MMP-19. In addition, transduction with AAV2, 5, 7 and 8 had no obvious effect on pyroptosis of chondrocytes. Comprehensive score analysis also showed that AAV2 had the highest score in intra-articular gene transfer. Collectively, our findings point to AAV2 as the best AAV serotype candidate for gene transfer on arthritic cartilage, resulting in minimal impact to ECM metabolism and pyroptosis of chondrocytes.

/pdf/comparative-intra-articular-gene-transfer-of-seven-adeno-4u1gi6ojxo.pdf

Comparative intra-articular gene transfer of seven adeno-associated virus serotypes reveals that AAV2 mediates the most efficient transduction to mouse arthritic chondrocytes.

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Comparative intra-articular gene transfer of seven adeno-associated virus serotypes reveals that AAV2 mediates the most efficient transduction to mouse arthritic chondrocytes.