Advances in Bone-Targeting Drug Delivery: Emerging Strategies Using Adeno-Associated Virus.

The development of bone-targeting drug delivery systems holds immense promise for improving the treatment of skeletal diseases. By precisely delivering therapeutic agents to the affected areas of bone, these strategies can enhance drug efficacy, minimize off-target effects, and promote patient adherence, ultimately leading to improved treatment outcomes and an enhanced quality of life for patients. This review aims to provide an overview of the current state of affinity-based bone-targeting agents and recent breakthroughs in innovative bone-targeting adeno-associated virus (AAV) strategies to treat skeletal diseases in mice. In particular, this review will delve into advanced AAV engineering, including AAV serotype selection for bone targeting and capsid modifications for bone-specific tropism. Additionally, we will highlight recent advancements in AAV-mediated gene therapy for skeletal diseases and discuss challenges and future directions of this promising therapeutic approach.

AAV-based gene editing of type 1 collagen mutation to treat osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a genetic disorder characterized by bone fragility, low bone mass, fractures, and extraskeletal manifestations. Since OI is commonly caused by single-nucleotide mutation(s) in the COL1A1 or COL1A2 genes encoding type I collagens, we developed a genome-editing strategy to correct a Col1a2 mutation in an OIM mouse model resembling a severe dominant form of human type III OI. Using a recombinant adeno-associated virus (rAAV), we delivered CRISPR-Cas9 to bone-forming osteoblast-lineage cells in the skeleton. Homology-directed repair (HDR)-mediated gene editing efficiency in these cells was improved when CRISPR-Cas9 was coupled with a donor AAV vector containing a promoterless partial mouse Col1a2 complementary DNA sequence. This approach effectively reversed the dysregulation of osteogenic differentiation by a Col1a2 mutation in vitro. Furthermore, systemic administration of dual rAAVs in OIM mice lowered bone matrix turnover rates by reducing osteoblast and osteoclast development while improving the cellular network of mechano-sensing osteocytes embedded in the bone matrix. This strategy significantly improved bone architecture/mass/mineralization, skeletal deformities, grip strength, and spontaneous fractures. Our study is the first demonstration that HDR-mediated gene editing via AAV-mediated delivery effectively corrects a collagen mutation in OI osteoblasts and reverses skeletal phenotypes in OIM mice.

Schnurri-3 inhibition suppresses bone and joint damage in models of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that leads to systemic and articular bone loss by activating bone resorption and suppressing bone formation. Despite current therapeutic agents, inflammation-induced bone loss in RA continues to be a significant clinical problem due to joint deformity and lack of articular and systemic bone repair. Here, we identify the suppressor of bone formation, Schnurri-3 (SHN3), as a potential target to prevent bone loss in RA. SHN3 expression in osteoblast-lineage cells is induced by proinflammatory cytokines. Germline deletion or conditional deletion of Shn3 in osteoblasts limits articular bone erosion and systemic bone loss in mouse models of RA. Similarly, silencing of SHN3 expression in these RA models using systemic delivery of a bone-targeting recombinant adenoassociated virus protects against inflammation-induced bone loss. In osteoblasts, TNF activates SHN3 via ERK MAPK-mediated phosphorylation and, in turn, phosphorylated SHN3 inhibits WNT/ß-catenin signaling and up-regulates RANKL expression. Accordingly, knock-in of a mutation in Shn3 that fails to bind ERK MAPK promotes bone formation in mice overexpressing human TNF due to augmented WNT/ß-catenin signaling. Remarkably, Shn3-deficient osteoblasts are not only resistant to TNF-induced suppression of osteogenesis, but also down-regulate osteoclast development. Collectively, these findings demonstrate SHN3 inhibition as a promising approach to limit bone loss and promote bone repair in RA.

Suppression of heterotopic ossification in fibrodysplasia ossificans progressiva using AAV gene delivery.

Heterotopic ossification is the most disabling feature of fibrodysplasia ossificans progressiva, an ultra-rare genetic disorder for which there is currently no prevention or treatment. Most patients with this disease harbor a heterozygous activating mutation (c.617 G > A;p.R206H) in ACVR1. Here, we identify recombinant AAV9 as the most effective serotype for transduction of the major cells-of-origin of heterotopic ossification. We use AAV9 delivery for gene replacement by expression of codon-optimized human ACVR1, ACVR1R206H allele-specific silencing by AAV-compatible artificial miRNA and a combination of gene replacement and silencing. In mouse skeletal cells harboring a conditional knock-in allele of human mutant ACVR1 and in patient-derived induced pluripotent stem cells, AAV gene therapy ablated aberrant Activin A signaling and chondrogenic and osteogenic differentiation. In Acvr1(R206H) knock-in mice treated locally in early adulthood or systemically at birth, trauma-induced endochondral bone formation was markedly reduced, while inflammation and fibroproliferative responses remained largely intact in the injured muscle. Remarkably, spontaneous heterotopic ossification also substantially decreased in in Acvr1(R206H) knock-in mice treated systemically at birth or in early adulthood. Collectively, we develop promising gene therapeutics that can prevent disabling heterotopic ossification in mice, supporting clinical translation to patients with fibrodysplasia ossificans progressiva.

Bone-Targeting AAV-Mediated Gene Silencing in Osteoclasts for Osteoporosis Therapy.

Improper activity of bone-resorbing osteoclasts results in low bone density and deterioration of bone structure, which increase the risk of fractures. Anti-resorptive therapies targeting osteoclasts have proven effective in preserving bone mass, but these therapeutic agents lead to defective new bone formation and numerous potential side effects. In this study, we demonstrate that recombinant adeno-associated virus, serotype 9 (rAAV9) can deliver to osteoclasts an artificial microRNA (amiR) that silences expression of key osteoclast regulators, RANK (receptor activator for nuclear factor κB) and cathepsin K (rAAV9.amiR-rank, rAAV9.amiR-ctsk), to prevent bone loss in osteoporosis. As rAAV9 is highly effective for the transduction of osteoclasts, systemic administration of rAAV9 carrying amiR-rank or amiR-ctsk results in a significant increase of bone mass in mice. Furthermore, the bone-targeting peptide motif (Asp)14 or (AspSerSer)6 was grafted onto the AAV9-VP2 capsid protein, resulting in significant reduction of transgene expression in non-bone peripheral organs. Finally, systemic delivery of bone-targeting rAAV9.amiR-ctsk counteracts bone loss and improves bone mechanical properties in mouse models of postmenopausal and senile osteoporosis. Collectively, inhibition of osteoclast-mediated bone resorption via bone-targeting rAAV9-mediated silencing of ctsk is a promising gene therapy that can preserve bone formation and mitigate osteoporosis, while limiting adverse off-target effects.

Methanolic extract of Euchelus asper exhibits in-ovo anti-angiogenic and in vitro anti-proliferative activities.

BACKGROUND: The marine environment is a rich source of bioactive natural products. Many of the marine bioactive compounds have been derived successfully from molluscs. Euchelus asper is a marine mollusc which is commonly found in the intertidal rocky regions of the Mumbai coast. The present study was focused on evaluating the anti-angiogenic and anti- proliferative activities of methanolic extract of Euchelus asper (EAME). METHODS: The anti-angiogenic activity of EAME (50-800 µg/mL) was assessed by chick chorio-allantoic membrane (CAM) model wherein multiple parameters in the CAM blood vessels were analysed through morphometric and histological investigations. In vitro testing of EAME (5-20 µg/mL) included its cytotoxicity against three different cancer cell lines, its effect on cell proliferation by wound healing assay as well as their relevant molecular mechanisms. Statistical analysis was carried out by two-tailed student's t test for two unpaired groups. RESULTS: Analysis of CAM revealed that the extract is effective in reducing the branching points of the 1st order blood vessels or capillaries of CAM. Histological analysis of CAM showed significant decrease in capillary plexus and compartmentalization along with increase in mesodermal blood vessels, thus establishing its anti-angiogenicity. Further, EAME exhibited moderate but significant cytotoxicity against A549 non-small cell lung carcinoma cell line. We also demonstrated that the cytotoxicity of EAME in A549 was associated with its apoptotic activity by subG1 phase arrest. Lastly, EAME significantly reduced A549 proliferation by reducing the expression of Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9). CONCLUSION: Overall, our study suggested that EAME has potential to inhibit tumour angiogenic and proliferative activity and may be a potential source for development of new anti-cancer pharmaceuticals.

Methanolic extract of Euchelus asper exhibits in-ovo anti-angiogenic and in vitro anti-proliferative activities

BACKGROUND: The marine environment is a rich source of bioactive natural products. Many of the marine bioactive compounds have been derived successfully from molluscs. Euchelus asper is a marine mollusc which is commonly found in the intertidal rocky regions of the Mumbai coast. The present study was focused on evaluating the anti-angiogenic and anti- proliferative activities of methanolic extract of Euchelus asper (EAME). METHODS: The anti-angiogenic activity of EAME (50-800 µg/mL) was assessed by chick chorio-allantoic membrane (CAM) model wherein multiple parameters in the CAM blood vessels were analysed through morphometric and histo-logical investigations. In vitro testing of EAME (5-20 µg/mL) included its cytotoxicity against three different cancer cell lines, its effect on cell proliferation by wound healing assay as well as their relevant molecular mechanisms. Statistical analysis was carried out by two-tailed student's t test for two unpaired groups. RESULTS: Analysis of CAM revealed that the extract is effective in reducing the branching points of the 1st order blood vessels or capillaries of CAM. Histological analysis of CAM showed significant decrease in capillary plexus and compartmentalization along with increase in mesodermal blood vessels, thus establishing its anti-angiogenicity. Further, EAME exhibited moderate but significant cytotoxicity against A549 non-small cell lung carcinoma cell line. We also demonstrated that the cytotoxicity of EAME in A549 was associated with its apoptotic activity by subG1 phase arrest. Lastly, EAME significantly reduced A549 proliferation by reducing the expression of Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9). CONCLUSION: Overall, our study suggested that EAME has potential to inhibit tumour angiogenic and proliferative activity and may be a potential source for development of new anti-cancer pharmaceuticals.