Targeting human osteoarthritic chondrocytes with ligand directed bacteriophage-based particles

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive deterioration and loss of articular cartilage. There is currently no treatment to reverse the onset of OA. Thus, we developed a targeted delivery strategy to transfer genes into primary human chondrocytes as a proof-of-concept study. We displayed a chondrocyte-affinity peptide (CAP) on the pIII minor coat protein of the M13 filamentous bacteriophage (phage)-based particle carrying a mammalian transgene cassette under cytomegalovirus CMV promoter and inverted terminal repeats (ITRs) cis elements of adeno-associated virus serotype 2 (AAV-2). Primary human articular chondrocytes (HACs) were used as an in vitro model, and the selectivity and binding properties of the CAP ligand in relation to the pathogenic conditions of HACs were characterized. We found that the CAP ligand is highly selective toward pathogenic HACs. Furthermore, the stability, cytotoxicity, and gene delivery efficacy of the CAP-displaying phage (CAP.Phage) were evaluated. We found that the phage particle is stable under a wide range of temperatures and pH values, while showing no cytotoxicity to HACs. Importantly, the CAP.Phage particle, carrying a secreted luciferase (Lucia) reporter gene, efficiently and selectively delivered transgene expression to HACs. In summary, it was found that the CAP ligand preferably binds to pathogenic chondrocytes, and the CAP.Phage particle successfully targets and delivers transgene to HACs

Systemically targeted cancer immunotherapy and gene delivery using transmorphic particles

Immunotherapy is a powerful tool for cancer treatment, but the pleiotropic nature of cytokines and immunological agents strongly limits clinical translation and safety. To address this unmet need, we designed and characterised a systemically targeted cytokine gene delivery system through transmorphic encapsidation of human recombinant adeno-associated virus DNA using coat proteins from a tumour-targeted bacteriophage (phage). We show that Transmorphic Phage/AAV (TPA) particles provide superior delivery of transgenes over current phage-derived vectors through greater diffusion across the extracellular space and improved intracellular trafficking. We used TPA to target the delivery of cytokine-encoding transgenes for interleukin-12 (IL12), and novel isoforms of IL15 and tumour necrosis factor alpha (TNFα) for tumour immunotherapy. Our results demonstrate selective and efficient gene delivery and immunotherapy against solid tumours in vivo, without harming healthy organs. Our transmorphic particle system provides a promising modality for safe and effective gene delivery, and cancer immunotherapies through cross-species complementation of two commonly used viruses

Inhibitory activities of sulfated proteoglycans on chondroitin sulfate A-mediated cytoadherence of Plasmodium falciparum isolates from Thailand.

Chondroitin sulfate A (CSA) is an important receptor for Plasmodium falciparum-infected erythrocytes in the placenta. To study the molecular interaction between parasitized erythrocytes (PE) to CSA, we performed in vitro cytoadherence inhibition assays of PE infected with wild and laboratory isolates of P. falciparum to CSA using various glycosaminoglycans (GAGs). Marked decrease in PE adhesion to immobilized CSA and CSA-expressed cells was achieved with soluble chondroitin sulfate D (CSD) and chondroitin sulfate E (CSE) at low concentrations. The effect was dose dependent with the degree of inhibition exceeded that of soluble CSA in certain clinical isolates. The results suggested the influence of oversulfation of CS variant chains on PE adherence to CSA. Interestingly, PE of the tested wild isolates could adhere to immobilized CSD and CSE at different levels while PE of CSA-selected laboratory lines could not. Partial inhibitory activity was observed when chondroitin sulfate C (CSC), chondroitin sulfate B (CSB), and polyolpolysulfate were used even at high concentrations. Keratan sulfate, colominic acid, and Suramine were unable to inhibit PE adherence. Taken together, the results confirm that the 4-sulfate amino sugar moiety, as well as the basic disaccharide structure of N-acetylgalactosamine linked to glucuronic acid, may influence the degree of this molecular interaction. However, other sulfation patterns that could influence the interaction could not be overlooked, as in the case of CSD which contains 2-O-sulfation at glucuronic acid. Studies using pentosan polysulfate, an oversulfated molecule with a xylan backbone, as an inhibitor also showed a reduction of PE adherence of most isolates tested. Thus, only the sulfate content and pattern of this molecule could affect the adhesive interactions. In addition, difference in capacity of low molecular weight heparins to inhibit CSA-mediated PE cytoadherence of clinical isolates was also observed, thereby providing evidence on the heterogeneity in cytoadherence characteristics of maternal parasite isolates as well as their therapeutic potentials

Raised chondroitin sulfate epitopes and hyaluronan in serum from rheumatoid arthritis and osteoarthritis patients

SummaryObjectivesSerum hyaluronan (HA) and chondroitin sulfate (CS) epitopes WF6 and 3B3 (+) were determined to investigate disease association in patients with osteoarthritis (OA), rheumatoid arthritis (RA) and healthy controls.MethodsSpecific assays for HA and CS epitopes WF6 and 3B3 (+) were established and applied to a cross-sectional study of serum samples from patients (96 OA, 57 RA and 50 healthy controls).ResultsBoth CS epitopes were increased in serum of many OA and RA patients and average levels were significantly above in healthy controls. In contrast serum HA was increased in RA, but only in few OA patients.ConclusionsCS epitopes WF6 and 3B3 (+) are raised in serum of patients with both OA and RA and were thus distinct from serum HA. The results suggest that OA may be detected systemically as well as RA. The range of levels of CS epitopes detected in OA and RA was wide and correlation with any aspect of disease activity is yet to be determined

Bacteriophage‐mediated therapy of chondrosarcoma by selective delivery of the tumor necrosis factor alpha (TNFα) gene

Chondrosarcoma is a cartilage‐forming bone tumor, well known for intrinsic resistance to chemotherapy and radiotherapy. We have designed a targeted chondrosarcoma gene therapy using a bacteriophage (phage) particle to deliver therapeutic genes. Phage has no tropism for mammalian cells, allowing engineered phage to be targeted to specific cell surface receptors in cancer. We modified the phage capsid to display the RGD4C ligand on the pIII minor coat proteins to specifically bind to αvβ3 or αvβ5 integrin receptors. The endosomal escape peptide, H5WYG, was also displayed on recombinant pVIII major coat proteins to enhance gene delivery. Finally, a human tumor necrosis factor alpha (TNFα) therapeutic transgene expression cassette was incorporated into the phage genome. First, we found that human chondrosarcoma cells (SW1353) have high expression of αvβ3, αvβ5 integrin receptors, and both TNFα receptors. Targeted particle encoding a luciferase reporter gene efficiently and selectively mediated gene delivery to these cells. When SW1353 cells were treated with the targeted particle encoding a TNFα transgene, significant cell killing was evident and was associated with high expression of TNFα and apoptosis‐related genes. In vivo, mice with established human chondrosarcoma showed suppression of tumors upon repetitive intravenous administrations of the targeted phage. These data show that our phage‐based particle is a promising, selective, and efficient tool for targeted chondrosarcoma therapy

Development of a mouse monoclonal antibody against the chondroitin sulfate-protein linkage region derived from shark cartilage

Glycosaminoglycans (GAGs) like chondroitin sulfate (CS) and heparan sulfate (HS) are synthesized on the tetrasaccharide linkage region, GlcAβ1-3Galβ1-3Galβ1-4Xylβ1-O-Ser, of proteoglycans. The Xyl can be modified by 2-O-phosphate in both CS and HS, whereas the Gal residues can be sulfated at C-4 and/or C-6 in CS but not in HS. To study the roles of these modifications, monoclonal antibodies were developed against linkage glycopeptides of shark cartilage CS proteoglycans, and one was characterized in detail. This antibody bound hexa- and pentasaccharide-peptides more strongly than tetrasaccharide-peptides, suggesting the importance of GalNAc. It did not react to the CS linkage region modified by 4-O-sulfation. Its reactivity was not affected by treatment with chondro-4-sulfatase or alkaline phosphatase. The results of an ELISA using various proteoglycans and glycopeptides with different modifications suggested the recognition of 6-O-sulfate on the GalNAc and/or Gal residues. Treatments with exopeptidases did not affect the reactivity of the hexasaccharide-peptide fraction, whereas weak alkali to cleave the Xyl-Ser linkage completely abolished the binding activity, suggesting the importance of the Xy-Ser linkage for the binding. Furthermore, the antibody stained wild-type CHO cells, but not mutant cells deficient in xylosyltransferase required for the synthesis of the linkage region. These results suggest that the antibody recognizes the structure GalNAc-GlcA-Gal-Gal-Xyl-Ser that is modified by 6-O-sulfation on GalNAc and/or Gal. The antibody will be a useful tool for investigating the significance of the linkage region in the biosynthesis and/or intracellular transport of different GAG chains