Enhancement of Adenovirus-Mediated Gene Delivery to Rheumatoid Arthritis Synoviocytes and Synovium by Fiber Modifications: Role of Arginine-Glycine-Aspartic Acid (RGD)- and Non-RGD-Binding Integrins

Abstract

Rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) do not express the coxsackie-adenovirus (Ad) receptor and are poorly permissive to Ad serotype 5 (Ad5). Genetically modified, coxsackie-Ad receptor-independent Ad5 vectors were studied for gene delivery in human RA FLS and synovium explants and murine collagen-induced arthritis. Short-fiber Ad5 vectors with seven fiber shaft repeats Ad5GFP-R7-knob, Ad5GFP-R7-arginine-glycine-aspartic acid (RGD) (RGD-liganded), and Ad5GFPΔknob (knob-deleted) were compared with Ad5GFP-FiWT, a conventional wild-type (WT) Ad5 vector. Gene transfer by Ad5GFP-R7-knob and Ad5GFP-R7-RGD was 40- to 50-fold and 25-fold higher, respectively, than Ad5GFP-FiWT in FLS. Ad5GFPΔknob was more efficacious than its knob-bearing version Ad5GFP-R7-knob in FLS transduction. Virus attachment and entry required RGD- and LDV-binding integrins including αv, αvβ3, avβ5, and β1. Ad5GFP-R7-knob infection of FLS was partially neutralized by synovial fluid (SF), but remained 30- to 40-fold higher than Ad5GFP-FiWT in the presence of SF. Ad5GFPΔknob was partially neutralized by SF at low virus input, but escaped viral neutralization by SF at higher virus input. Gene transfer to human synovium ex vivo explants and murine collagen-induced arthritis in vivo was also more efficient with short fiber-modified vectors (with and without the knob domain) than Ad5GFPFiWT. Gene transfer by short fiber-modified vectors was enhanced by inflammatory cytokines in vitro and in the presence of inflammation in murine synovium in vivo. Our data indicated that the highly efficient gene delivery RA was mediated by RGD- and non-RGD-binding integrins and enhanced by inflammation. Short fiber modifications with knob ablation may be a strategy to enhance gene delivery, reducing vector dose and vector-induced inflammation and toxicity.

Essential Role for the Lectin Pathway in Collagen Antibody–Induced Arthritis Revealed through Use of Adenovirus Programming Complement Inhibitor MAp44 Expression

“…Furthermore, an Ad gene delivery system is highly efficient at transferring genes to a variety of proliferating and quiescent cells both in vitro and in vivo (61). It has also been shown that genetically modified Ad5 vectors with short-shafted fibers are highly efficient in transduction of RA fibroblast-like synoviocytes (FLS) and of human and murine synovium (62). The marked prevention of disease using AdhMAp44 sequences provides evidence that Ad vectors containing complement inhibitor genes and/or use of recombinant MAp44 alone or along with nanoparticles or fused to targeting molecules, should be evaluated as a treatment for inflammatory arthritis in humans.…”

Improved Adenovirus Type 5 Vector-Mediated Transduction of Resistant Cells by Piggybacking on Coxsackie B-Adenovirus Receptor-Pseudotyped Baculovirus

“…Several types of human primary cells have been reported to be refractory to or poorly transduced by Ad5 vectors, e.g., dermal fibroblasts and synoviocytes (76). These cells were transduced with aliquots of virus inoculum containing increasing MOIs of Ad5GFP mixed with a constant BV CAR input (500 vp/cell) and analyzed by fluorescence microscopy and flow cytometry.…”

“…Human dermal fibroblasts were obtained from the skin of a patient undergoing joint surgery for osteoarthritis. Both synoviocytes and dermal fibroblasts were isolated by enzyme digestion and cultured in Dulbecco modified Eagle medium supplemented with 10% FBS and antibiotics (76) and were used between passages 4 and 9. Human MSC cultures were established using the French National protocol of the SFGM-TC Society.…”

Human ESC vs. iPSC—Pros and Cons

“…The ranks of epigenetic modulators and the hierarchy of contribution to reprogramming, DNA methylation, histone deacetylation, and RNA-associated silencing need to be elucidated. Third, gene delivery techniques not requiring or causing genomic integrations must be investigated, including adenoviral and nonviral methods, such as purified DNA delivered via voltage sequences using nucleofector devices (Amaxa Biosystems, Cologne, Germany) and purified mRNA or protein delivered using chemical agents including polyethyleneimine or conjugation to arginine-glycine-aspartic acid peptides [13,14]. Finally, an additional concern regarding the number and nature of genetic-epigenetic alterations that the initial somatic cells' genome may have incurred during the life of the donor, due to multiple factors including age, predisposition and lifestyle, and how they will affect the efficacy and safety of the derived iPSC must also be resolved.…”