Abstract
The technology of virus-based genetic modification in tissue engineering has provided the opportunity to produce more flexible and versatile biomaterials for transplantation. Localizing the transgene expression with increased efficiency is critical for tissue engineering as well as a challenge for virus-based gene delivery. In this study, we tagged the VP2 protein of type 2 adeno-associated virus (AAV) with a 3×FLAG plasmid at the N-terminus and packaged a FLAG-tagged recombinant AAV2 chimeric mutant. The mutant AAVs were immobilized onto the tissue engineering scaffolds with crosslinked anti-FLAG antibodies by N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP). Cultured cells were seeded to scaffolds to form 3D transplants, and then tested for viral transduction both in vitro and in vivo. The results showed that our FLAG-tagged AAV2 exerted similar transduction efficiency compared with the wild type AAV2 when infected cultured cells. Following immobilization onto the scaffolds of PLGA or gelatin sponge with anti-FLAG antibodies, the viral mediated transgene expression was significantly improved and more localized. Our data demonstrated that the mutation of AAV capsid targeted for antibody-based immobilization could be a practical approach for more efficient and precise transgene delivery. It was also suggested that the immobilization of AAV might have attractive potentials in applications of tissue engineering involving the targeted gene manipulation in 3D tissue cultures.
Highlights
Adeno-associated virus (AAV) is used as a common vector for the gene therapy of a broad range of human genetic or metabolic diseases
In AAV clinical applications, the viruses are introduced through different routes of administration, including intramuscular, intravenous and intraocular administrations, where skeletal muscles, liver, ocular, heart and central nervous system are intended as the tissue delivery targets [1,2,3,4,5]
To test whether the FLAG-VP2 proteins could be expressed in eukaryotic cells, the AAV producer line 293T cells were transfected with the constructed plasmids
Summary
Adeno-associated virus (AAV) is used as a common vector for the gene therapy of a broad range of human genetic or metabolic diseases. In recent studies in the bioengineering field, various scaffold materials have been extensively applied, some of which are involved in genetic manipulation of the implanted cells Viral vectors, such as adenovirus or AAV were tested with lyophilized scaffolds [10, 11] and improved transgene expression with reduced immune reactions was indicated. In these reports, certain antibody crosslinking system mediated by SPDP linker for the viral immobilization onto the collagen coated scaffold surface was the most commonly used strategy, and was promisingly demonstrated to anchor adenoviruses in the scaffolds to embed into and infect heart tissues. The crosslinking reaction using SPDP is robust and easy to operate, and can be extensively used for both laboratory or industrial purposes [15,16,17]
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