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Hyaluronic acid-binding insulin-like growth factor-1: Creation of a gene encoding a bifunctional fusion protein

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Abstract

Chondrogenic growth factors are promising therapeutic agents for articular cartilage repair. A persistent impediment to fulfilling this promise is a limited ability to apply and retain the growth factors within the region of cartilage damage that is in need of repair. Current therapies successfully deliver cells and/or matrices, but growth factors are subject to diffusion into the joint space and then loss from the joint. To address this problem, we created a novel gene that encodes a bifunctional fusion protein comprised by a matrix binding domain and a growth factor. The gene encodes the hyaluronic acid binding region of the cartilage matrix molecule, versican, and the chondrogenic growth factor, insulin-like growth factor-1 (IGF-1). We delivered the gene in an adeno-associated virus-based plasmid vector to articular chondrocytes. The cells synthesized and secreted the fusion protein gene product. The fusion protein bound to hyaluronic acid and retained the anabolic and mitogenic actions of IGF-1 on the chondrocytes. This proof-of-concept study suggests that the bifunctional fusion protein, in concert with chondrocytes and a hyaluronic acid-based delivery vehicle, may serve as an intra-articular therapy to help achieve articular cartilage repair.

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Abbreviations

AAV:

Adeno-associated virus

AG1:

Aggrecan G1 domain

AU:

Arbitrary unit

CM:

Conditioned medium

DMB:

Dimethylmethlyene blue

DMEM:

Dulbecco’s minimum essential medium

EDTA:

Ethylenediaminetetraacetic acid

F:

Forward primer

FBS:

Fetal bovine serum

G1:

Globular domain 1

GAG:

Glycosaminoglycan

HA:

Hyaluronic acid

HB-EGF:

Heparin-binding epidermal-like growth factor

HEK-293:

Human embryonic kidney 293 cells

HRP:

Horseradish peroxidase

IGF-1:

Insulin-like growth factor-1

Ka :

Equilibrium association constant

PBS:

Phosphate buffered saline

R:

Reverse primer

SDS:

Sodium dodecyl sulfate/sulphate

TBST:

Tris-buffered saline/Tween

VG1:

Versican G1 domain

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Funding

This research was funded in part by the National Institute of Arthritis and Musculoskeletal and Skin Diseases, Grant/Award Number: AR047702; U.S. Department of Veterans Affairs, Grant/Award Number: I01 BX000447; Indiana University School of Medicine Research Infrastructure Fund; and Indiana University Department of Orthopaedic Surgery. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Author notes

  1. Shuiliang Shi

    Present address: , MaAnShan, BeiCheng, HuangYan, TaiZhou, 318020, ZheJiang, China

Authors and Affiliations

  1. Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, IN, USA

    Shuiliang Shi, Congrong Wang & Stephen B. Trippel

  2. Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA

    Stephen B. Trippel

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  1. Shuiliang Shi
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Contributions

Conceptualizaton: SS and ST. Methodology: SS, ST, and CW. Validation: SS, CW and ST. Formal analysis: SS and ST. Investigation: SS and CW. Resources: ST. Writing – original draft: SS and ST. Writing-review & editing: SS, ST, and CW. Visualization: SS. Project administration: SS and ST. Funding acquisition: ST.

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Correspondence to Shuiliang Shi.

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The authors declare no conflicts of interest. The funders had no role in the design of the study, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

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Shi, S., Wang, C. & Trippel, S.B. Hyaluronic acid-binding insulin-like growth factor-1: Creation of a gene encoding a bifunctional fusion protein. Mol Biol Rep 47, 9749–9756 (2020). https://doi.org/10.1007/s11033-020-06034-w

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