A novel nucleic acid delivery system for skin gene therapy

On November 7, 2022, Professor Xu Fujian and Associate Professor Hu Yang of Beijing University of Chemical Technology collaborated with Professor Cheng Gang of the University of Illinois at Chicago to publish an article entitled “One ternary nucleic acid delivery system with smart dextran-peptide coating enables in vivo and ex vivo wound” in the journal Matter therapy”.

This achievement reported that the dextran-electronegative polypeptide coupling was prepared by the efficient chemical reaction of polysaccharides and peptides, and then a ternary nucleic acid delivery system (HPD) with intelligent shell layer was constructed, which realized in vitro nucleic acid delivery with high serum stability and dose independence, as well as in vivo and ex vivo skin wound gene therapy.

The corresponding authors of the paper are Xu Fujian, Cheng Gang, and Hu Yang; The first author is Fan Yaqian.

The skin has a complex structure and function, which is important for maintaining homeostasis in the internal environment of the human body. Skin damage caused by surgery, trauma or metabolic diseases has become a common disease, and the need for efficient and high-quality wound healing is urgent. Gene therapy is a promising therapeutic strategy in skin wounds that offers the advantage of in situ and continuous delivery of growth factors by delivering plasmid DNA that encodes growth factors to the wound. However, widely developed cationic polymer-based nucleic acid delivery systems are often accompanied by defects such as easy interaction of serum proteins and rapid/nonlinear reduction of transfection efficiency at low doses, which are insufficient to effectively deal with the obstacles faced by skin wound gene therapy such as large wound exudate and difficult transfection of primary cells. Natural polysaccharides, peptides and other materials have flexible material design space in gene delivery system research in recent years due to their good biocompatibility and structural regulation.

The team of Professor Xu Fujian and Associate Professor Hu Yang of Beijing University of Chemical Technology and Professor Cheng Gang of the University of Illinois at Chicago first designed a negatively charged polypeptide (CH2E5), grafted onto the modified dextran skeleton through Michael addition reaction, and constructed a stably synthesized dextran-electronegative polypeptide coupling (Dex-H2E5). Then, Dex-H2E5 was coated in the outer layer of polyhexamethylene biguanide (PHMB)/plasmid binary positive nucleic acid composite system, and a ternary nucleic acid delivery system (HPD) with intelligent shell was prepared. Based on in vitro experiments to verify the efficient delivery of nucleic acids by HPD and its mechanism of action, HPD was applied to both in vivo and ex vivo modes of skin defect gene therapy (delivery of functional plasmid phEGF encoding human epidermal growth factor to treat full-thickness defect wounds), and Yang Peiying and Liu Yan, collaborators of Ruijin Hospital affiliated to Shanghai Jiao Tong University School of Medicine, provided guidance on the construction and analysis of animal experimental models.

Figure 1: Schematic diagram of the preparation of the ternary gene delivery system HPD and its gene therapy in a skin defect model.

HPD can overcome multiple barriers in nucleic acid delivery.Compared with the binary system, the Dex-H2E5 shell can effectively reduce the surface potential of HPD and appropriately increase its particle size. In vitro simulation conditions, it was found that the Dex-H2E5 shell could effectively reduce the surface non-specific protein adsorption of HPD, making it stable in a high serum environment. The shell also exhibits proper pH responsiveness, which is conducive to improving the content/lysosomal escape ability of HPD in cells and promoting the efficient release of DNA.

Fig. 2: Physical and chemical structure characterization of HPD and performance analysis of overcoming multiple obstacles during nucleic acid delivery under simulated conditions.

HPD enables efficient delivery of reporter plasmids and therapeutic plasmids in multiple modes.The results of reporter plasmid transfection of cell lines showed that HPD had excellent performance with low toxicity and high transfection efficiency under standard serum/dose, high serum, and low dose conditions. In addition, HPD can effectively transport phEGF in L929 cells, upregulate the expression of EGF protein and promote cell proliferation. It also enables low-toxic, efficient delivery of reporter plasmids in primary rat fibroblasts (RFBs).

Figure 3: Performance analysis of HPD delivery reporter plasmid in cell lines.

Figure 4: Performance analysis of HPD delivery of therapeutic plasmids and primary cell delivery reporter plasmids in cell lines.

HPD can achieve good results with both in vivo and ex vivo modes of skin wound gene therapy.In vivo gene therapy model A (i.e., HPD was used to deliver a low plasmid dose of phEGF in a single pass and transfect residual cells in the wound, In vivo) in the rat full-thickness skin defect model, and the wound was rapidly healed and the healing quality was high. For potential applications with few residual cells on large-scale burns or ulcer wounds, the ex vivo gene therapy model B (i.e., phEGF is delivered in vitro to allogeneic RFB cells in vitro by HPD, and then gene-edited RFB cell suspension is administered on the wound, Ex vivo) to achieve rapid and efficient healing of skin wounds.

Figure 5: Pro-healing effects of HPD in vivo and ex vivo gene therapy in a full-thickness defect skin model.

This work provides an efficient and stable synthesis of polymer-based nucleic acid delivery system, which provides a reference treatment model for the clinical application of gene therapy for skin diseases, and is expected to be applied to other clinical scenarios that require efficient gene therapy. The research work was supported by the National Key Research and Development Program of China (2021YFB3800900), the National Natural Science Foundation of China (51829301, 51703008 and 51733001), and the Beijing Outstanding Young Scientists Program (BJJWZYJH01201910010024). (Source: Web of Science)

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