Gene therapy to enhance angiogenesis in chronic wounds

Abstract

Skin injuries and chronic non-healing wounds are one of the major global burdens on the healthcare systems worldwide due to their difficult-to-treat nature, associated co-morbidities, and high health care costs. Angiogenesis has a pivotal role in the wound-healing process, which becomes impaired in many chronic non-healing wounds, leading to several healing disorders and complications. Therefore, induction or promotion of angiogenesis can be considered a promising approach for healing of chronic wounds. Gene therapy is one of the most promising upcoming strategies for the treatment of chronic wounds. It can be classified into three main approaches: gene augmentation, gene silencing, and gene editing. Despite the increasing number of encouraging results obtained using nucleic acids (NAs) as active pharmaceutical ingredients of gene therapy, efficient delivery of NAs to their site of action (cytoplasm or nucleus) remains a key challenge. Selection of the right therapeutic cargo and delivery methods is crucial for a favorable prognosis of the healing process. This article presents an overview of gene therapy and non-viral delivery methods for angiogenesis induction in chronic wounds.

Keywords: MT: Oligonucleotides; Therapies and Applications; angiogenesis; chronic wounds; gene therapy; non-viral methods; nucleic acids; wound healing.

Graphical abstract

Figure 1

Schematic representation of gene augmentation and protein expression upon cell transfection with mRNA or pDNA (1) First, therapeutic nucleic acids (pDNA or mcDNA) are internalized into the cells via viral or non-viral methods, and then (2) released into the cytoplasm. (3) The released pDNA or mcDNA is transported into the nucleus (4) where the gene expression cassette is transcribed in mRNA. (5) The transcribed mRNA is exported from the nucleus to cytoplasm (6), where the cellular translation machinery (ribosomes) is located, and translated into protein. (7) Finally, the nascent polypeptide is then folded to become the functional protein. For mRNA, (a) after cell internalization and (b) release into the cytoplasm, (c and d) it is translated directly into the corresponding protein.

Figure 2

Schematic illustrations of scaffold formation and preparation of composite nanofibers for increased angiogenesis in a wound model (A) Schematic illustration of scaffold formation and its application in a splinted excisional wound model. The scaffold is made of modified hyaluronic acid functionalized with methacrylic anhydride (HAMA), dextran modified with N-hydroxyethylacrylamide (Dex-HEAA), and β-cyclodextrin modified with N-hydroxyethylacrylamide, and simply mixed with resveratrol (β-CD-526-Res). Hydrogels with different ratios of HAMA, Dex-HEAA, and 526-β-CD-Res were formed by photo-polymerization under a UV initiator at 365 nm for 10 min. Then, the freeze-dried hydrogel was soaked in a dispersion of PEI/pDNA complexes for 4 h in the dark. Finally, the hydrogel was freeze dried again to obtain a scaffold with PEI/pDNA-VEGF complexes. Resveratrol and VEGF DNA plasmid are integrated into the scaffold for their anti-inflammatory and pro-angiogenic effects. (B) The general procedure employed for the preparation of the PLGA/CNC/Cur/pDNA-ANG composite nanofibers. First, the PEI-CMCS copolymer was preformed using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling reagents and then mixed with pDNA-ANG to form PEI-CMCS/pDNA complexes. Finally, PEI-CMCS/pDNA complexes and Cur were added to the PLGA/CNC electrospinning solution at room temperature to create electrospun composite nanofibers. ANG, angiogenin; CNCs, cellulose nanocrystals, CMCS, carboxymethyl carbodiimide; NHS, N-hydroxysuccinimide; PEI, polyethyleneimine; PLGA, polylactic-co-glycolic acid.

Figure 3

Schematic illustration of porous nanofibers for controlled release of pAng nanovectors The PLLA/POSS porous nanofibers loaded with pANG-TMC could release pAng in a sustained manner. After transfection of endothelial cells by pAng, cells can express angiopoietin (Ang) and vinculin, which are important growth factors involved in angiogenesis and regulating migration, adhesion, and survival of endothelial cells. pAng, pDNA encoding angiopoietin-1, TMC, N-trimethyl chitosan chloride, HUVECs, human umbilical vein endothelial cells. Adapted from Li et al. with permission from Elsevier.

Figure 4

A schematic illustration of the animal study in rat skin wounds for ESCs transfected in a 3D scaffold (A) Preparation of the CYD-PEI/pDNA polyplexes and gelatin/β-TCP matrix scaffold. (B) Isolation and culture of ESCs from skin tissue biopsy and then culture and transfection of the ESCs in the gene-activated matrix. (C) Establishment of rat full-thickness skin wound. Application of transfected ESCs with the 3D scaffold to the wound site. Wound healing is stimulated by the recombinant ESCs.

Figure 5

Effect of mRNA lipoplexes loaded onto mineral-coated microparticles (MCMs) in wound healing (A) Schematic overview of the sequester and release mechanism of mRNA lipoplexes loaded onto mineral-coated microparticles (MCMs). Lipoplexes containing basic growth factor (bFGF) mRNA are loaded onto MCMs. After treatment, mRNA lipoplexes are released from MCMs and taken up by the cells. After the endosomal escape, the bFGF mRNAs are translated into proteins, which are secreted into the extracellular space. The secreted proteins are bound and sequestered by the MCMs, which sustain growth factor release over time and prolong the biological response. (B) MCM-mediated mRNA delivery in wound healing. Representative gross and histological images show improved wound closure and resolution after the application of chemically modified bFGF mRNA (CM-mRNA)-loaded MCMs compared with recombinant bFGF protein with MCMs to wounds in mice. Scale bar, 5 mm. (C) Co-delivery of B18R via MCM-mediated mRNA delivery in wound healing. Representative gross and histological images show improved wound closure and resolution for wild-type (WT) mRNA delivered via MCMs with B18R compared with WT-mRNA alone delivered via MCMs. Scale bar, 5 mm. Adapted from Khalil et al.⁷²).

Figure 6

Schematic representation of the mechanism of RNAi (a) miRNA is transcribed from DNA sequences into primary miRNA (pri-mRNA) and is later processed into precursor miRNA (pre-miRNA) by the Drosha-DGCR8 complex. The pre-miRNA is then actively transported to the cytoplasm by exportin-5. Next, the pre-miRNA is further processed by the Dicer-TRBP complex, usually generating two paired, partially complementary, mature miRNAs. One of the mature miRNA strands then guides the RNA-induced silencing complex (RISC) to cognate target genes and represses target gene expression by either destabilizing target mRNAs or repressing their translation. (b) When siRNA enters the cytosol, the passenger strand is removed from the guide strand and directly incorporated into RISC to guide the RISC complex to its complementary mRNA target for degradation. (c) The plasmid encoding shRNA is transcribed in the nucleus and shRNA is subsequently exported to the cytoplasm via exportin-5. Similar to miRNA, the enzyme Dicer cleaves shRNA into siRNA, which then combines with RISC to degrade the targeted mRNA sequence.

Figure 7

Effect of outgrowth endothelial cell (OEC) transplantation incubated with miR-dsDNA-AuNR and sequenced irradiation in an in vivo acute wound mouse model (A) Schematic representation of outgrowth endothelial cell (OEC) transplantation in an in vivo acute wound mouse model. OECs were incubated for 4 h with a mixture of miRNA-dsDNA-AuNRs. After incubation, cells were trypsinized and transplanted subcutaneously at the border of the dorsal wounds created in nude mice. After transplantation, the site of injection was irradiated for 2 min at 1.25 W cm⁻². After 24 h, a second stimulus was applied (2 min at 2 W cm⁻²). (B) Wound closure in nude mice treated with cells previously incubated with miR-dsDNA-AuNR. (i) OECs without treatment. (ii) OECs incubated with miRNA-155-dsDNA-AuNR and miR302a-dsDNA-AuNR. The site of injection was irradiated for 2 min at 1.25 W cm⁻² to release miRNA-155 conjugated with ssDNA. After 24 h, miRNA-302a conjugated with ssDNA was released by a second stimulus (2 min at 2 W cm⁻²). (iii) The order of delivery was inverted by changing the DNA strands conjugated to each miRNA so that OECs first incubated with miRNA-302a-dsDNA-AuNR and after 24 h miRNA-155-dsDNA-AuNR was released. Reprinted with permission from Lino et al. Copyright 2018 American Chemical Society. (C) The wound-healing kinetics was monitored during 10 days by the quantification of wound area. Results are expressed as average ±SEM (n = 7, 8). Statistical significance,∗p < 0.05 and ∗∗p < 0.01 assessed by unpaired t test. Reprinted with permission from Lino et al. Copyright 2018 American Chemical Society.

Figure 8

Schematic representation of how HIF-1 activates gene transcription in response to normoxic versus hypoxic conditions Under normoxic conditions, HIF-1α is subjected to hydroxylation by prolyl hydroxylase domain protein 2 (PHD-2). This hydroxylation is required for binding of the von Hippel-Lindau protein (VHL), the recognition subunit of a ubiquitin protein ligase that targets HIF-1α for ubiquitination and proteasomal degradation. Under hypoxic conditions, hydroxylation is inhibited and HIF-1α is stabilized, which dimerizes with HIF-1β and binds to target genes at the consensus sequence hypoxia-responsive element (HRE). The target gene DNA sequence is then transcribed into mRNA. Under hyperglycemic conditions in the diabetic wound, the function and stability of HIF-1α are impaired by high levels of glucose and reactive oxygen species (ROS), so that fewer new vessels are formed and wound healing becomes impaired. Reproduced from Shaabani et al.

Figure 9

siPHD-2 LbL nanoformulation for the transfection of NIH3T3cells (A) Schematic representation of the LbL nanoformulations with either Chitosan (CS) (AuNPs@CS) or PLA (AuNPs@PLA) as the third and final layer. (B) Knockdown of PHD-2 and its effect on the expression of angiogenic growth factors in NIH3T3cells. NIH3T3cells were incubated for 4 h with the nanoformulations loaded with 30 nM non-specific siRNA (siCtrl) or siPHD-2, followed by 20-h incubation with cell culture medium without or with 20 μm desloratadine (DES). After another 24-h incubation in fresh cell medium, mRNA was isolated and subjected to real-time reverse transcriptase-PCR analysis to determine expression of vascular endothelial growth factor (VEGF), and fibroblast growth factor 2 (FGF2). The results were normalized to the mRNA expression level of β-actin and shown relative to cells transfected with siCtrl. Adapted from Shaabani et al. Data are represented as the mean ± the standard deviation for a minimum of three independent experiments. (ns = not significant p > 0.05, ∗ p ≤ 0.05, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001).

Figure 10

Schematic illustration of a hydrogel containing anti-miRNA for improvement of angiogenesis in wound healing (A) Schematic representation of the fabrication process of the PCN-miR/Col hydrogel. A nanozyme-reinforced self-protecting hydrogel (PCN-miR/Col) composed of 25-kDa polyethylenimine (PEI25K), functionalized ceria nanoclusters (PCN), and antagomiRNA-26a (miR) nanocomplexes (PCN-miR). This PCN-miR/Col matrix is designed to simultaneously reshape the hostile wound microenvironment by creation of a pro-regenerative wound microenvironment and providing simultaneous self-protecting delivery of pro-angiogenic miRNA cues. Reprinted with permission from Wu et al. Copyright 2019 American Chemical Society. (B) Schematic representation of Gel/Alg@ori/HA-PEI@siRNA-29a hydrogel preparation and wound repair. A novel hydrogel (Gel/Alg@ori/HA-PEI@siRNA-29a) prepared with a hierarchical micro/macro structure crosslinked via Schiff base bonds between adipic dihydrazide-modified hyaluronic acid (HA-ADH) and oxidized hydroxymethyl propyl cellulose (OHMPC) was combined with oridonin (ori)-loaded alginate microspheres (Alg@ori) and HA-PEI@siRNA-29a gene complexes (HA-PEI@siRNA-29a) in the interior of the hydrogels. The addition of ori and siRNA-29a is aimed at downregulating pro-inflammatory factors and enhancing the vascularization. Reproduced from Yang et al. with permission from Elsevier.

Figure 11

Aptamer approaches enhance angiogenesis in wound healing (A) A schematic illustration of bifunctional hydrogel synthesis. (1) The thiolated hyaluronic acid (tHA) is reacted for a short time with an acrydite-modified DNA aptamer sequence to introduce the aptamer. (2) The gel is formed by the addition of the polyethylene glycol diacrylate (PEGDA) crosslinker. The PEGDA needs to be added in excess so that there are a number of free acrylate groups remaining for the final step. (3) After the gel has partially crosslinked, it is incubated in a solution of the thiol-modified arginine-glycine-aspartate (RGD) peptide to react with the remaining free acrylate groups on the PEGDA. Adapted from Roy et al. with permission from Wiley Materials. (B) Angiogenic aptamer-modified tetrahedral framework nucleic acid (tFNA) stimulated angiogenesis in vivo. Schematic diagram of tFNA, tFNA-Apt02, and tFNA-AptVEGF formation, which can promote angiogenesis in vivo. For evaluation of in vivo angiogenesis, a Matrigel plug assay was used. The mixture of HUVECs and Matrigel was subcutaneously injected into the right ventral side of the nude mice and then, for the next 7 days, different nanoparticle treatments were injected locally into Matrigel plugs every day. Finally, the Matrigel plugs were harvested for evaluation with different methods. (C) Photographs of a Matrigel plug assay after treatment with nanoparticles (ssDNA, Apt02, AptVEGF, tFNA, tFNA-Apt02, and tFNA-AptVEGF). Scale bars, 500 μm. Reprinted with permission from Zhao et al. Copyright 2021 American Chemical Society.

Figure 12

The four major genome-editing platforms and their working principles (A) Homing endonucleases-meganucleases are engineered restriction enzymes that recognize long stretches of DNA sequences through protein-DNA binding. (B) Zinc-finger nuclease (ZFN) recognizes triple DNA code specificity through protein-DNA binding. (C) Transcription activator-like effector nuclease (TALEN) recognize individual base specificity through protein-DNA binding. (D) The clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease Cas9 (CRISPR/Cas9) derives its specificity from Watson-Crick RNA-DNA base pairing. (E) All these tools result in DNA double-strand breaks, which are repaired either by error-prone non-homology end joining (NHEJ) or homology-directed repair (HDR).