Localized delivery of CRISPR/dCas9 via layer-by-layer self-assembling peptide coating on nanofibers for neural tissue engineering

The clustered regularly interspaced short palindromic repeat (CRISPR) systems have a wide variety of applications besides precise genome editing. In particular, the CRISPR/dCas9 system can be used to control specific gene expression by CRISPR activation (CRISPRa) or interference (CRISPRi). However, the safety concerns associated with viral vectors and the possible off-target issues of systemic administration remain huge concerns to be safe delivery methods for CRISPR/Cas9 systems. In this study, a layer-by-layer (LbL) self-assembling peptide (SAP) coating on nanofibers is developed to mediate localized delivery of CRISPR/dCas9 systems. Specifically, an amphiphilic negatively charged SAP− is first coated onto PCL nanofibers through strong hydrophobic interactions, and the pDNA complexes and positively charged SAP+-RGD are then absorbed via electrostatic interactions. The SAP-coated scaffolds facilitate efficient loading and sustained release of the pDNA complexes, while enhancing cell adhesion and proliferation. As a proof of concept, the scaffolds are used to activate GDNF expression in mammalian cells, and the secreted GDNF subsequently promotes neurite outgrowth of rat neurons. These promising results suggest that the LbL self-assembling peptide coated nanofibers can be a new route to establish a bioactive interface, which provides a simple and efficient platform for the delivery of CRISPR/dCas9 systems for regenerative medicine.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)Accepted versio

Scaffold-mediated non-viral delivery of nucleic acids for sustained gene silencing in regenerative medicine

Treatments with nucleic acid therapeutics have emerged as a promising approach since it addresses the molecular causes of hindered tissue repair by manipulating gene expression profiles in targeted cells within the injured tissue system. Although nucleic acid-based therapy has seen significant advancement in various tissue repair applications, its success has largely been hindered by the mode of delivery. Highly efficient viral gene vectors and carriers are typically used for effective transfection to occur. However, significant safety issues and complications have also arisen out of such viral delivery strategies. Non-viral nucleic acid delivery strategies offer improved safety profiles and are promising alternatives but unfortunately, the limited transfection efficiencies of non-viral delivery platforms must first be addressed before achieving functional tissue repair outcomes. Moreover, existing non-viral delivery of nuclei acids largely rely on hydrogels and particles. None of which mimics the injury environment nor can they sustain gene knockdown. Accordingly, the aim of this thesis was to design and fabricate electrospun fibers that mimic the injury environment while incorporating small nucleic acids that silence genes upregulated upon injury. Specifically, such synergistic delivery of topographical and biochemical cues aims to enhance tissue repair in skin and spinal cord. While both tissue environments are vastly different, the key barrier that prevents healing in chronic wounds and after a spinal cord injury (SCI) is the chronic and sustained high levels of inflammation. Silencing genes upregulated during inflammation provides an alternative to mitigating the damage in chronic wounds and after SCI. Wet electrospinning and air gap electrospinning were the two different techniques used to fabricate poly(ε-caprolactone) (PCL), rat-tail type 1 collagen and poly (caprolactone-co-ethyl ethylene phosphate) (PCLEEP) scaffolds that mimicked the native extracellular matrix in the skin and axons, respectively. Wet electrospun PCL scaffolds promoted cell migration, vascularization and re-epithelialisation with minimal inflammatory response. These scaffolds were then incorporated with Connexin 43 (Cx43) - specific antisense oligodeoxynucleotides (asODNs). However, due to the limitations of the wet electrospinning technique and the choice of polymer, the mass of Cx43 asODNs delivered to wound bed was insufficient to downregulate Cx43 expression at the epithelial tongue. Hence, no significant difference in epithelial tongue thickness or migration distance was observed. These findings were consolidated and then extended into a more complex SCI environment where a fiber-hydrogel scaffold for sustained delivery of Cx43 asODN, while providing synergistic topographical cues to guide axonal ingrowth was fabricated. Here, as an extension of my previous work on delivering nucleic acids to SCI injury environment, PCLEEP, instead of PCL, was used to accelerate degradation. Materials used in wounds could ideally be rapidly integrated or pushed out along with the scab as the wound heals. In contrast, accessibility to scaffolds implanted at SCI sites is low and hence, scaffolds cannot be easily removed. Thus, the need to be bioresorbable is more apparent in SCI applications. These scaffolds demonstrated the sustained release of Cx43 asODN for up to 25 days. In addition, Cx43 up-regulation after complete transection SCI in rats was supressed, preserving neurons around the injury site, promoting axonal extension while decreasing glial scarring and microglial activation after SCI. Beyond non-viral delivery of nucleic acids, CRISPR/Cas9 components were also delivered in a localized and non-viral manner via electrospun scaffolds. Specifically, using mussel-inspired bioadhesive coating, polyDOPA-melanin (pDOPA), Cas9: sgRNA lipofectamine complexes were adsorbed onto bio-mimicking fiber scaffolds. As this is a proof of concept, PCL was chosen as the choice of polymer to be electrospun especially since no specific application nor injury environment was decided. U2OS.EGFP cells took up Cas9: sgRNA lipofectamine complexes directly from the scaffolds via reverse transfection and expression of EGFP in these cells was successfully knockdown. In vitro studies use cells derived from animals or cell lines which have an infinite lifespan. While these model systems are relatively cheap and simple to purchase, they fail to capture the inherent complexity of organ systems. The use of animals in in vivo studies addresses many of the shortcomings of in vitro studies. Hence, the bulk of this thesis relies on animal models to evaluate nucleic acids loaded scaffolds. However, the problem of translatability remains. Given the considerable physiological differences between human and animals, the animal models used for evaluation might not truly reflect the functional outcome when translated to humans. In this thesis, a novel perturbed wound model that more accurately recapitulates features of human chronic wounds for more accurate testing of biofunctionalized scaffolds was established. Numerous features such as hyper-thickened epithelial tongue, delayed wound closure, chronic inflammatory environment, overexpression of Cx43, presence of senescent cells and ECM degradation at wound edges were observed in the perturbed wounds. These features accurately recapitulated the elements that hinder chronic wound healing in humans. Given its relevance to chronic wounds, this perturbed wound model potentially serves as a more relevant platform for testing of wound healing therapeutics. Altogether, this thesis demonstrated the feasibility of electrospinning to fabricate scaffolds that mimic vastly different injury environments while non-virally delivering gene-silencing nucleic acids for regenerative medicine. Importantly, the methodology can be extended to more complicated gene editing techniques (e.g. CRISPR). Additionally, a new perturbed model that more accurately recapitulates features of human chronic wounds was established in hopes of serving as a more relevant platform for testing of wound healing therapeutics and biofunctionalized scaffolds.Doctor of Philosoph

Establishing an aligned nanofibre platform to deliver synergistic topographical and biochemical signalling

Given the complexity of the nervous system, mechanisms to promote functional recovery at injury sites have yet been completely understood by both clinicians and researchers. Tissue engineering approaches serve as a potential therapy for neural regeneration. In particular, aligned sub-micron and nan-scale fibrous scaffolds which accurately mimic the topography of natural extracellular matrix can function as prospective scaffold candidates to facilitate recovery. In this study, a fiber fabrication technique, Electrospinning, was adopted and electrospun fibers were collected across an electrically charged air gap. Parameters were optimized to achieve aligned poly (Ɛ-caprolactone) (PCL) fiber with diameters ranging from 390nm to 1.14um. Though this fabrication technique has been proven efficient, the use of organic solvents, which are highly cytotoxic, to dissolve PCL is unfavorable for both in vitro and in vivo use. Hence, a solvent-free electrospinning technique, melt electrospinning, was established. Several configurations were explored and a stable heating system was obtained through a combination of proportional integral derivative (PID) controlling system coupled with the use of a solid state relay switching mechanism. This established setup was subsequently evaluated and proven to be feasible for melting low molecular weight PCL pallets to achieve a stable melt flow at high flow rates along with high heating temperature. Bio-functionality of aligned fibrous scaffolds can be established through incorporation of biochemical cue delivery. Gene silencing through RNA interference can be achieved with the use of small-interfering RNA (siRNA) and this technique has revealed immense potential in silencing inhibitory intrinsic factors of axonal regeneration at injury sites. Micellar Nanoparticles (MNP) has recently been developed and engaged in delivery of nucleic acids. Two copolymers, poly (Ɛ-caprolactone)-poly (ethylene glycol) (PCL-PEG) and poly (Ɛ-caprolactone)-poly (2-aminoethyl ethylene phosphate) (PCL-PPEEA), were used for MNP synthesis weight ratios of both copolymers were varied to obtain a range of MNPs exhibiting different particle sizes and charges. Upon siRNA loading, siRNA/MNP complexes were formed and these complexes had a general reduction in overall size as well as charge. Combination of MNPs with aligned fibrous scaffolds could serve as an efficient scaffold-mediated delivery system to induce functional recovery at injury sites especially within the non-permissive environment of the central nervous system (CNS).Bachelor of Engineering (Chemical and Biomolecular Engineering

Localised non-viral delivery of nucleic acids for nerve regeneration in injured nervous systems

Axons damaged by traumatic injuries are often unable to spontaneously regenerate in the adult central nervous system (CNS). Although the peripheral nervous system (PNS) has some regenerative capacity, its ability to regrow remains limited across large lesion gaps due to scar tissue formation. Nucleic acid therapy holds the potential of improving regeneration by enhancing the intrinsic growth ability of neurons and overcoming the inhibitory environment that prevents neurite outgrowth. Nucleic acids modulate gene expression by over-expression of neuronal growth factor or silencing growth-inhibitory molecules. Although in vitro outcomes appear promising, the lack of efficient non-viral nucleic acid delivery methods to the nervous system has limited the application of nucleic acid therapeutics to patients. Here, we review the recent development of efficient non-viral nucleic acid delivery platforms, as applied to the nervous system, including the transfection vectors and carriers used, as well as matrices and scaffolds that are currently used. Additionally, we will discuss possible improvements for localised nucleic acid delivery.NRF (Natl Research Foundation, S’pore)ASTAR (Agency for Sci., Tech. and Research, S’pore)NMRC (Natl Medical Research Council, S’pore)Accepted versio

Biofunctional scaffolds with high packing density of aligned electrospun fibers support neural regeneration

Neurons of the central nervous system do not regenerate spontaneously after injury. As such, biofunctional tissue scaffolds have been explored to provide a growth‐promoting environment to enhance neural regeneration. In this regard, aligned electrospun fibers have proven invaluable for regeneration by offering guidance for axons to cross the injury site. However, a high fiber density could potentially limit axonal ingrowth into the scaffold. Here, we explore which fiber density provides the optimal environment for neurons to regenerate. By changing fiber electrospinning time, we generated scaffolds with different fiber densities and implanted these in a rat model of spinal cord injury (SCI). We found that neurons were able to grow efficiently into scaffolds with high fiber density, even if the gaps between fiber bundles were very small (<1 μm). Scaffolds with high fiber density showed good host‐implant integration. Cell infiltration was not affected by fiber density. Efficient blood vessel ingrowth likely requires larger gaps between fibers or faster degrading fibers. We conclude that scaffolds with high fiber densities, and thus a large number of small gaps in between fiber bundles, provide the preferred environment for nerve regeneration after SCI.National Medical Research Council (NMRC)National Research Foundation (NRF)Accepted versionPartial funding support from the Singapore National Research Foundation under its NMRC-CBRG grant (NMRC/CBRG/0096/2015) and administered by the Singapore Ministry of Health’s National Medical Research Council and RRIS Rehabilitation Research Grant (RRG1/16004) are acknowledged

Targeting connexin 43 expression via scaffold mediated delivery of antisense oligodeoxynucleotide preserves neurons, enhances axonal extension, reduces astrocyte and microglial activation after spinal cord injury

Injury to the central nervous system (CNS) provokes an inflammatory reaction and secondary damage that result in further tissue damage and destruction of neurons away from the injury site. Upon injury, expression of connexin 43 (Cx43), a gap junction protein, upregulates and is responsible for the spread and amplification of cell death signals through these gap junctions. In this study, we hypothesise that the downregulation of Cx43 by scaffold-mediated controlled delivery of antisense oligodeoxynucleotide (asODN), would minimise secondary injuries and cell death, and thereby support tissue regeneration after nerve injuries. Specifically, using spinal cord injury (SCI) as a proof-of-principle, we utilised a fibre-hydrogel scaffold for sustained delivery of Cx43asODN, while providing synergistic topographical cues to guide axonal ingrowth. Correspondingly, scaffolds loaded with Cx43asODN, in the presence of NT-3, suppressed Cx43 up-regulation after complete transection SCI in rats. These scaffolds facilitated the sustained release of Cx43asODN for up to 25 days. Importantly, asODN treatment preserved neurons around the injury site, promoted axonal extension, decreased glial scarring, and reduced microglial activation after SCI. Our results suggest that implantation of such scaffold-mediated asODN delivery platform could serve as an effective alternative SCI therapeutic approach.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)Nanyang Technological UniversityNational Research Foundation (NRF)Skin Research Institute of Singapore (SRIS)Published versionThe author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: The experiments in this work were supported by the Ministry of Education Tier 1 (RG38/19, RG37/20) and the National Research Foundation, Singapore, under its Intra-CREATE Thematic Grant (Award number: NRF2019-THE002-0001). We acknowledge the Agency for Science, Technology and Research (A*STAR) under its Industry Alignment Fund – Pre-Positioning Programme (IAF-PP) (Grant number H17/01/a0/0C9 and H1701a0004). We thank the Skin Research Institute of Singapore, Phase 2: SRIS@Novena for providing this work with the floor infrastructure and core equipment. JS Chin was also supported by IGS’s studentship

A laser microdissection-based axotomy model incorporating the use of biomimicking fiber scaffolds reveals microRNAs promote axon regeneration over long injury distances

The regeneration of injured neurons over long injury distances remains suboptimal. In order to successfully stimulate nerve regrowth, potent biomolecules are necessary. Furthermore, reproducible and translatable methods to test the potency of candidate drugs in enhancing nerve regeneration over long axotomy distances are also needed. To address these issues, we report a novel laser microdissection-based axotomy model that involves the use of biomimicking aligned fiber substrates to precisely control neuronal axotomy distances. Correspondingly, we demonstrate that in the absence of therapeutics, dorsal root ganglion (DRG) explants (consisting of neurons) axotomized within short distances from the main cell somas regenerated significantly longer than axons that were injured more distally (p < 0.05). However, when treated with a cocktail of microRNAs (miR-132/miR-222/miR-431), robust neurite outgrowth was observed (p < 0.05). Specifically, microRNA treatment promoted neurite outgrowth by ~2.2-fold as compared to untreated cells and this enhancement was more significant under the less conducive regeneration condition of a long axotomy distance (i.e. 1000 µm from the cell soma). Besides that, we demonstrated that the treatment of miR-132/miR-222/miR-431 led to longer length of nerve regeneration as well as a bigger nerve extension area after sciatic nerve transection injury. These results indicate that distance effects on axonal regrowth may be overcome by the effects of microRNAs and that these microRNAs may serve as promising therapeutics for nerve injury treatment.National Research Foundation (NRF)Accepted versionPartial financial support was received from Singapore National Research Foundation under its NMRC-CBRG grant (NMRC/CBRG/0096/2015) and the Ministry of Education Tier 1 grant (RG38/19). Na Zhang would like to acknowledge the support of NTU by providing Nanyang Research Scholarship to carry out these research works

Challenges faced in developing an ideal chronic wound model

Introduction: Chronic wounds are a major drain on healthcare resources and can lead to substantial reductions in quality of life for those affected. Moreover, they often precede serious events such as limb amputations and premature death. In the long run, this burden is likely to escalate with an ageing population and lifestyle diseases such as obesity. Thus far, the identification of beneficial therapeutics against chronic wounds have been hindered by the lack of an ideal chronic wound animal model. Although animal models of delayed healing have been developed, none of these models fully recapitulate the complexity of the human chronic wound condition. Furthermore, most animals do not develop chronic wounds. Only the thoroughbred racehorse develops chronic ulcers. Areas covered: In this review, the different characteristics of chronic wounds that highlight its complexity are described. In addition, currently available models reflecting different aspects of chronic wound pathology and their relevance to human chronic wounds are discussed. This article concludes by listing relevant features representative of an ideal chronic wound model. Additionally, alternative approaches for the development of chronic wound models are discussed. Expert opinion: Delayed models of healing, including the streptozotocin diabetic model, skin flap model and magnet-induced IR models have emerged. While these models have been widely adopted for preclinical therapeutic testing, their relevance towards human chronic wounds remains debatable. In particular, current delayed healing models often fail to fully incorporate the key characteristics of chronic ulcers. Ultimately, more representative models are required to expedite the advancement of novel therapeutics to the clinic.Agency for Science, Technology and Research (A*STAR)Ministry of Education (MOE)Nanyang Technological UniversityPublished versionThis research is supported by the Agency for Science, Technology and Research (A*STAR) under its Industry Alignment Fund–Pre-Positioning Programme (IAF-PP) grant number H17/01/a0/0C9 as part of the Wound Care Innovation for the Tropics (WCIT) Programme. This research is also supported by the Agency for Science, Technology and Research (A*STAR) under its Industry Alignment Fund–Pre-Positioning Programme (IAF-PP) grant number H1701a0004 and the Skin Research Institute of Singapore, Phase 2: SRIS@Novena. Nanyang Technological University (Start-up grant) and the Ministry of Education (Tier 1 T1-002-098 and T1-002-013) also supported this research

Scaffold-mediated non-viral delivery platform for CRISPR/Cas9-based genome editing

Genome editing, especially via the simple and versatile type II CRISPR/Cas9 system, offers an effective avenue to precisely control cell fate, an important aspect of tissue regeneration. Unfortunately, most CRISPR/Cas9 non-viral delivery strategies only utilise micro-/nano-particle delivery methods. While these approaches provide reasonable genomic editing efficiencies, their systemic delivery may lead to undesirable off-target effects. For in vivo applications, a more localized and sustained delivery approach may be useful, particularly in the context of tissue regeneration. Here, we developed a scaffold that delivers the CRISPR/Cas9 components (i.e. single guide RNA (sgRNA) and Cas9 protein complexes) in a localized and non-viral manner. Specifically, using mussel-inspired bioadhesive coating, polyDOPA-melanin (pDOPA), we absorbed Cas9:sgRNA lipofectamine complexes onto bio-mimicking fiber scaffolds. To evaluate the genome-editing efficiency of this platform, U2OS.EGFP cells were used as the model cell type. pDOPA coating was essential in allowing Cas9:sgRNA lipofectamine complexes to adhere onto the scaffolds with a higher loading efficiency, while laminin coating was necessary for maintaining cell viability and proliferation on the pDOPA-coated fibers for effective gene editing (21.5% editing efficiency, p < 0.001). Importantly, U2OS.EGFP cells took up Cas9:sgRNA lipofectamine complexes directly from the scaffolds via reverse transfection. Overall, we demonstrate the efficacy of such fiber scaffolds in providing localized, sustained and non-viral delivery of Cas9:sgRNA complexes. Such genome editing scaffolds may find useful applications in tissue regeneration.Accepted versio

Bio-Mimicking Acellular Wet Electrospun Scaffolds Promote Accelerated Integration and Re-Epithelialization of Full-Thickness Dermal Wounds

Scaffolds can promote the healing of burns and chronic skin wounds but to date have suffered from issues with achieving full skin integration. Here, we characterise the wound response by both tissue integration and re-epithelialization to a scaffold using wet electrospinning to fabricate 3D fibrous structures. Two scaffold materials were investigated: poly(&epsilon;-caprolactone) (PCL) and PCL + 20% rat tail type 1 collagen (PCL/Coll). We assessed re-epithelisation, inflammatory responses, angiogenesis and the formation of new extracellular matrix (ECM) within the scaffolds in rat acute wounds. The 3D PCL/Coll scaffolds impeded wound re-epithelisation, inducing a thickening of wound-edge epidermis as opposed to a thin tongue of migratory keratinocytes as seen when 3D PCL scaffolds were implanted in the wounds. A significant inflammatory response was observed with 3D PCL/Coll scaffolds but not with 3D PCL scaffolds. Enhanced fibroblast migration and angiogenesis into 3D PCL scaffolds was observed with a significant deposition of new ECM. We observed that this deposition of new ECM within the scaffold was key to enabling re-epithelialization over the scaffold. Such scaffolds provide a biocompatible environment for cell integration to lay down new ECM and encourage re-epithelisation over the implanted scaffold