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

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR) systems have 4 a wide variety of applications besides precise genome editing. In particular, the CRISPR/dCas9 5 system can be used to control specific gene expression by CRISPR activation (CRISPRa) or 6 interference (CRISPRi). However, the safety concerns associated with viral vectors and the 7 possible off-target issues of systemic administration remain huge concerns to be safe delivery 8 methods for CRISPR/Cas9 systems. In this study, a layer-by-layer (LbL) self-assembling peptide 9(SAP) coating on nanofibers is developed to mediate localized delivery of CRISPR/dCas9 10 systems. Specifically, an amphiphilic negatively charged SAPis first coated onto PCL 11 nanofibers through strong hydrophobic interactions, and the pDNA complexes and positively 12 charged SAP + -RGD are then absorbed via electrostatic interactions. The SAP-coated scaffolds 13 facilitate efficient loading and sustained release of the pDNA complexes, while enhancing cell 14 adhesion and proliferation. As a proof of concept, the scaffolds are used to activate GDNF 15 expression in mammalian cells, and the secreted GDNF subsequently promotes neurite 16 outgrowth of rat neurons. These promising results suggest that the LbL self-assembling peptide 17 coated nanofibers can be a new route to establish a bioactive interface, which provides a simple 18 and efficient platform for the delivery of CRISPR/dCas9 systems for regenerative medicine.

Neuroprotective Effects of VEGF-A Nanofiber Membrane and FAAH Inhibitor URB597 Against Oxygen–Glucose Deprivation-Induced Ischemic Neuronal Injury

“…The LBL self-assembling electrospun NF is a promising medium for the continuous and controlled delivery of active substances for neural injury repair and tissue engineering. 29 , 37 Our team previously reported that a biomimetic scaffold was deposited on the surface of PCL NFs to enable the local delivery of proteins to diseased tissues. 28 In this study, we successfully fabricated a nanofibrous of VEGF-A loaded PCL membrane by using LBL.…”

Enhanced efficiency of nonviral direct neuronal reprogramming on topographical patterns

“…103,104 Other genetic engineering techniques such as CRISPR/Cas9 have been combined with biomaterials to create substrates that can mediate localized delivery of CRISPR/Cas9 systems for neuronal regeneration. 105,106 This demonstrates the potential to translate the use of topographical cues to enhance direct neuronal reprogramming from in vitro to in vivo.…”

Nanotechnology‐Assisted RNA Delivery: From Nucleic Acid Therapeutics to COVID‐19 Vaccines

“…[ 131 , 132 , 133 ] Moreover, recent investigations on the architectural design of polymeric nanoplatforms can facilitate tissue integration, promote localized delivery of genetic molecules, and finally affect the development of fibrous capsules. [ 134 , 135 ] Finally, and in order to address the unbalanced relationships between cytotoxicity and transfection efficiency of polymeric nanocarriers, the emergence of green nanoparticles will be discussed in this section. [ 136 ]…”

“…successfully fabricated a layer‐by‐layer self‐assembling peptide coated on PCL nanofibers. [ 135 ] The proposed system was able to mediate the localized delivery to promote neural regeneration. In another study, miRNA‐gelatin polyplexes were introduced into PCL nanofibers.…”