Precisely Designed Peptides With Multifunctional Fragments As Gene-delivery Vectors

AbstractGene-based therapy is the intentional modulation of gene expression in specific cells to treat pathological conditions. This modulation is accomplished by introducing exogenous nucleic acids such as DNA, m RNA, antisense oligomucleotides, mi RNA or si RNA. More recently, the genome editing system CRISPER-Cas9 provide a more precise way to repair disease-causing genes. However, the cellular uptake of free nucleic acids is hindered by the large size and negative charge of these complexes. Moreover, these complexes are rapidely degraded by ribozymes in an acidic environment. Thus, a safe and effective gene delivery system has remained a barrier to effective gene therapy for many years. Although viral vectors were used in about 70% of gene therapy clinical trials due to their high transfection efficiency, several limitations are associated with viral vecors, including immunogenicity, carcinogenesis, limitated DNA packaging capacity and difficulty to scale-up. Non-viral vectors offer potential routes for gene delivery. Nevertheless, few of these vectors have so far been developed clinically owing their low delivery efficicency relative to virial vectors. In vitro and in vivo barriers such as DNA complexation, cellular uptake, endosomal escape, nuclear entry, enzymatice degradation and tissue targeting permeabilization remain to be addressed. In recent years, peptides have been developed as an alternative to non-viral vectors due to their ability to mediate various biological functions, including cell penetration, targeting and endosomal escape. We focused on deisigning and synthesizing safe and effective peptide-based vectors that can avoid both in vitro and in vivo barriers for gene delivery.In the present study, we precisely designed a series of pepides containing different functional segments to conque the major barriers in gene delivery. It will be illustrated in the following three sections:1. To overcome barriers associated with gene delivery, a series of peptides consisting of multifunctional fragmentswere designed and synthesized for evaluation as non-viral gene vectorsThe peptide-based vectorsincludea cationic amphiphilic α-helical antimicrobial peptide(AMP), the cell penetrating peptide(CPP), TAT, a stearyl moiety, and cysteine residues.. TAT and AMP segments were utilized to mediate cellular uptake and endosomal escape, respectively. Stearyl moieties provide an intramolecular hydrophobic environment to promote AMPs to form an α-helical conformation in PBS, and this is beneficial for DNA binding, cellular uptake, and endosomal escape. The α-helical content of the peptides, as well as the particle size, zeta potential, and morphology of the peptide/DNA complexes, were characterized. Fluorescence activated cell sorting(FACS) and confocal microscopy data showed that the peptides were able to efficiently translocate a p GL3 control plasmid across the plasma membrane via endocytosis, and then they successfully evaded endosomal entrapment and possible metabolic degradation. Moreover, one of the peptide(C18-C(LLKK)3C-TAT) vectors exhibited a high transfection efficiency similar to that of Lipofectamine 2000, concomitant with lower cytotoxicity.To avoid enzymatice degradation, C18-c(llkk)3c-tat with D-amino acids were synthesized. The transfection of C18-c(llkk)3c-tat in NIH-3T3 cell increased to 3.3-, 4-times compared to C18-C(LLKK)3C-TAT and Lipofectamine 2000, repectively. In vivo distribution and tranfection efficiency of C18-c(llkk)3c-tat were evaluated in tumor-bearing mice by in vivo imaging, and C18-c(llkk)3c-tat exhibited comparabale efficiency to that of Lipofectamine 2000.Overall, a combination of the four functional segments tested was used to generate a non-viral gene vector that synergistically promoted cellular uptake, endosomal escape, and gene expression.2. Incorporating histidine in the vectors to further enhance endosomal escape efficiencyEndosomal entrapment is a major barrier to gene delivery, to enhance the endosomal escape efficiency of the peptide-based vectors above, we incorporate histidine in the peptide. The imidazole group of hisdine has a p Ka of ~6.0 and can thus absorb protons in the acidic envirionment of the endosome, leading to osmotic swelling, membrane disruption and eventrually DNA escape. We synthesized for peptide compounds, C18-C(LLHH)3C-TAT, C18-C(LLHH)3C-R9,C18-C(LLKK)3CH6-TAT, C18-C(LLKK)3CH6-R9.The particle size, zeta potential, and morphology of the peptide/DNA complexes were characterized. The transfection efficiency of the four peptide-based vectors was higher than those of without histines. The luciferase expression level of C18-C(LLHH)3C-TAT(1.33×109 RLU/mg protein) in NIH-3T3 cells was 7.8-, 21.9- and 17.2- fold higher than that of C18-c(llkk)3c-tat,Lipofectamine 2000 and PEI 25 k Da. The data showed that the strategy of combination histines and cationic amphiphilic α-helical antimicrobial peptide can overcoming the ensosomal obstacle and increase the transfection efficiency.3. RVG-mediated neural targeting gene vectorsThe intrinsic property of cell-penetrating peptides(CPPs) to deliver therapeutic molecules(nucleic acids, drugs, imaging agents) to cells and tissues in a nontoxic manner has indicated that they may be potential components of future drugs and disease diagnostic agents. Nevertheless, cardinal problems i.e. poor specificity to target selected cells, especially neurons, and low cellular traffic are not sufficiently solved. A short peptide derived from rabies virus glycoprotein(RVG) enables the transvascular delivery of nucleic acids to the brain. This 29-amino-acid peptide specifically binds to the acetylcholine receptor expressed by neuronal cells.Two chimaeric peptides RVG-c(llkk)3-tat and RVG-c(llhh)3-tat were synthesized by natural ligation reation. The two peptide-based vectors were able to bind and transducer DNA to C6 cells in vitro, resulting in high transfection efficiency. The luciferase expression level of RVG-c(llhh)3-tat is 5-fold higher than that of Lipofectamine 2000. Thus, RVG-c(llhh)3-tat provides a safe and noninvasive approach of the delivery of DNA to neuronal cells. It is anticipated that the structural and multifunctional features evaluated in the present study will provide a promising gene delivery system for further investigation.ii...