Cell Penetrating Peptide Sequence Research Articles

Structure based peptide design, molecular dynamics and MM-PBSA studies for targeting C terminal dimerization of NFAT5 DNA binding domain

NFAT5 as a transcription factor with an established role in osmotic stress response, has also been revealed to be active under numerous settings, including pathological conditions such as diabetic microvascular complications, chronic arthritis and cancer. Despite these links, current strategies for downregulating NFAT5 activity only relies on indirect modulators, not directly targeting NFAT5, itself. With this study, through using a computational approach, an original peptide was explored to directly target C terminal dimerization of NFAT5 RHR, located in its DNA binding domain. At first, homodimeric NFAT5 RHR bound to its consensus DNA was used for prediction of a preliminary peptide sequence. Possible amino acid replacements for this preliminary peptide were predicted for optimization, which was followed by addition of a cell penetrating peptide sequence. These attempts yielded a small peptide library, which was further investigated for peptide affinities towards C terminal of NFAT5 RHR through molecular docking, 50 ns and 250 ns molecular dynamics simulations, followed by estimation of MM-PBSA based relative binding free energies. Results indicated that after receiving mutations on the preliminary peptide sequence for optimization, a unique peptide could target C terminal dimerization region of NFAT5 RHR through using its cell penetrating peptide sequence. In conclusion, this is the first study presenting computational evidence on identification of a novel peptide capable of directly targeting NFAT5 dimerization. Besides, future implications of these observations were also discussed in terms of methodology and possible applications.

Activation of cell-penetrating peptide fragments by disulfide formation

Three cell-penetrating peptides (CPPs), Tat, Pep-3 and penetratin, were split into two parts and each fragment was terminated with a cysteine residue, to allow disulfide bridge formation, as well as a fluorescent label, for visualization and quantitative analysis. After disulfide formation between two complementary CPP fragments, cellular uptake of the resulting conjugates was observed. As confirmed by in vitro experiments, the conjugated peptides showed uptake activity comparable to the native CPP sequences, while the truncated peptides were hardly active. Until now, this split CPP strategy has only been demonstrated for oligo-arginine CPPs, but here we demonstrate that it is also applicable to other cell-penetrating peptides. This wider applicability may help in the design of new activatable cell-penetrating peptides for, e.g., targeted drug delivery.

A new biology of cell penetrating peptides

AbstractAs pharmacokinetic modifiers, cell penetrating peptides (CPPs) have proven utility for the delivery of otherwise impermeable cargoes into the discrete intracellular compartments of eukaryotic cells. Methods for the molecular optimisation of CPP sequences can significantly increase the performance of novel vectors matched to a specific delivery function. Moreover, the ‘information rich’ composition and inherent molecular flexibility of many CPPs can also facilitate their interaction with intracellular proteins and other cellular structures. This proteomimetic property of CPPs has been exploited in the design of bioportides, bioactive CPPs that regulate cellular functions often by binding relatively flat PPI interfaces to achieve a dominant negative action. This new biology of CPPs is rapidly gathering momentum through the design and synthesis of a bewildering variety of peptides that may be, in whole or part, linear, helical, cyclic and/or chimeric in nature. A particular emphasis of contemporary CPP‐centred drug discovery is the unmet medical need of cancer, though the biomedical scope of bioportide applications is impressively broad including cellular signalling and reprogramming. We are hopeful that one or more of the fascinating studies described herein will translate into the clinic to establish bioportide technologies as a viable option for drug discovery.

Photostable NIR emitting ruthenium(II) conjugates; uptake and biological activity in live cells

A photostable Ru(2,2-biquinoline)2(3-(2-pyridyl)-5-(4-carboxyphenyl)-1,2,4-triazolate) (Ru(biq)2(trzbenzCOOH)) complex that exhibits near-infrared (NIR) emission centred at 786 nm is reported. The parent complex was conjugated via amide coupling to a cell-penetrating peptide sequence octa-arginine (R8), and two signal peptide sequences; the nuclear localizing sequence (NLS) VQRKRQKLMP and the mitochondria penetrating peptide (MPP) FrFKFrFK(Ac) (r = D isomer of arginine, Ac = terminal lysine amine acetyl blocked). Notably, none of the peptide conjugates were cell-permeable as chloride salts but efficient and rapid membrane permeation was observed post ion exchange with perchlorate counterion. Also, surprisingly, all three peptide conjugates exhibited potent dark cytotoxicity in both CHO and HeLa cell lines. The peptide conjugates induce cell death through a caspase dependent apoptotic pathway. At the minimum concentration of dye (approx. 15 μM) required for cell imaging, only 20% of the cells were viable after a 24 h incubation period. To overcome cytotoxicity, the parent complex was PEGylated; this dramatically decreased cytotoxicity, where 50% of cells were viable even at 150 μM concentration after 24 h. Confocal luminescence microscopy indicated that all four bioconjugates, peptides in perchlorate form and polyethylene glycol (PEG) in chloride form, were rapidly internalized within the cell. However, interestingly the precise localisation by the signal peptides observed in related complexes was not observed here and the peptide conjugates were unsuitable as luminescent probes for cell microscopy due to their high cell toxicity. The poor targeting of signal peptides in this instance is attributed to the high lipophilicity of the metal centre.

SMR peptide antagonizes mortalin promoted release of extracellular vesicles and affects mortalin protection from complement-dependent cytotoxicity in breast cancer cells and leukemia cells.

Background: Mortalin/GRP-75/mt-hsp70 is a mitochondrial chaperone protein, found in the cytoplasm, endoplasmic reticulum and cytoplasmic vesicles. It functions in many cellular processes such as mitochondrial biogenesis, intracellular trafficking, cell proliferation, signaling, immortalization and tumorigenesis. Thus, inhibition of mortalin is a promising avenue for cancer therapy. Previous studies in our lab have suggested that mortalin contributes to breast cancer development and progression. We showed that tumor extracellular vesicle secretion was decreased by knockdown of mortalin expression using HIV-1 Nef SMR peptides. Specifically, these peptides can block extracellular vesicle secretion and mediate cell cycle arrest in MDA-MB-231 and MCF-7 breast cancer cells. Aims: This study aims to investigate further the function and mechanism of interaction of PEG-SMR-CLU and SMR-CPP peptides with the chaperone protein mortalin and to explore the effect of SMR-derived peptides and mortalin expression on extracellular vesicle release and complement dependent cell toxicity in human breast cancer and leukemia cell lines. Results: Our results demonstrated additional effects reversing the tumorigenicity of these cells. First, the modified SMRwt peptides reduced the expression of the mesenchymal marker vimentin (VIM). Second, exposure to the SMRwt peptide inhibited mortalin and complement C9 expression in MDA-MB-231, MCF-7 breast cancer cells and K562 leukemia cells as measured by the Western blot analysis. Third, the SMRwt peptides blocked the cancer cells’ ability to release extracellular vesicles, which we observed blocked extracellular vesicle-mediated release of complement, re-establishing complements mediated cell death in those peptide-treated cells. Methods: We developed a series of peptides derived from the Secretion Modification Region (SMR) of HIV-1 Nef protein, modified by the addition of either a cell-penetrating peptide (CPP), a positively charged arginine-rich peptide derived from HIV-1 regulatory protein Tat, or a Clusterin-binding peptide (CLU), a molecular chaperone involved in protein secretion. Both CPP and CLU peptide sequences were added at the C-terminus of the Nef SMR peptide. The CLU-containing peptides were also modified with polyethylene glycol (PEG) to enhance solubility. After treatment of cells with the peptides, we used the MTT cell viability and complement-mediated cytotoxicity assays to confirm the inhibitory role of modified SMRwt peptides on the proliferation of MDA-MB-231 and MCF-7 breast cancer cells and K562 leukemia cells. Flow cytometry was used to determine complement mediated cell apoptosis and death. Western blot analysis was used to track SMR peptides impact on expression of mortalin, vimentin and complement C9 and to measure the expression of extracellular vesicle proteins. NanoSight analysis and acetylcholinesterase (AChE) assay were used for measuring extracellular vesicles particle size and concentration and acetylcholinesterase. Conclusions: Mortalin promotes cell proliferation, metastasis, angiogenesis, downregulate apoptotic signaling. Thus, mortalin is a potential therapeutic target for cancer immunotherapy. The novel SMRwt peptides antagonize the functions of mortalin, blocking tumor extracellular vesicle release and extracellular vesicle-mediated release of complement. This leads to decreases in breast cancer cell metastasis and allows standard treatment of these late stage tumor cells, thus having important clinical implications for late stage breast cancer chemotherapy. These findings support further investigation into the therapeutic value of the SMR peptide in cancer metastasis.

G-DipC: An Improved Feature Representation Method for Short Sequences to Predict the Type of Cargo in Cell-Penetrating Peptides.

Cell-penetrating peptides (CPPs) are functional short peptides with high carrying capacity. CPP sequences with targeting functions for the highly efficient delivery of drugs to target cells. In this paper, which is focused on the prediction of the cargo category of CPPs, a biocomputational model is constructed to efficiently distinguish the category of cargo carried by CPPs as macromolecular carriers among the seven known deliverable cargo categories. Based on dipeptide composition (DipC), an improved feature representation method, general dipeptide composition (G-DipC) is proposed for short peptide sequences and can effectively increase the abundance of features represented. Then linear discriminant analysis (LDA) is applied to mine some important low-dimensional features of G-DipC and a predictive model is built with the XGBoost algorithm. Experimental results with five-fold cross validation show that G-DipC improves accuracy by 25 and 5 percent compared with amino acid composition (AAC) and DipC, respectively. G-DipC is even found to be better than tripeptide composition (TipC). Thus, the proposed model provides a novel resource for the study of cell-penetrating peptides, and the improved dipeptide composition G-DipC can be widely adapted to determine the feature representation of other biological sequences.

Cell-penetrating peptide sequence and modification dependent uptake and subcellular distribution of green florescent protein in different cell lines

Protein therapy holds great promise for treating a variety of diseases. To act on intracellular targets, therapeutic proteins must cross the plasma membrane. This has previously been achieved by covalent attachment to a variety of cell-penetrating peptides (CPPs). However, there is limited information on the relative performance of CPPs in delivering proteins to cells, specifically the cytosol and other intracellular locations. Here we use green fluorescent protein (GFP) as a model cargo to compare delivery capacity of five CPP sequences (Penetratin, R8, TAT, Transportan, Xentry) and cyclic derivatives in different human cell lines (HeLa, HEK, 10T1/2, HepG2) representing different tissues. Confocal microscopy analysis indicates that most fusion proteins when incubated with cells at 10 µM localise to endosomes. Quantification of cellular uptake by flow cytometry reveals that uptake depends on both cell type (10T1/2 > HepG2 > HeLa > HEK), and CPP sequence (Transportan > R8 > Penetratin≈TAT > Xentry). CPP sequence cyclisation or addition of a HA-sequence increased cellular uptake, but fluorescence was still contained in vesicles with no evidence of endosomal escape. Our results provide a guide to select CPP for endosomal/lysosomal delivery and a basis for developing more efficient CPPs in the future.

Oligoarginine-Bearing Tandem Repeat Penetration-Accelerating Sequence Delivers Protein to Cytosol via Caveolae-Mediated Endocytosis.

To facilitate the cytosolic delivery of larger molecules such as proteins, we developed a new cell-penetrating peptide sequence, named Pas2r12, consisting of a repeated Pas sequence (FFLIG-FFLIG) and d-dodeca-arginine (r12). This peptide significantly enhanced the cellular uptake and cytosolic release of enhanced green fluorescent protein and immunoglobulin G as cargos. We found that simply mixing Pas2r12 with cargos could generate cytosolic introducible forms. The cytosolic delivery of cargos by Pas2r12 was found to be an energy-requiring process, to rely on actin polymerization, and to be suppressed by caveolae-mediated endocytosis inhibitors (genistein and methyl-β-cyclodextrin) and small interfering RNA against caveolin-1. These results suggest that Pas2r12 enhances membrane penetration of cargos without the need for cross-linking and that caveolae-mediated endocytosis may be the route by which cytosolic delivery is enhanced.

Population-based analysis of cell-penetrating peptide uptake using a microfluidic droplet trapping array.

Cell-penetrating peptides (CPPs) have garnered significant attention as a method to introduce reporters and therapeutics into intact cells. While numerous studies have been performed identifying new CPP sequences, relatively little is known about their uptake efficiency at the single-cell level. Here, a droplet microfluidic trapping array was used to characterize CPP uptake across a population of single intact cells. The microfluidic device allowed for facile and rapid isolation and analysis of single-cell fluorescence in a 787-member overhead trapping array with > 99% droplet trapping efficiency. The permeability efficiencies of four different CPPs were studied and compared in HeLa cells. Population analysis was performed using linkage hierarchical cluster analysis by R programming to bin cells into subpopulations expressing very low to very high peptide uptake efficiencies. CPP uptake was observed to be heterogeneous across the population of cells with peptide concentration and sequence both playing important roles in the diversity of CPP uptake, the overall peptide uptake efficiency, and the intracellular homogeneity of peptide distribution. This microfluidic-based analytical approach finds application in personalized medicine and provides new insight in the heterogeneity of CPP uptake which has the potential to affect both biosensor and drug internalization in intact cells. Graphical abstract .

Effective in Vivo Targeting of Influenza Virus through a Cell-Penetrating/Fusion Inhibitor Tandem Peptide Anchored to the Plasma Membrane.

The impact of influenza virus infection is felt each year on a global scale when approximately 5-10% of adults and 20-30% of children globally are infected. While vaccination is the primary strategy for influenza prevention, there are a number of likely scenarios for which vaccination is inadequate, making the development of effective antiviral agents of utmost importance. Anti-influenza treatments with innovative mechanisms of action are critical in the face of emerging viral resistance to the existing drugs. These new antiviral agents are urgently needed to address future epidemic (or pandemic) influenza and are critical for the immune-compromised cohort who cannot be vaccinated. We have previously shown that lipid tagged peptides derived from the C-terminal region of influenza hemagglutinin (HA) were effective influenza fusion inhibitors. In this study, we modified the influenza fusion inhibitors by adding a cell penetrating peptide sequence to promote intracellular targeting. These fusion-inhibiting peptides self-assemble into ∼15-30 nm nanoparticles (NPs), target relevant infectious tissues in vivo, and reduce viral infectivity upon interaction with the cell membrane. Overall, our data show that the CPP and the lipid moiety are both required for efficient biodistribution, fusion inhibition, and efficacy in vivo.

β-Lactamase Tools for Establishing Cell Internalization and Cytosolic Delivery of Cell Penetrating Peptides.

The ability of cell penetrating peptides (CPPs) to deliver biologically relevant cargos into cells is becoming more important as targets in the intracellular space continue to be explored. We have developed two assays based on CPP-dependent, intracellular delivery of TEM-1 β-lactamase enzyme, a functional biological molecule comparable in size to many protein therapeutics. The first assay focuses on the delivery of full-length β-lactamase to evaluate the internalization potential of a CPP sequence. The second assay uses a split-protein system where one component of β-lactamase is constitutively expressed in the cytoplasm of a stable cell line and the other component is delivered by a CPP. The delivery of a split β-lactamase component evaluates the cytosolic delivery capacity of a CPP. We demonstrate that these assays are rapid, flexible and have potential for use with any cell type and CPP sequence. Both assays are validated using canonical and novel CPPs, with limits of detection from <500 nM to 1 µM. Together, the β-lactamase assays provide compatible tools for functional characterization of CPP activity and the delivery of biological cargos into cells.

Personalized Cancer Vaccine Platform for Clinically Relevant Oncolytic Enveloped Viruses.

The approval of the first oncolytic virus for the treatment of metastatic melanoma and the compiling evidence that the use of oncolytic viruses can enhance cancer immunotherapies targeted against various immune checkpoint proteins has attracted great interest in the field of cancer virotherapy. We have developed a novel platform for clinically relevant enveloped viruses that can direct the virus-induced immune response against tumor antigens. By physically attaching tumor-specific peptides onto the viral envelope of vaccinia virus and herpes simplex virus 1 (HSV-1), we were able to induce a strong T cell-specific immune response toward these tumor antigens. These therapeutic peptides could be attached onto the viral envelope by using a cell-penetrating peptide sequence derived from human immunodeficiency virus Tat N-terminally fused to the tumor-specific peptides or, alternatively, therapeutic peptides could be conjugated with cholesterol for the attachment of the peptides onto the viral envelope. We used two mouse models of melanoma termed B16.OVA and B16-F10 for testing the efficacy of OVA SIINFEKL-peptide-coated viruses and gp100-Trp2-peptide-coated viruses, respectively, and show that by coating the viral envelope with therapeutic peptides, the anti-tumor immunity and the number of tumor-specific CD8+ T cells in the tumor microenvironment can be significantly enhanced.

Cell-Penetrating Peptides Containing Fluorescent d-Cysteines.

A series of fluorescent d-cysteines (Cys) has been synthesized and their optical properties were studied. The key synthetic step is the highly diastereoselective 1,4-conjugate addition of aryl thiols to a chiral bicyclic dehydroalanine recently developed by our group. This reaction is fast at room temperature and proceeds with total chemo- and stereoselectivity. The Michael adducts were easily transformed into the corresponding amino acids to study their optical properties and, in some selected cases, into the corresponding N-Fmoc-d-cysteine derivatives to be used in solid-phase peptide synthesis (SPPS). To further demonstrate the utility of these non-natural Cys-derived fluorescent amino acids, the coumaryl and dansyl derivatives were incorporated into cell-penetrating peptide sequences through standard SPPS and their optical properties were studied in different cell lines. The internalization of these fluorescent peptides was monitored by fluorescence microscopy.

Machine Learning To Predict Cell-Penetrating Peptides for Antisense Delivery.

Cell-penetrating peptides (CPPs) can facilitate the intracellular delivery of large therapeutically relevant molecules, including proteins and oligonucleotides. Although hundreds of CPP sequences are described in the literature, predicting efficacious sequences remains difficult. Here, we focus specifically on predicting CPPs for the delivery of phosphorodiamidate morpholino oligonucleotides (PMOs), a compelling type of antisense therapeutic that has recently been FDA approved for the treatment of Duchenne muscular dystrophy. Using literature CPP sequences, 64 covalent PMO–CPP conjugates were synthesized and evaluated in a fluorescence-based reporter assay for PMO activity. Significant discrepancies were observed between the sequences that performed well in this assay and the sequences that performed well when conjugated to only a small-molecule fluorophore. As a result, we envisioned that our PMO–CPP library would be a useful training set for a computational model to predict CPPs for PMO delivery. We used the PMO activity data to fit a random decision forest classifier to predict whether or not covalent attachment of a given peptide would enhance PMO activity at least 3-fold. To validate the model experimentally, seven novel sequences were generated, synthesized, and tested in the fluorescence reporter assay. All computationally predicted positive sequences were positive in the assay, and one sequence performed better than 80% of the tested literature CPPs. These results demonstrate the power of machine learning algorithms to identify peptide sequences with particular functions and illustrate the importance of tailoring a CPP sequence to the cargo of interest.

A High-Throughput Synthetic Platform Enables the Discovery of Proteomimetic Cell Penetrating Peptides and Bioportides

Collectively, cell penetrating peptide (CPP) vectors and intrinsically active bioportides possess tremendous potential for drug delivery applications and the discrete modulation of intracellular targets including the sites of protein–protein interactions (PPIs). Such sequences are usually relatively short (< 25 AA), polycationic in nature and able to access the various intracellular compartments of eukaryotic cells without detrimental influences upon cellular biology. The high-throughput platform for bioportide discovery described herein exploits the discovery that many human proteins are an abundant source of potential CPP sequences which are reliably predicted using QSAR algorithms or other methods. Subsequently, microwave-enhanced solid phase peptides synthesis provides a high-throughput source of novel proteomimetic CPPs for screening purposes. By focussing upon cationic helical domains, often located within the molecular interfaces that facilitate PPIs, bioportides which act by a dominant-negative mechanism at such sites can be reliably identified within small number libraries of CPPs. Protocols that employ fluorescent peptides, routinely prepared by N-terminal acylation with carboxytetramethylrhodamine, further enable both the quantification of cellular uptake kinetics and the identification of specific site(s) of intracellular accretion. Chemical modifications of linear peptides, including strategies to promote and stabilise helicity, are compatible with the synthesis of second-generation bioportides with improved drug-like properties to further exploit the inherent selectivity of biologics.

Modulation of UVB-induced Carcinogenesis by Activation of Alternative DNA Repair Pathways

The molecular basis for ultraviolet (UV) light-induced nonmelanoma and melanoma skin cancers centers on cumulative genomic instability caused by inefficient DNA repair of dipyrimidine photoproducts. Inefficient DNA repair and subsequent translesion replication past these DNA lesions generate distinct molecular signatures of tandem CC to TT and C to T transitions at dipyrimidine sites. Since previous efforts to develop experimental strategies to enhance the repair capacity of basal keratinocytes have been limited, we have engineered the N-terminally truncated form (Δ228) UV endonuclease (UVDE) from Schizosaccharomyces pombe to include a TAT cell-penetrating peptide sequence with or without a nuclear localization signal (NLS): UVDE-TAT and UVDE-NLS-TAT. Further, a NLS was engineered onto a pyrimidine dimer glycosylase from Paramecium bursaria chlorella virus-1 (cv-pdg-NLS). Purified enzymes were encapsulated into liposomes and topically delivered to the dorsal surface of SKH1 hairless mice in a UVB-induced carcinogenesis study. Total tumor burden was significantly reduced in mice receiving either UVDE-TAT or UVDE-NLS-TAT versus control empty liposomes and time to death was significantly reduced with the UVDE-NLS-TAT. These data suggest that efficient delivery of exogenous enzymes for the initiation of repair of UVB-induced DNA damage may protect from UVB induction of squamous and basal cell carcinomas.

Novel cell-penetrating-amyloid peptide conjugates preferentially kill cancer cells.

The goal of this study was to develop a peptide which could use the toxic effects of amyloid, a substance which is the hallmark of over 25 known human diseases, to selectively kill cancer cells. Here we demonstrate that two separate amyloid-forming hexapeptides, one from the microtubule associated protein Tau involved in formation of paired helical filaments of Alzheimer's disease, and the other an amyloid forming sequence from apolipoprotein A1, when conjugated to a cell penetrating peptide (CPP) sequence, form toxic oligomers which are stable for up to 14 h and able to enter cells by a combination of endocytosis and transduction. The amyloid peptide conjugates showed selective cytotoxicity to breast cancer, neuroblastoma and cervical cancer cells in culture compared to normal cells. Fluorescence imaging experiments showed the CPP-amyloid peptide oligomers formed intracellular fibrous amyloid, visible in the endosomes/lysosomes, cytosol and nucleus with thioflavin S (ThS) staining. Further experiments with rhodamine-conjugated Dextran, propidium iodide (PI), and acridine orange (AO) suggested the mechanism of cell death was the permeability of the lysosomal membrane brought about by the formation of amyloid pores. Cytotoxicity could be abrogated by inhibitors of lysosomal hydrolases, consistent with a model where lysosomal hydrolases leak into the cytosol and induce cytotoxicity in subsequent downstream steps. Taken together, our data suggest that CPP-amyloid peptide conjugates show potential as a new class of anti-cancer peptides (ACPs).

Cell-Penetrating Peptide Spirolactam Derivative as a Reversible Fluorescent pH Probe for Live Cell Imaging.

A colorless and nonfluorescent spirolactam derivative, RhB-R12K, was synthesized by amide condensation between the carboxyl group of rhodamine B (RhB) and the amino group of cell-penetrating peptide (CPP). The fluorescence intensity of RhB-R12K sharply increased as the pH value decreased from 8.0 to 4.9, demonstrating sensitive and reversible response to intracellular pH distribution. This CPP probe was completely water soluble, had low cytotoxicity, was membrane permeable, and was suitable for pH measurement in various organelles by choosing organelle-specific CPP sequences. Interestingly, CPPs acted not only as carriers but also as indispensable parts of fluorophores here. The presence of active groups on the peptides potentially allows for modification with additional dyes to construct multifunctional and ratiometric probes for cell imaging.

Identification of a Short Cell-Penetrating Peptide from Bovine Lactoferricin for Intracellular Delivery of DNA in Human A549 Cells.

Cell-penetrating peptides (CPPs) have been shown to deliver cargos, including protein, DNA, RNA, and nanomaterials, in fully active forms into live cells. Most of the CPP sequences in use today are based on non-native proteins that may be immunogenic. Here we demonstrate that the L5a CPP (RRWQW) from bovine lactoferricin (LFcin), stably and noncovalently complexed with plasmid DNA and prepared at an optimal nitrogen/phosphate ratio of 12, is able to efficiently enter into human lung cancer A549 cells. The L5a CPP delivered a plasmid containing the enhanced green fluorescent protein (EGFP) coding sequence that was subsequently expressed in cells, as revealed by real-time PCR and fluorescent microscopy at the mRNA and protein levels, respectively. Treatment with calcium chloride increased the level of gene expression, without affecting CPP-mediated transfection efficiency. Zeta-potential analysis revealed that positively electrostatic interactions of CPP/DNA complexes correlated with CPP-mediated transport. The L5a and L5a/DNA complexes were not cytotoxic. This biomimetic LFcin L5a represents one of the shortest effective CPPs and could be a promising lead peptide with less immunogenic for DNA delivery in gene therapy.

Targeting the Nrf2 Signaling Pathway in the Retina With a Gene-Delivered Secretable and Cell-Penetrating Peptide.

PurposeOxidative stress has been linked to several ocular diseases, initiating an inflammatory response that increases tissue injury. The Nrf2 transcription factor regulates expression of antioxidant genes and is tightly regulated by Kelch-Like ECH-Associated Protein 1 (Keap-1). We evaluate the antioxidant and anti-inflammatory properties of an adeno-associated virus (AAV) vector delivering an Nrf2-derived peptide that binds Keap-1.MethodsThe sequence of the Nrf2 peptide was fused to a cell-penetrating peptide (Tat-peptide) sequence (TatNrf2mer). The effects of lentiviral-delivered TatNrf2mer were studied in vitro. Transcript (quantitative [q] RT-PCR) and protein levels (ELISA and immunofluorescence) were quantified. Cell viability was measured by MTT and Cell Titer assays. The AAV vectors were packaged with the TatNrf2mer fused to secretable green fluorescent protein (GFP) under the control of the small chicken β actin promoter. The protective effects of this vector were evaluated in a model of RPE oxidative injury and in a mouse model of uveitis after intravitreal injection.ResultsExpression of TatNrf2mer peptide induced antioxidant gene expression, blocked IL-1β secretion, and protected cells from oxidative injury. In mice, TatNrf2mer expression partially protected photoreceptor function based on ERG responses and optical coherence tomography measurements in the sodium iodate (NaIO3) model. Furthermore, sGFP-TatNrf2mer expression decreased IL-1β and IL-6 in the NaIO3-treated mice, and resulted in a 54% decrease in the number of inflammatory cells in the vitreous body of the endotoxin-induced uveitis mouse model.ConclusionsThe intravitreally delivered AAV-TatNrf2mer has antioxidant and anti-inflammatory effects in widely-used models of ocular injury, suggesting it also could be useful in ocular diseases associated with oxidative stress and inflammasome activation.