skip to main content

Title: Cell Penetrating Peptides, Novel Vectors for Gene Therapy
Cell penetrating peptides (CPPs), also known as protein transduction domains (PTDs), first identified ~25 years ago, are small, 6–30 amino acid long, synthetic, or naturally occurring peptides, able to carry variety of cargoes across the cellular membranes in an intact, functional form. Since their initial description and characterization, the field of cell penetrating peptides as vectors has exploded. The cargoes they can deliver range from other small peptides, full-length proteins, nucleic acids including RNA and DNA, liposomes, nanoparticles, and viral particles as well as radioisotopes and other fluorescent probes for imaging purposes. In this review, we will focus briefly on their history, classification system, and mechanism of transduction followed by a summary of the existing literature on use of CPPs as gene delivery vectors either in the form of modified viruses, plasmid DNA, small interfering RNA, oligonucleotides, full-length genes, DNA origami or peptide nucleic acids.
Authors:
;
Award ID(s):
1944130 1739308
Publication Date:
NSF-PAR ID:
10215368
Journal Name:
Pharmaceutics
Volume:
12
Issue:
3
Page Range or eLocation-ID:
225
ISSN:
1999-4923
Sponsoring Org:
National Science Foundation
More Like this
  1. Protein therapeutics represent a significant and growing component of the modern pharmacopeia, but their potential to treat human disease is limited because most proteins fail to traffic across biological membranes. Recently, we discovered a class of cell-permeant miniature proteins (CPMPs) containing a precisely defined, penta-arginine (penta-Arg) motif that traffics readily to the cytosol and nucleus of mammalian cells with efficiencies that rival those of hydrocarbon-stapled peptides active in animals and man. Like many cell-penetrating peptides (CPPs), CPMPs enter the endocytic pathway; the difference is that CPMPs containing a penta-Arg motif are released efficiently from endosomes, while other CPPs are not.more »Here, we seek to understand how CPMPs traffic from endosomes into the cytosol and what factors contribute to the efficiency of endosomal release. First, using two complementary cell-based assays, we exclude endosomal rupture as the primary means of endosomal escape. Next, using an RNA interference screen, fluorescence correlation spectroscopy, and confocal imaging, we identifyVPS39—a gene encoding a subunit of the homotypic fusion and protein-sorting (HOPS) complex—as a critical determinant in the trafficking of CPMPs and hydrocarbon-stapled peptides to the cytosol. Although CPMPs neither inhibit nor activate HOPS function, HOPS activity is essential to efficiently deliver CPMPs to the cytosol. CPMPs localize within the lumen of Rab7+and Lamp1+endosomes and their transport requires HOPS activity. Overall, our results identify Lamp1+late endosomes and lysosomes as portals for passing proteins into the cytosol and suggest that this environment is prerequisite for endosomal escape.

    « less
  2. Cationic liposomes (CLs) are effective carriers of a variety of therapeutics. Their applications as vectors of nucleic acids (NAs), from long DNA and mRNA to short interfering RNA (siRNA), have been pursued for decades to realize the promise of gene therapy, with approvals of the siRNA therapeutic patisiran and two mRNA vaccines against COVID-19 as recent milestones. The long-term goal of developing optimized CL-based NA carriers for a broad range of medical applications requires a comprehensive understanding of the structure of these vectors and their interactions with cell membranes and components that lead to the release and activity of themore »NAs within the cell. Structure–activity relationships of lipids for CL-based NA and drug delivery must take into account that these lipids act not individually but as components of an assembly of many molecules. This review summarizes our current understanding of how the choice of the constituting lipids governs the structure of their CL–NA self-assemblies, which constitute distinct liquid crystalline phases, and the relation of these structures to their efficacy for delivery. In addition, we review progress toward CL–NA nanoparticles for targeted NA delivery in vivo and close with an outlook on CL-based carriers of hydrophobic drugs, which may eventually lead to combination therapies with NAs and drugs for cancer and other diseases.« less
  3. Simultaneous delivery of small molecules and nucleic acids using a single vehicle can lead to novel combination treatments and multifunctional carriers for a variety of diseases. In this study, we report a novel library of aminoglycoside-derived lipopolymers nanoparticles (LPNs) for the simultaneous delivery of different molecular cargoes including nucleic acids and small-molecules. The LPN library was screened for transgene expression efficacy following delivery of plasmid DNA, and lead LPNs that showed high transgene expression efficacies were characterized using hydrodynamic size, zeta potential, 1 H NMR and FT-IR spectroscopy, and transmission electron microscopy. LPNs demonstrated significantly higher efficacies for transgene expressionmore »than 25 kDa polyethyleneamine (PEI) and lipofectamine, including in presence of serum. Self-assembly of these cationic lipopolymers into nanoparticles also facilitated the delivery of small molecule drugs ( e.g. doxorubicin) to cancer cells. LPNs were also employed for the simultaneous delivery of the small-molecule histone deacetylase (HDAC) inhibitor AR-42 together with plasmid DNA to cancer cells as a combination treatment approach for enhancing transgene expression. Taken together, our results indicate that aminoglycoside-derived LPNs are attractive vehicles for simultaneous delivery of imaging agents or chemotherapeutic drugs together with nucleic acids for different applications in medicine and biotechnology.« less
  4. The discovery of cell penetrating peptides (CPPs) with unique membrane activity has inspired the design and synthesis of a variety of cell penetrating macromolecules, which offer tremendous opportunity and promise for intracellular delivery of a variety of imaging probes and therapeutics. While cell penetrating macromolecules can be designed and synthesized to have equivalent or even superior cell penetrating activity compared with natural CPPs, most of them suffer from moderate to severe cytotoxicity. Inspired by recent advances in peptide self-assembly and cell penetrating macromolecules, in this work, we demonstrated a new class of peptide assemblies with intrinsic cell penetrating activity andmore »excellent cytocompatibility. Supramolecular assemblies were formed through the self-assembly of de novo designed multidomain peptides (MDPs) with a general sequence of K x (QW) 6 E y in which the numbers of lysine and glutamic acid can be varied to control supramolecular assembly, morphology and cell penetrating activity. Both supramolecular spherical particles and nanofibers exhibit much higher cell penetrating activity than monomeric MDPs while supramolecular nanofibers were found to further enhance the cell penetrating activity of MDPs. In vitro cell uptake results suggested that the supramolecular cell penetrating nanofibers undergo macropinocytosis-mediated internalization and they are capable of escaping from the lysosome to reach the cytoplasm, which highlights their great potential as highly effective intracellular therapeutic delivery vehicles and imaging probes.« less
  5. Intercalating ds-DNA/RNA with small molecules can play an essential role in controlling the electron transmission probability for molecular electronics applications such as biosensors, single-molecule transistors, and data storage. However, its applications are limited due to a lack of understanding the nature of intercalation and electron transport mechanisms. We addressed this long-standing problem by studying the effect of intercalation on both the molecular structure and charge transport along the nucleic acids using molecular dynamics simulations and first-principle calculations coupled with Green’s function method, respectively. The study on anthraquinone and anthraquinone-neomycin conjugate intercalation into short nucleic acids reveals some universal features: 1)more »the intercalation affects the transmission by two mechanisms: a) inducing energy levels within the bandgap and b) shifting the location of the Fermi energy with respect to the molecular orbitals of the nucleic acid, 2) the effect of intercalation was found to be dependent on the redox state of the intercalator: while oxidized anthraquinone decreases, reduced anthraquinone increases the conductance, and 3) the sequence of intercalated nucleic acid further affects the transmission: lowering the AT-region length was found to enhance the electronic coupling of the intercalator with GC bases, hence yielding an increase of more than four times in conductance. We anticipate our study to inspire designing intercalator-nucleic acid complexes for potential use in molecular electronics via creating a multi-level gating effect.« less