Peptide Vectors for the Nonviral Delivery of Nucleic Acids

Abstract

Over the past two decades, gene therapy has garnered tremendous attention and is heralded by many as the ultimate cure to treat diseases such as cancer, viral infections, and inherited genetic disorders. However, the therapeutic applications of nucleic acids extend beyond the delivery of double-stranded DNA and subsequent expression of deficient gene products in diseased tissue. Other strategies include antisense oligonucleotides and most notably RNA interference (RNAi). Antisense strategies bear great potential for the treatment of diseases that are caused by misspliced mRNA, and RNAi is a universal and extraordinarily efficient tool to knock down the expression of virtually any gene by specific degradation of the desired target mRNA. However, because of the hurdles associated with effective delivery of nucleic acids across a cell membrane, the initial euphoria surrounding siRNA therapy soon subsided. The ability of oligonucleotides to cross the plasma membrane is hampered by their size and highly negative charge. Viral vectors have long been the gold standard to overcome this barrier, but they are associated with severe immunogenic effects and possible tumorigenesis. Cell-penetrating peptides (CPPs), cationic peptides that can translocate through the cell membrane independent of receptors and can transport cargo including proteins, small organic molecules, nanoparticles, and oligonucleotides, represent a promising class of nonviral delivery vectors. This Account focuses on peptide carrier systems for the cellular delivery of various types of therapeutic nucleic acids with a special emphasis on cell-penetrating peptides. We also emphasize the clinical relevance of this research through examples of promising in vivo studies. Although CPPs are often derived from naturally occurring protein transduction domains, they can also be artificially designed. Because CPPs typically include many positively charged amino acids, those electrostatic interactions facilitate the formation of complexes between the carriers and the oligonucleotides. One drawback of CPP-mediated delivery includes entrapment of the cargo in endosomes because uptake tends to be endocytic: coupling of fatty acids or endosome-disruptive peptides to the CPPs can overcome this problem. CPPs can also lack specificity for a single cell type, which can be addressed through the use of targeting moieties, such as peptide ligands that bind to specific receptors. Researchers have also applied these strategies to cationic carrier systems for nonviral oligonucleotide delivery, such as liposomes or polymers, but CPPs tend to be less cytotoxic than other delivery vehicles.

Effective small interfering RNA delivery in vitro via a new stearylated cationic peptide

“…Ideally, to target tumors, the diameter of CPPs-siRNA particles should not exceed 100-300 nm for efficient uptake. 30 In this report, the particle size of STR-HK-siRNA complexes at a molar ratio of .15/1 was in the range of 100-160 nm and fulfilled the criteria of size required for an efficient transfection agent. At a molar ratio of 10/1, STR-HK-siRNA complexes displayed a slightly negative surface charge (-13 mV), indicating the surface of the complex was not fully covered by positive charged peptide.…”

“…19,20,[30][31][32] However, siRNA therapeutics were limited by their low bioavailability due to their physicochemical properties (negative charges, large molecular weight, and size) and instability with plasma half-life of about 10 minutes, [33][34][35] immune stimulation when administered systemically, 36 and possible off-target effects due to partial nucleotide sequence match between the siRNA and off-target mRNA. 37 Seeking efficient delivery systems for these molecules is a prerequisite for successful gene therapy.…”

“…CPPs as one group of non-viral peptide-based delivery vectors have attracted more and more attention due to their high internalization efficiency, low cytotoxicity, and flexible structural design. 30,38,39 Recently, a handful of papers have shown that stearylation is a successful strategy to enhance the transfection efficiency of CPPs mostly for DNA delivery. Several delivery systems have incorporated this strategy for siRNA delivery.…”

Cell‐penetrating Peptides as a Novel Transdermal Drug Delivery System

“…Although viral vector delivery of oligonucleotides such as siRNA first appeared very promising in gene therapy of various diseases, its serious carcinogenic and immunogenic consequences casted doubt upon its therapeutic values. The positively charged or amphipathic CPPs are effective and less-toxic alternatives to the viral vectors for systemic and topical delivery of oligonucleotides such as siRNA (99)(100)(101). Recently, a Peptide Transduction Domain-ds (double-stranded) RNA Binding Domain (PTD-DRBD) fusion protein has been proposed for more efficient and non-cytotoxic siRNA delivery in variety of cellular models.…”

Reduction-responsive cross-linked stearyl peptide for effective delivery of plasmid DNA

“…10,11 It was reported that stearylation could improve intracellular uptake and endosomal escape. [12][13][14] A stearyl moiety could increase the buffering capacity of histidine, thus increasing the efficiency of transfection by the hydrophobic moiety of stearylation on the N-terminus of the peptide and facilitating absorption of the complex through the membrane. 15,16 Additionally, the disulfide bonds in C-SHR could be cleaved rapidly by thiol-disulfide exchange reactions with intracellular reducing molecules, especially with glutathione.…”