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Noncovalently Associated Cell Penetrating Peptides for Nonviral Gene Delivery
Alhakamy, Nabil A.
Alhakamy, Nabil A.
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Abstract
Gene therapy has become a promising strategy for treatment of numerous diseases such as cancer, hemophilia, and neurodegenerative diseases. Glybera® (alipogene tiparvovec) became the first gene therapy approved in the European Union, for the treatment of lipoprotein lipase deficiency (LPLD). Promising late-stage clinical trials of this drug may herald the first gene therapy to be approved in the United States. The advancement of vectors (viral and nonviral) for efficient and safe gene delivery has garnered significant attention recently. Although viral vectors (e.g., retroviruses and adenoviruses) are the most effective vectors, applied in 70% of gene therapy clinical trials, they present several notable challenges including safety concerns (e.g., immunogenicity and pathogenicity), production difficulties, and rapid clearance from circulation. In contrast, nonviral vectors could be promising gene carriers due to their low cost, ease of synthesis, and decreased immunogenicity relative to viral vectors. Key progress has been made in the development of several nonviral gene delivery vectors. The use of cell penetrating peptides (CPPs) to deliver genetic materials for gene therapy has been a topic of interest for more than 20 years. One strategy is through covalent conjugation of CPPs with genetic materials, which requires complex synthesis procedures. In contrast, electrostatic complexation of CPPs with genetic materials is relatively simple and has been demonstrated to improve gene delivery both in vitro and in vivo. Reported herein, a simple method to generate small CPP complexes (100-200 nm) capable of high transfection efficiency was explored. Positively charged CPPs (e.g., polyarginine 9 [R9] and polylysine 9 [K9]) were complexed with plasmid DNA (pDNA), which resulted in unstable large particles (~1 micron). These were then condensed into small nanoparticles using Ca2+. CPPs also displayed negligible cytotoxicity. These CPP-pDNA-Ca2+ complexes showed high transfection efficiency and low cytotoxicity in vitro (human and mouse cell lines) and in vivo (syngeneic mice). Thus, Ca2+-condensed CPP complexes emerged as simple, attractive candidates for future studies on nonviral gene delivery. Futhermore, the relationships between transfection efficiency and polyarginine molecular weight, polyarginine-pDNA charge ratios, and calcium concentrations were studied. Polyarginine 7 was significantly more effective than other polyarginines under most formulation conditions, suggesting a link between molecular weight and transfection efficiency. Furthermore, Polylysine 9 was complexed with angiotensin II type 2 receptor (AT2R) plasmid DNA (pAT2R). The polylysine 9 complexe (K9-pAT2R-Ca2+) showed high transfection efficiency and negligible in vitro cytotoxicity towards human and mouse cell lines. This complex demonstrated cancer-targeted gene delivery in vivo when administered via intravenous injection or intratracheal spray. A single administration of this complex markedly attenuated lung cancer growth. Mechanistic understanding of CPP-mediated membrane insertion and intracellular translocation of nonviral gene complexes would allow rational design of next-generation CPPs for gene delivery. To this aim, we employed zwitterionic and anionic phospholipid monolayers as models to mimic the membrane composition of the outer leaflet of cell plasma and intracellular vesicular membranes at relevant intracellular pH. Subsequently, we investigated the membrane insertion potential of CPPs and gene complexes (CPP-pDNA-Ca2+ complexes) into model membranes. The insertion potential of CPPs and complexes were recorded using a Langmuir monolayer approach that records peptides and complexes adsorption to model membranes. Results showed that small changes to amino acids and peptide sequences resulted in dramatically different insertion potentials and membrane reorganization. Lastly, the effect of CPP charge type, charge spacing, and hydrophobicity on transfection efficiency was investigated by replacing three residues of the polyarginine 9 with a hydrophilic residue (histidine) or hydrophobic residues (alanine, leucine, and tryptophan) at positions 3, 4, and 7. R9 and RW9 complexes appeared especially effective compared to other CPP complexes, whereas RH9, RA9, and RL9 complexes seemed to have moderate- to low-gene expression. Initially, this suggested CPPs with better membrane penetration yielded higher gene expression. After further exploration, we discovered the charge spacing of CPPs affected the ability of CPPs to complex with nucleic acids and this property correlated to gene expression levels. In conclusion, our complexes emeraged as simple, attractive candidates for further in vivo studies on nonviral gene delivery.
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Date
2016-08-31
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University of Kansas
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Keywords
Pharmaceutical sciences, calcium, cell penetrating peptides, DNA, gene delivery, polyarginine, polylysine