CPP-ZFN: A potential DNA-targeting anti-malarial drug

Abstract

BackgroundMultidrug-resistant Plasmodium is of major concern today. Effective vaccines or successful applications of RNAi-based strategies for the treatment of malaria are currently unavailable. An unexplored area in the field of malaria research is the development of DNA-targeting drugs that can specifically interact with parasitic DNA and introduce deleterious changes, leading to loss of vital genome function and parasite death.Presentation of the hypothesisAdvances in the development of zinc finger nuclease (ZFN) with engineered DNA recognition domains allow us to design and develop nuclease of high target sequence specificity with a mega recognition site that typically occurs only once in the genome. Moreover, cell-penetrating peptides (CPP) can cross the cell plasma membrane and deliver conjugated protein, nucleic acid, or any other cargo to the cytoplasm, nucleus, or mitochondria. This article proposes that a drug from the combination of the CPP and ZFN systems can effectively enter the intracellular parasite, introduce deleterious changes in its genome, and eliminate the parasite from the infected cells.Testing the hypothesisAvailability of a DNA-binding motif for more than 45 triplets and its modular nature, with freedom to change number of fingers in a ZFN, makes development of customized ZFN against diverse target DNA sequence of any gene feasible. Since the Plasmodium genome is highly AT rich, there is considerable sequence site diversity even for the structurally and functionally conserved enzymes between Plasmodium and humans. CPP can be used to deliver ZFN to the intracellular nucleus of the parasite. Signal-peptide-based heterologous protein translocation to Plasmodium-infected RBCs (iRBCs) and different Plasmodium organelles have been achieved. With successful fusion of CPP with mitochondrial- and nuclear-targeting peptides, fusion of CPP with 1 more Plasmodium cell membrane translocation peptide seems achievable.Implications of the hypothesisTargeting of the Plasmodium genome using ZFN has great potential for the development of anti-malarial drugs. It allows the development of a single drug against all malarial infections, including multidrug-resistant strains. Availability of multiple ZFN target sites in a single gene will provide alternative drug target sites to combat the development of resistance in the future.

Gene transport and expression by arginine-rich cell-penetrating peptides in Paramecium

“…This re-Tat-Tp2 fusion peptide possessed a superior ability to stimulate CD8 + T cell responses. A Plasmodium-targeting peptide containing the fusion of a signal peptide for infected red blood cell (RBC) penetration, a CPP, an organelle targeting peptide and a peptide of zinc finger nuclease (ZFN) with an engineered DNA recognition domain was used for the development of anti-malarial drugs (Nain et al, 2010). The Plasmodium-targeting peptide could cross the membrane barriers of infected RBC and Plasmodium, ZFN introduced a DNA break in the Plasmodium genome and finally parasite death.…”

“…Images were captured from the Fujifilm LAS-3000 luminescent image analyzer system. (Ryves and Harwood, 2006), Theileria parva (Tineg et al, 2009) and Plasmodium (Nain et al, 2010). A CPP, penetratin, was fused with the cAMP-dependent protein kinase inhibitor (PKI) peptide to form the PenPKI fusion peptide (Ryves and Harwood, 2006).…”

“…Recently, three arginine-rich CPPs (SR9, HR9 and PR9) were investigated in our group (Liu et al, 2011). However, limited study of CPPs was presented in the field of eukaryotic protista (Nain et al, 2010;Ryves and Harwood, 2006;Tineg et al, 2009).…”

Targeted genome engineering in human induced pluripotent stem cells by penetrating TALENs

“…The concept of CPP-ZFN proteins has been proposed previously [ 21 , 22 ]. However, we failed to utilize TAT-ZFN to modify the endogenous gene because of a purification problem.…”

Zinc finger nucleases: Tailor-made for gene therapy

“…Carried by a cell penetration peptide (CPP), the ZFN has been hypothesized to be delivered to infected red blood cells to target the Plasmodium genome (93). This approach may result in functional gene knockout in the parasite and lead to the death of Plasmodium if the delivery system is effective.…”