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Targeting Protein Translation, RNA Splicing, and Degradation by Morpholino-Based Conjugates in Plasmodium Falciparum

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Targeting Protein Translation, RNA Splicing, and Degradation by Morpholino-Based Conjugates in Plasmodium Falciparum Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum Aprajita Garga, Donna Wesolowskib, Dulce Alonsob,1, Kirk W. Deitschc, Choukri Ben Mamouna, and Sidney Altmanb,2 aDepartment of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520; bDepartment of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520; and cDepartment of Microbiology and Immunology, Weill Medical College of Cornell University, New York, NY 10065 Contributed by Sidney Altman, August 11, 2015 (sent for review May 27, 2015; reviewed by Ron Dzikowski and Rima Mcleod) Identification and genetic validation of new targets from available same targets, MO conjugates have also been used to inhibit RNA genome sequences are critical steps toward the development of splicing and initiation of protein translation (9, 17, 18). new potent and selective antimalarials. However, no methods are To enhance cellular uptake of morpholino oligomers and other currently available for large-scale functional analysis of the Plasmo- drug-like molecules, arginine-rich peptides and polyguanidino dium falciparum genome. Here we present evidence for successful dendrimers have been used (19–23). For morpholino-based anti- use of morpholino oligomers (MO) to mediate degradation of target microbial activity, two types of conjugates have been developed, mRNAs or to inhibit RNA splicing or translation of several genes of PPMOs and vivo morpholino oligomers (VMOs, octa-guanidinium P. falciparum involved in chloroquine transport, apicoplast biogen- dendrimer-conjugated MOs; Materials and Methods). PPMOs are esis, and phospholipid biosynthesis. Consistent with their role in the produced following conjugation of a specific MO to a cell-pen- parasite life cycle, down-regulation of these essential genes resulted etrating, arginine-rich peptide, whereas VMOs are synthesized in inhibition of parasite development. We show that a MO conju- as conjugates between a MO molecule and an octa-guanidinium PfCRT gate that targets the chloroquine-resistant transporter is head group (13, 17, 24). To date, VMOs have been used to down- effective against chloroquine-sensitive and -resistant parasites, regulate gene expression in human fibroblasts, mice, zebrafish, MICROBIOLOGY causes enlarged digestive vacuoles, and renders chloroquine-resis- Xenopus oocytes, Toxoplasma gondii, and other model organisms tant strains more sensitive to chloroquine. Similarly, we show that (20, 25–29). However, these conjugates have not yet been assessed a MO conjugate that targets the PfDXR involved in apicoplast bio- for their use in functional analysis of P. falciparum genes. genesis inhibits parasite growth and that this defect can be res- We report here the successful use of VMO or PPMO conju- cued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of gates designed to target translation, splicing, and degradation of the P. falciparum genome. target RNAs in P. falciparum. Using these conjugates, we have targeted the PfDXR, PfPMT, and PfCRT genes that play a critical malaria | intraerythrocytic development | peptide conjugated morpholino role in apicoplast biogenesis, membrane biosynthesis, and drug/ – oligomer | vivo morpholino oligomer | gene expression metabolite transport (30 32). We show that VMO (PfPMT, PfCRT) and PPMO (PfDXR) conjugates reduce endogenous levels of their target RNAs and inhibit parasite growth. PfCRT- f the ∼5,300 genes encoded by the Plasmodium falciparum Ogenome, only a small number of genes have been success- VMO was effective against drug-sensitive and -resistant strains fully targeted for genetic modification using available genetic tools. With the lack of RNAi technology in this parasite, forward Significance genetic approaches suitable for large-scale functional analysis are needed to validate possible drug targets and to gain a better Malaria remains a major public health issue worldwide and understanding of P. falciparum pathophysiology (1). Recent ef- world health organization estimates ∼198 million cases and forts aimed to develop such tools include the use of Piggy-Bac, ∼584,000 deaths in the year of 2013 alone due to malaria. peptide-conjugated morpholino oligomers (PPMO), zinc-finger Lack of an effective vaccine and rapid emergence of drug re- nucleases, glmS ribozyme, CRISPR-Cas9–mediated genome sistance are two major causes of this persistent problem. Un- editing, peptide nucleic acids, and the Tet-R aptamer system (2–8). derstanding the biology of the parasite and studies of its Each of these methods requires further development and op- gene function are essential to identify potential drug targets. timization to be used in a large-scale format to assess the Here we report a morpholino oligomer (MO)-based approach function of P. falciparum genes. Morpholino oligomers (MO) to alter gene expression via inhibition of post-transcriptional have identical Watson–Crick base-pair characteristics as DNA or processes or by targeting mRNAs for degradation. The ease in RNA. They are resistant to degradation by nucleases due to the design of the MO molecules presents a possibility for their use presence of a morpholine ring and have no charge because of the in large-scale genome functional analyses and possibly in phosphorodiamidate bond in the backbone. The MO-based malaria therapy. RNA-targeting approach has been shown to be an excellent al- ternative to RNAi as morpholinos can bind to RNA with high Author contributions: A.G. and C.B.M. designed research; A.G., D.W., and K.W.D. per- formed research; D.W., D.A., and S.A. contributed new reagents/analytic tools; A.G., specificity (9). The sequence of the MO thereby decides the fate C.B.M., and S.A. analyzed data; and A.G., D.W., C.B.M., and S.A. wrote the paper. of the endogenous transcript. For example, MOs with External Reviewers: R.D., Hebrew University–Hadassah Medical School; and R.M., University of Guide Sequence (EGS) conjugated to peptides (PPMOs) have Chicago. been designed to target essential genes of several pathogenic The authors declare no conflict of interest. – bacteria and have displayed strong antibacterial activity (10 13). 1Present address: Department of Molecular Biosciences, Northwestern University, Evanston, Similarly, PPMOs targeting the P. falciparum PfGyrA and PfPAT IL 60208. RNAs for RNase P-mediated cleavage inhibit parasite growth in 2To whom correspondence should be addressed. Email: [email protected]. – the low micromolar range (4, 14 16). Because binding of MOs to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. their target RNAs can prevent binding of other molecules to the 1073/pnas.1515864112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1515864112 PNAS | September 22, 2015 | vol. 112 | no. 38 | 11935–11940 Downloaded by guest on October 1, 2021 growth. As expected, no differences in growth could be detected after one or two cycles between Luc-VMO– and Ctrl-VMO– treated parasites (see Fig. S1 A and B). However, treatment with 2 μM Luc-VMO resulted in a 17 and 30% reduction in luciferase activity compared with Ctrl-VMO after one and two cycles, re- spectively (Fig. 1D). Higher concentrations of VMO conjugates altered parasite growth most likely due to the inherent toxicity of the targeting dendrimer as has been previously reported in mice (Fig. S1B) (34). Gene-Specific Transcript Expression Is Reduced After VMO Treatment. To assess the possible use of VMO conjugates in down-regula- tion of P. falciparum gene expression, two VMO conjugates were designed to target splicing of PfPMT and PfCRT RNAs, encod- ing the phosphoethanolamine methyltransferase and digestive vacuole transporter of the parasite, respectively (35, 36). The sequences of PfCRT and PfPMT VMOs are shown in Table 1. These VMOs were designed to bind to the first exon–intron junction in each gene’s pre-mRNA to prevent splicing, resulting in accumulation of unspliced RNAs. Parameters for morpholino design were similar to those described for Luc-VMO. Both VMOs were conjugated to a dendritic molecular transporter with guanidine headgroups to facilitate delivery. The possible out- comes of the VMO treatment on the parasite endogenous transcript are illustrated in Fig. 1 B and C. PCR analyses using primers specific to the first intron region were performed to compare levels of unspliced transcripts between control and treated parasites (Fig. 1 B and C). Levels of steady-state mRNA were determined by using primer pairs specific to exon regions. A highly synchronized culture of P. falciparum was treated with Fig. 1. Inhibition of translation and splicing using VMOs (A–C) Schematic the VMOs for 6 h after which total RNA was isolated for cDNA representation of the binding sites of luciferase-VMO. (A), PfPMT-VMO (B), preparation. Real-time PCR analysis showed 24 and 60% re- and PfCRT-VMO (C) on their respective sites on the mRNA or pre-mRNA. Arrows indicate the sites and orientation of the primers used for qPCR. cDNA duction in PfPMT and PfCRT steady-state mRNA levels, re- was made from VMO-treated 3D7 parasites. (D) Luciferase-expressing par- spectively (Fig. 1 E and G), whereas the levels of unspliced asites were treated with 1 or 2 μM Ctrl-VMO and luciferase-VMO; 72 h (one transcripts (Fig. 1 F and H) were 38% higher in both PfPMT- cycle) and 96 h (two cycles) later parasites lysates were used for luciferase VMO or PfCRT-VMO–treated parasites compared with para- assay. Luciferase activity is plotted after normalization to Ctrl-VMO. (E and G) sites treated with Ctrl-VMO. The observed decrease in the qPCR studies with primers 2F and 2R to amplify PfPMT or PfCRT steady-state steady-state mRNA levels could be due to nonsense-mediated transcripts. (F and H) qPCR analyses carried out using primers 1F and 1R, which mRNA decay of mis-spliced transcripts (37). PCR amplification amplify PfPMT or PfCRT unspliced transcripts. The results represent three in- – dependent experiments, and the error bars indicate SE of mean.
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