Delivery Of Antisense Oligonucleotides Research Articles

Systemic delivery of microRNA-21 antisense oligonucleotides to the brain using T7-peptide decorated exosomes

Antisense miRNA oligonucleotides against miR-21 (AMO-21) have a therapeutic potential for treatment of glioblastoma. However, glioblastoma-targeted delivery through systemic injection requires development of an efficient targeting carrier. For this purpose, a glioblastoma-targeting carrier was developed using the T7 peptide and exosomes. The transferrin receptor is overexpressed on the surface of glioblastoma cells, and T7 is a transferrin receptor-binding peptide. A T7 peptide-decorated exosome (T7-exo) was produced by incorporation of T7 into the exosome membrane as a fusion protein of T7 and Lamp2b. As a control, rabies virus glycoprotein (RVG) peptide targeting brain neuron cells was incorporated into the exosome membrane. AMO-21 was loaded into the exosomes by electroporation. In vitro studies of AMO-21 delivery showed that T7-exo had a higher delivery efficiency to C6 glioblastoma cells than unmodified exosome (Unmod-exo) and RVG-decorated exosome (RVG-exo). For in vivo delivery studies, T7-exo with AMO-21 was delivered into intracranial glioblastoma rat models by intravenous injection through the tail vein. The results showed that T7-exo delivered AMO-21 into the brain more efficiently than Unmod-exo and RVG-exo. In addition, delivery of AMO-21 using T7-exo reduced the miR-21 level in the glioblastoma efficiently. Reduction of miR-21 by AMO-21 induced the expression of PDCD4 and PTEN in tumors, resulting in reduction of tumor sizes. Taken together, these findings indicate that T7-exo is an efficient carrier of AMO-21 into the glioblastoma and may be useful in development of glioblastoma therapy.

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System.

The blood brain barrier (BBB) is an important defense against the entrance of potentially toxic or pathogenic agents from the blood into the central nervous system (CNS). However, its existence also dramatically lowers the accessibility of systemically administered therapeutic agents to the CNS. One method to overcome this, is to inject those agents directly into the cerebrospinal fluid (CSF), thus bypassing the BBB. This can be done via implantation of a catheter for either continuous infusion using an osmotic pump, or for single bolus delivery. In this article, we describe a surgical protocol for delivery of CNS-targeting antisense oligonucleotides (ASOs) via a catheter implanted directly into the cauda equina space of the adult rat spine. As representative results, we show the efficacy of a single bolus ASO intrathecal (IT) injection via this catheterization system in knocking down the target RNA in different regions of the rat CNS. The procedure is safe, effective and does not require expensive equipment or surgical tools. The technique described here can be adapted to deliver drugs in other modalities as well.

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

Intrathecal Delivery of Antisense Oligonucleotides in the Rat Central Nervous System

Brain pharmacology of intrathecal antisense oligonucleotides revealed through multimodal imaging.

Intrathecal (IT) delivery and pharmacology of antisense oligonucleotides (ASOs) for the CNS have been successfully developed to treat spinal muscular atrophy. However, ASO pharmacokinetic (PK) and pharmacodynamic (PD) properties remain poorly understood in the IT compartment. We applied multimodal imaging techniques to elucidate the IT PK and PD of unlabeled, radioactively labeled, or fluorescently labeled ASOs targeting ubiquitously expressed or neuron-specific RNAs. Following lumbar IT bolus injection in rats, all ASOs spread rostrally along the neuraxis, adhered to meninges, and were partially cleared to peripheral lymph nodes and kidneys. Rapid association with the pia and arterial walls preceded passage of ASOs across the glia limitans, along arterial intramural basement membranes, and along white-matter axonal bundles. Several neuronal and glial cell types accumulated ASOs over time, with evidence of probable glial accumulation preceding neuronal uptake. IT doses of anti-GluR1 and anti-Gabra1 ASOs markedly reduced the mRNA and protein levels of their respective neurotransmitter receptor protein targets by 2 weeks and anti-Gabra1 ASOs also reduced binding of the GABAA receptor PET ligand 18F-flumazenil in the brain over 4 weeks. Our multimodal imaging approaches elucidate multiple transport routes underlying the CNS distribution, clearance, and efficacy of IT-dosed ASOs.

Efficient Delivery of Antisense Oligonucleotides by an Amphipathic Cell-Penetrating Peptide in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii (A. baumannii) was on the top of the list of the most threatening bacteria published by the WHO in 2017. Antisense oligonucleotides (ASOs) based therapy is a promising strategy for combating Multi-Drug Resistant (MDR) bacteria because of its high specificity, easy design and lower induction of resistance, but poor cellular uptake by bacteria has restricted the further utilization of this therapy. Here, we used CADY, a secondary amphipathic peptide of 20 residues that could successfully carry siRNA into mammalian cells, to prepare CADY/ASOs nanoparticles (CADY-NPs) targeting acpP (encoding acyl carrier protein), and evaluated the uptake features, the inhibitory effects of CADY-NPs on gene expression and the growth of MDR-A. baumannii. We found that CADY-NPs could be quickly internalized by drug-sensitive and MDR-A. baumannii in an energy independent manner, which could be restrained by chlorpromazine (an inhibitor of clathrin mediated endocytosis) significantly. In addition, CADY-NPs targeting acpP concentrationdependently retarded the growth of MDR-A. baumannii, which was associated with the decreased expression of targeted genes in A. baumannii. In conclusion, our research is the first to demonstrate that CADY can deliver ASOs into bacteria and provide a novel strategy for the treatment of MDR-A. baumannii.

Ethylcellulose nanoparticles as a new “in vitro” transfection tool for antisense oligonucleotide delivery

Oil-in-water nano-emulsions have been obtained in the HEPES 20 mM buffer solution / [Alkylamidoammonium:Kolliphor EL = 1:1] / [6 wt% ethylcellulose in ethyl acetate] system over a wide oil-to-surfactant range and above 35 wt% aqueous component at 25 °C. The nano-emulsion with an oil-to-surfactant ratio of 70/30 and 95 wt% aqueous component was used for nanoparticles preparation. These nanoparticles (mean diameter around 90 nm and zeta potential of +22 mV) were non-toxic to HeLa cells up to a concentration of 3 mM of cationic species. Successful complexation with an antisense phosphorothioate oligonucleotide targeting Renilla luciferase mRNA was achieved at cationic/anionic charge ratios above 16, as confirmed by zeta potential measurements and an electrophoretic mobility shift assay, provided that no Fetal Bovine Serum is present in the cell culture medium. Importantly, Renilla luciferase gene inhibition shows an optimum efficiency (40%) for the cationic/anionic ratio 28, which makes these complexes promising for “in vitro” cell transfection.

Peptide-conjugated oligonucleotides evoke long-lasting myotonic dystrophy correction in patient-derived cells and mice.

Antisense oligonucleotides (ASOs) targeting pathologic RNAs have shown promising therapeutic corrections for many genetic diseases including myotonic dystrophy (DM1). Thus, ASO strategies for DM1 can abolish the toxic RNA gain-of-function mechanism caused by nucleus-retained mutant DMPK (DM1 protein kinase) transcripts containing CUG expansions (CUGexps). However, systemic use of ASOs for this muscular disease remains challenging due to poor drug distribution to skeletal muscle. To overcome this limitation, we test an arginine-rich Pip6a cell-penetrating peptide and show that Pip6a-conjugated morpholino phosphorodiamidate oligomer (PMO) dramatically enhanced ASO delivery into striated muscles of DM1 mice following systemic administration in comparison with unconjugated PMO and other ASO strategies. Thus, low-dose treatment with Pip6a-PMO-CAG targeting pathologic expansions is sufficient to reverse both splicing defects and myotonia in DM1 mice and normalizes the overall disease transcriptome. Moreover, treated DM1 patient–derived muscle cells showed that Pip6a-PMO-CAG specifically targets mutant CUGexp-DMPK transcripts to abrogate the detrimental sequestration of MBNL1 splicing factor by nuclear RNA foci and consequently MBNL1 functional loss, responsible for splicing defects and muscle dysfunction. Our results demonstrate that Pip6a-PMO-CAG induces long-lasting correction with high efficacy of DM1-associated phenotypes at both molecular and functional levels, and strongly support the use of advanced peptide conjugates for systemic corrective therapy in DM1.

Targeting RyR Activity Boosts Antisense Exon 44 and 45 Skipping in Human DMD Skeletal or Cardiac Muscle Culture Models

Systemic delivery of antisense oligonucleotides (AO) for DMD exon skipping has proven effective for reframing DMD mRNA, rescuing dystrophin expression, and slowing disease progression in animal models. In humans with Duchenne muscular dystrophy treated with AOs, low levels of dystrophin have been induced, and modest slowing of disease progression has been observed, highlighting the need for improved efficiency of human skipping drugs. Here, we demonstrate that dantrolene and Rycals S107 and ARM210 potentiate AO-mediated exon skipping of exon 44 or exon 45 in patient-derived myotube cultures with appropriate mutations. Further, dantrolene is shown to boost AO-mediated exon skipping in patient-derived, induced cardiomyocyte cultures. Our findings further validate the ryanodine receptors (RyR) as the likely target responsible for exon skip boosting and demonstrate potential applicability beyond exon 51 skipping. These data provide preclinical support of dantrolene trial as an adjuvant to AO-mediated exon-skipping therapy in humans and identify a novel Rycal, ARM210, for development as a potential exon-skipping booster. Further, they highlight the value of mutation-specific DMD culture models for basic discovery, preclinical drug screening and translation of personalized genetic medicines.

Gene Therapy for ALS-A Perspective.

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease (MND) with no cure. Recent advances in gene therapy open a new perspective to treat this disorder—particularly for the characterized genetic forms. Gene therapy approaches, involving the delivery of antisense oligonucleotides into the central nervous system (CNS) are being tested in clinical trials for patients with mutations in SOD1 or C9orf72 genes. Viral vectors can be used to deliver therapeutic sequences to stably transduce motor neurons in the CNS. Vectors derived from adeno-associated virus (AAV), can efficiently target genes and have been tested in several pre-clinical settings with promising outcomes. Recently, the Food and Drug Administration (FDA) approved Zolgensma, an AAV-mediated treatment for another MND—the infant form of spinal muscular atrophy. Given the accelerated progress in gene therapy, it is potentially a promising avenue to develop an efficient and safe cure for ALS.

I-Motif-Based in Situ Bipedal Hybridization Chain Reaction for Specific Activatable Imaging and Enhanced Delivery of Antisense Oligonucleotides.

The efficient and precise delivery of antisense oligonucleotides (ASOs) to target cells is of great value in gene silencing. However, the specificity and packaging capacity of delivery system still remains challenging. Here, we designed an i-motif forming-initiated in situ bipedal hybridization chain reaction (pH-Apt-BiHCR) amplification strategy for specific target cells imaging and enhanced gene delivery of ASOs. As a proof of concept, an 8-nt ASO modified with locked nucleic acid (LNA) which is complementary to the seed region of microRNA21 (miR-21) was used for gene silencing studies. Benefiting from the design of hairpin-contained i-motif, the stimuli-responsive assembly of pH-Apt-BiHCR was successfully achieved on MCF-7 cells surface based on the specific recognition of aptamer. Using this strategy, the pH-Apt-BiHCR not only contains repeated fluorescence resonance energy transfer (FRET) units for activatable tumor imaging with high contrast but also arrays with plenty of LNA ASOs as interference molecules for cancer cells inhibition. An in vitro assay showed that this strategy presented an excellent response ability in buffer within a narrow pH range (6.0-7.0) with a transition midpoint (pHT) of 6.44 ± 0.06. Moreover, live cell studies revealed that it realized a specific activatable imaging of target cells, while the ASOs arrayed pH-Apt-BiHCR exhibited improved internalization via an endocytosis pathway and enhanced gene silencing to MCF-7 cells compared to single ASO alone. We believe that this design will inspire the development of novel probes for early diagnosis and therapy of cancer cells.

Targeted mitochondrial delivery of antisense RNA-containing nanoparticles by a MITO-Porter for safe and efficient mitochondrial gene silencing

Mitochondrial gene therapy will be needed to treat mitochondrial diseases. We previously demonstrated mitochondrial gene silencing by the mitochondrial delivery of antisense RNA oligonucleotide (ASO) targeting mtDNA-encoded mRNA using a MITO-Porter, a liposomal nano carrier system designed for mitochondrial delivery. Here, we report on the efficient packaging of ASO in the MITO-Porter via a nanoparticle packaging method, which showed a 10-fold higher packaging efficiency than the conventional method. The constructed carrier showed a decrease in the target mRNA levels and ATP production. These results indicate that such a MITO-Porter has potential for use in therapies designed to regulate mitochondrial function.

Dually Stabilized Triblock Copolymer Micelles with Hydrophilic Shell and Hydrophobic Interlayer for Systemic Antisense Oligonucleotide Delivery to Solid Tumor

For intravenous delivery of antisense oligonucleotides (ASOs) to solid tumors, a triblock copolymer was synthesized from poly(2-ethyl-2-oxazoline) (PEtOx), poly(2-n-propyl-2-oxazoline) (PnPrOx), and poly(l-lysine) (PLL) segments. The triblock copolymer, PEtOx-PnPrOx-PLL, was utilized to fabricate a compartmentalized polymeric micelle featuring a hydrophilic PEtOx shell, thermoresponsive PnPrOx interlayer, and ASO/PLL polyion complex (PIC) core. In this formulation, the PnPrOx-derived interlayer underwent the phase transition from hydrophilic elongated state to hydrophobic collapsed state at a lower critical solution temperature (LCST) to enhance the micelle stability. Three triblock copolymers comprising varying lengths of PEtOx segment (2k, 7k, and 12 kDa) were compared to investigate the effect of hydrophilic chain length on the micelle properties. The triblock copolymer micelles (TCMs) were prepared in a two-step manner: mixing between triblock copolymer and ASO in a buffer solution at 4 °C and then increasing the temperature of the solution up to 37 °C. This protocol was crucial for the fabrication of TCMs with both smaller size and narrower size distribution, probably due to the formation of the well-compartmentalized hydrophobic interlayer in the micelle structure. The presence of the PnPrOx segment dramatically enhanced the stability of TCMs in serum-containing media and elicited more efficient cellular uptake of ASO payloads, resulting in higher gene silencing efficiency in cultured prostate cancer (PC-3) cells, compared with a control diblock copolymer micelle (DCM). The blood circulation property of TCMs was prolonged with an increase in the length of PEtOx segment, permitting the efficient accumulation of ASO payloads in a subcutaneous PC-3 tumor model. Ultimately, the systemic delivery of ASO targeting a long noncoding RNA (lncRNA) by the TCMs significantly reduced the expression level of lncRNA in the subcutaneous PC-3 tumor in a sequence-specific manner. These results demonstrate the superiority of TCMs equipped with the hydrophilic shell and hydrophobic interlayer to the cancer-targeted systemic ASO delivery.

Specific Delivery of Oligonucleotides to the Cell Nucleus via Gentle Compression and Attachment of Polythymidine.

Nonviral delivery of nucleic acids to the cell nucleus typically requires chemical methods that do not guarantee specific delivery (e.g., transfection agent) or physical methods that may require extensive fabrication (e.g., microfluidics) or an elevated pressure (e.g., 105 Pa for microneedles). We report a method of delivering oligonucleotides to the nucleus with high specificity (relative to the cytosol) by synergistically combining chemical and physical approaches. Particularly, we demonstrate that DNA oligonucleotides appended with a polythymidine [poly(T)] segment (chemical) profusely accumulate inside the nucleus when the cells are under gentle compression imposed by the weight of a single glass coverslip (physical; ∼2.2 Pa). Our "compression-cum-poly(T)" delivery method is simple, can be generalizable to three "hard-to-transfect" cell types, and does not induce significant levels of cytotoxicity or long-termoxidative stress to the treated cells when provided the use of suitable compression times and oligonucleotide concentrations. In bEnd.3 endothelial cells, compression-aided intranuclear delivery of poly(T) is primarily mediated by importin β and nucleoporin 62. Our method significantly enhances the intranuclear delivery of antisense oligonucleotides to bEnd.3 endotheliomacells and the inhibition of two target genes, including a reporter gene encoding the enhanced green fluorescent protein and an intranuclear lncRNA oncogene (metastasis-associated lung adenocarcinoma transcript 1), when compared with delivery without gentle compression or poly(T) attachment. Our data underscore the critical roles of pressure and nucleotide sequence on the intranuclear delivery of nucleic acids.

Enhanced Potency of GalNAc-Conjugated Antisense Oligonucleotides in Hepatocellular Cancer Models.

Antisense oligonucleotides (ASOs) are a novel therapeutic approach to target difficult-to-drug protein classes by targeting their corresponding mRNAs. Significantly enhanced ASO activity has been achieved by the targeted delivery of ASOs to selected tissues. One example is the targeted delivery of ASOs to hepatocytes, achieved with N-acetylgalactosamine (GalNAc) conjugation to ASO, which results in selective uptake by asialoglycoprotein receptor (ASGR). Here we have evaluated the potential of GalNAc-conjugated ASOs as a therapeutic approach to targeting difficult-to-drug pathways in hepatocellular carcinoma (HCC). The activity of GalNAc-conjugated ASOs was superior to that of the unconjugated parental ASO in ASGR (+) human HCC cells in vitro, but not in ASGR (−) cells. Both human- and mouse-derived HCC displayed reduced levels of ASGR, however, despite this, GalNAc-conjugated ASOs showed a 5- to 10-fold increase in potency in tumors. Systemically administered GalNAc-conjugated ASOs demonstrated both enhanced antisense activity and antitumor activity in the diethylnitrosamine-induced HCC tumor model. Finally, GalNAc conjugation enhanced ASO activity in human circulating tumor cells from HCC patients, demonstrating the potential of this approach in primary human HCC tumor cells. Taken together, these results provide a strong rationale for a potential therapeutic use of GalNAc-conjugated ASOs for the treatment of HCC.

Pharmacokinetics and Clinical Pharmacology Considerations of GalNAc3-Conjugated Antisense Oligonucleotides

ABSTRACTIntroduction: Triantennary N-acetyl galactosamine (GalNAc3) – conjugated antisense oligonucleotides (ASOs) have demonstrated improved hepatocyte uptake and pharmacologic activity over their parent unconjugated ASOs in animals and humans.Areas covered: In this review, the ADME (absorption, distribution, metabolism, and excretion) characteristics of GalNAc3-conjugated ASOs in animals and in humans are summarized, and their clinical relevance is evaluated from the clinical pharmacology perspectives.Expert opinion: ASOs distribute to tissues via receptor-mediated processes, and conjugation to a ligand specific to certain cell types can improve target tissue delivery. GalNAc3-conjugation represents a good example on this regard and has demonstrated ideal characteristics of a prodrug to target delivery of ASOs to hepatocytes via the asialoglycoprotein receptor (ASGPR). The improved potency and safety margin permit more flexible dosing (e.g. monthly or less frequently if needed) taking full advantage of the long half-life of the parent ASO in humans. However, while still speculative, it should be noted that ASGPR-mediated uptake could become nonlinear with dose and factors that impact ASGPR expression or compete with ASGPR-mediated uptake could potentially affect the uptake of GalNAc3-conjugated ASOs, further studies are warranted.

Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle.

Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma Cmax and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders.

Delivery of Antisense Oligonucleotide LOR-2501 Using Transferrin-conjugated Polyethylenimine-based Lipid Nanoparticle.

Efficient delivery of antisense oligonucleotide (ASO) by nanoparticle vectors is critical for its clinical application. The aim of this study was to design and evaluate a novel ASO vector TPSH-LP consisting of a reduction-sensitive cationic polymer PEI-SS-HA (PSH), lipids and transferrin (Tf) as a targeting ligand. PSH was synthesized based on PEI 25 kDa. Nanoparticles containing PSH and Tf (TPSH-LP) were prepared and used to deliver an ASO LOR-2501 targeting ribonucleotide reductase R1. The physical and chemical properties of TPSH-LP and cellular uptake in HepG2 cells were studied. TPSH-LP formed a complex with LOR-2501 (L-TPSH-LP) which showed suitable particle size (267.77±16.20 nm) and zeta potential (4.87±0.52 mV). TPSH-LP showed lower cytotoxicity and higher transfection efficiency than PEI 25 kDa in HepG2 cells. The addition of Tf enhanced the targeting and delivery efficiency of PSH-LP. TPSH-LP transported LOR-2501 and down-regulated the levels of R1 protein efficiently by 64.15%. Data demonstrated the potential utility of TPSH-LP for oligonucleotide delivery in cancer therapy.

Antisense Oligonucleotides for the Treatment of Inner Ear Dysfunction.

Antisense oligonucleotides (ASOs) have shown potential as therapeutic molecules for the treatment of inner ear dysfunction. The peripheral sensory organs responsible for both hearing and equilibrium are housed within the inner ear. Hearing loss and vestibular balance problems affect a large portion of the population and limited treatment options exist. Targeting ASOs to the inner ear as a therapeutic strategy has unique pharmacokinetic and drug delivery opportunities and challenges. Here, we review ASO technology, delivery, disease targets, and other key considerations for development of this therapeutic approach.

Delivery of GalNAc-Conjugated Splice-Switching ASOs to Non-hepatic Cells through Ectopic Expression of Asialoglycoprotein Receptor

Splice-switching antisense oligonucleotides (ASOs) are promising therapeutic tools to target various genetic diseases, including cancer. However, in vivo delivery of ASOs to orthotopic tumors in cancer mouse models or to certain target tissues remains challenging. A viable solution already in use is receptor-mediated uptake of ASOs via tissue-specific receptors. For example, the asialoglycoprotein receptor (ASGP-R) is exclusively expressed in hepatocytes. Triantennary N-acetylgalactosamine (GalNAc) (GN3)-conjugated ASOs bind to the receptor and are efficiently internalized by endocytosis, enhancing ASO potency in the liver. Here we explore the use of GalNAc-mediated targeting to deliver therapeutic splice-switching ASOs to cancer cells that ectopically express ASGP-R, both in vitro and in tumor mouse models. We found that ectopic expression of the major isoform ASGP-R1 H1a is sufficient to promote uptake and increase GN3-ASO potency to various degrees in four of five tested cancer cells. We show that cell-type-specific glycosylation of the receptor does not affect its activity. In vivo, GN3-conjugated ASOs specifically target subcutaneous xenograft tumors that ectopically express ASGP-R1, and modulate splicing significantly more strongly than unconjugated ASOs. Our work shows that GN3-targeting is a useful tool for proof-of-principle studies in orthotopic cancer models, until endogenous receptors are identified and exploited for efficiently targeting cancer cells.

The Triticum Mosaic Virus Internal Ribosome Entry Site Relies on a Picornavirus-Like YX-AUG Motif To Designate the Preferred Translation Initiation Site and To Likely Target the 18S rRNA.

Several viruses encode an internal ribosome entry site (IRES) at the 5' end of their RNA, which, unlike most cellular mRNAs, initiates translation in the absence of a 5' m7GpppG cap. Here, we report a uniquely regulated translation enhancer found in the 739-nucelotide (nt) sequence of the Triticum mosaic virus (TriMV) leader sequence that distinguishes the preferred initiation site from a plethora of IRES-encoded AUG triplets. Through deletion mutations of the TriMV 5' untranslated region (UTR), we show that the TriMV 5' UTR encodes a cis-acting picornaviral Y16-X11-AUG-like motif with a 16-nt polypyrimidine CU-tract (Y16), at a precise, 11-nt distance (X11) from the preferred 13th AUG. Phylogenetic analyses indicate that this motif is conserved among potyviral leader sequences with multiple AUGs. Consistent with a broadly conserved mechanism, the motif could be functionally replaced with known picornavirus YX-AUG motifs and is predicted to function as target sites for the 18S rRNA by direct base pairing. Accordingly, mutations that disrupted overall complementarity to the 18S rRNA markedly reduced TriMV IRES activity, as did the delivery of antisense oligonucleotides designed to block YX-AUG accessibility. To our knowledge, this is the first report of a plant viral IRES YX-AUG motif, and our findings suggest that a conserved mechanism regulates translation for multiple economically important plant and animal positive single-stranded RNA viruses.IMPORTANCE Uncapped viral RNAs often rely on their 5' leader sequences to initiate translation, and the Triticum mosaic virus (TriMV) devotes an astonishing 7% of its genome to directing ribosomes to the correct AUG. Here we uncover a novel mechanism by which a TriMV cis-regulatory element controls cap-independent translation. The upstream region of the functional AUG contains a 16-nt polypyrimidine tract located 11 nt from the initiation site. Based on functional redundancy with similar motifs derived from human picornaviruses, the motif is likely to operate by directing ribosome targeting through base pairing with 18S rRNA. Our results provide the first report of a broad-spectrum mechanism regulating translation initiation for both plant- and animal-hosted picornaviruses.