Rafal Goraczniak

The regulatory element in the 3'-untranslated region of human papillomavirus 16 inhibits expression by binding CUG-binding protein 1.

The 3′-untranslated regions (UTRs) of human papillomavirus 16 (HPV16) and bovine papillomavirus 1 (BPV1) contain a negative regulatory element (NRE) that inhibits viral late gene expression. The BPV1 NRE consists of a single 9-nucleotide (nt) U1 small nuclear ribonucleoprotein (snRNP) base pairing site (herein called a U1 binding site) that via U1 snRNP binding leads to inhibition of the late poly(A) site. The 79-nt HPV16 NRE is far more complicated, consisting of 4 overlapping very weak U1 binding sites followed by a poorly understood GU-rich element (GRE). We undertook a molecular dissection of the HPV16 GRE and identify via UV cross-linking, RNA affinity chromatography, and mass spectrometry that is bound by the CUG-binding protein 1 (CUGBP1). Reporter assays coupled with knocking down CUGBP1 levels by small interfering RNA and Dox-regulated shRNA, demonstrate CUGBP1 is inhibitory in vivo. CUGBP1 is the first GRE-binding protein to have RNA interfering knockdown evidence in support of its role in vivo. Several fine-scale GRE mutations that inactivate GRE activity in vivo and GRE binding to CUGBP1 in vitro are identified. The CUGBP1·GRE complex has no activity on its own but specifically synergizes with weak U1 binding sites to inhibit expression in vivo. No synergy is seen if the U1 binding sites are made weaker by a 1-nt down-mutation or made stronger by a 1-nt up-mutation, underscoring that the GRE operates only on weak sites. Interestingly, inhibition occurs at multiple levels, in particular at the level of poly(A) site activity, nuclear-cytoplasmic export, and translation of the mRNA. Implications for understanding the HPV16 life cycle are discussed.

Ectopic 5′ Splice Sites Inhibit Gene Expression by Engaging RNA Surveillance and Silencing Pathways in Plants

The quality control of mRNA maturation is a highly regulated process that surveys pre-mRNA integrity and eliminates improperly matured pre-mRNAs. In nature, certain viruses regulate the expression of their genes by hijacking the endogenous RNA quality control machinery. We demonstrate that the inclusion of 5′ splice sites within the 3′-untranslated region of a reporter gene in plants alters the pre-mRNA cleavage and polyadenylation process, resulting in pre-mRNA degradation, exemplifying a regulatory mechanism conserved between kingdoms. Altered pre-mRNA processing was associated with an inhibition of homologous gene expression in trans and the preferential accumulation of 24-nucleotide (nt) short-interfering RNAs (siRNAs) as opposed to 21-nt siRNA subspecies, suggesting that degradation of the aberrant pre-mRNA involves the silencing machinery. However, gene expression was not restored by coexpression of a silencing suppressor or in an RNA-dependent RNA polymerase (RDR6)-deficient background despite reduced 24-nt siRNA accumulation. Our data highlight a complex cross talk between the quality control RNA machinery, 3′-end pre-mRNA maturation, and RNA-silencing pathways capable of discriminating among different types of aberrant RNAs.

U1 Adaptor Oligonucleotides Targeting BCL2 and GRM1 Suppress Growth of Human Melanoma Xenografts In Vivo

U1 Adaptor is a recently discovered oligonucleotide-based gene-silencing technology with a unique mechanism of action that targets nuclear pre-mRNA processing. U1 Adaptors have two distinct functional domains, both of which must be present on the same oligonucleotide to exert their gene-silencing function. Here, we present the first in vivo use of U1 Adaptors by targeting two different human genes implicated in melanomagenesis, B-cell lymphoma 2 (BCL2) and metabotropic glutamate receptor 1 (GRM1), in a human melanoma cell xenograft mouse model system. Using a newly developed dendrimer delivery system, anti-BCL2 U1 Adaptors were very potent and suppressed tumor growth at doses as low as 34 µg/kg with twice weekly intravenous (iv) administration. Anti-GRM1 U1 Adaptors suppressed tumor xenograft growth with similar potency. Mechanism of action was demonstrated by showing target gene suppression in tumors and by observing that negative control U1 Adaptors with just one functional domain show no tumor suppression activity. The anti-BCL2 and anti-GRM1 treatments were equally effective against cell lines harboring either wild-type or a mutant V600E B-RAF allele, the most common mutation in melanoma. Treatment of normal immune-competent mice (C57BL6) indicated no organ toxicity or immune stimulation. These proof-of-concept studies represent an in-depth (over 800 mice in ~108 treatment groups) validation that U1 Adaptors are a highly potent gene-silencing therapeutic and open the way for their further development to treat other human diseases.

U1 Adaptors suppress the KRAS-MYC oncogenic axis in human pancreatic cancer xenografts

Targeting KRAS and MYC has been a tremendous challenge in cancer drug development. Genetic studies in mouse models have validated the efficacy of silencing expression of both KRAS and MYC in mutant KRAS-driven tumors. We investigated the therapeutic potential of a new oligonucleotide-mediated gene silencing technology (U1 Adaptor) targeting KRAS and MYC in pancreatic cancer. Nanoparticles in complex with anti-KRAS U1 Adaptors (U1-KRAS) showed remarkable inhibition of KRAS in different human pancreatic cancer cell lines in vitro and in vivo. As a nanoparticle-free approach is far easier to develop into a drug, we refined the formulation of U1 Adaptors by conjugating them to tumor-targeting peptides (iRGD and cRGD). Peptides coupled to fluorescently tagged U1 Adaptors showed selective tumor localization in vivo. Efficacy experiments in pancreatic cancer xenograft models showed highly potent (>90%) antitumor activity of both iRGD and (cRGD)2-KRAS Adaptors. U1 Adaptors targeting MYC inhibited pancreatic cancer cell proliferation caused by apoptosis in vitro (40%–70%) and tumor regressions in vivo. Comparison of iRGD-conjugated U1 KRAS and U1 MYC Adaptors in vivo revealed a significantly greater degree of cleaved caspase-3 staining and decreased Ki67 staining as compared with controls. There was no significant difference in efficacy between the U1 KRAS and U1 MYC Adaptor groups. Our results validate the value in targeting both KRAS and MYC in pancreatic cancer therapeutics and provide evidence that the U1 Adaptor technology can be successfully translated using a nanoparticle-free delivery system to target two undruggable genes in cancer. Mol Cancer Ther; 16(8); 1445–55. ©2017 AACR.

262. Therapeutic Suppression of the KRAS-MYC Oncogenic Axis in Human Pancreatic Cancer Xenografts with U1 Adaptor Oligonucleotide / RGD Peptide Conjugates

U1 Adaptors are synthetic oligonucleotides that enable the U1 small nuclear ribonucleoprotein (U1 snRNP) complex to stably bind within the terminal exon of a specific pre-mRNA. This interferes with the obligatory polyadenylation step in mRNA maturation, causing selective destruction of the targeted mRNA inside the nucleus. In contrast to siRNA or antisense oligos, U1 Adaptors can accept extensive covalent modifications for nuclease resistance, targeted delivery or in-vivo imaging without loss of silencing activity, offering important advantages as therapeutic agents. A panel of candidate U1 Adaptors targeting human KRAS (KRAS Adaptors) was screened in vitro using the human pancreatic cancer cell line MIA-PaCa2. The best candidates reduced KRAS mRNA expression by up to 76% - as effectively as siRNA controls. Reduced KRAS protein expression was confirmed by western blot. Inhibition of cell growth in vitro and increased apoptosis were seen for both the MIA-PaCa2 (KRASG12C) and Panc1(KRASG12D) cell lines, but not in BxPC3, a KRASwildtype pancreatic cancer cell line. In a parallel project, U1 Adaptors targeting human MYC mRNA were designed and screened in B-cell lymphoma lines, where the best candidates reduced MYC mRNA levels by over 95%. Because of the observed relationship between activating KRAS mutation and MYC overexpression in pancreatic cancers, MYC Adaptors were tested in MIA-PaCa2 cells. MYC Adaptors also inhibited cell growth and increased apoptosis in vitro. U1 Adaptors were tested for efficacy in mice bearing subcutaneous MIA-PaCa2 xenograft tumors. For in-vivo delivery, Adaptor oligos were directly conjugated to a cyclic RGD-motif peptide (cRGD), which is a targeting ligand for specific integrin-family receptors overexpressed on parenchyma and endothelial cells of many solid tumors. Alternately, U1 Adaptor oligos were linked to “internalizing” RGD (iRGD), a variant RGD peptide that also triggers permeabilization of tumor endothelium and internalization by cells through secondary binding to neuropilin-1. KRAS Adaptors linked to cRGD or iRGD were administered by tail vein injections twice weekly for three to four weeks. Over a series of experiments, tumor growth was inhibited by averages of 68% to 93%. Tumor stasis or regression occurred in some treated mice. In a pilot study, MYC Adaptors conjugated to iRGD peptide were also highly effective in suppressing tumor growth and inducing tumor regression. Excised tumors were analyzed by qPCR and western blot, which confirmed reductions of the targeted mRNAs and proteins. We have shown that U1 Adaptors conjugated to tumor-targeting / tumor-penetrating peptides can effectively target human KRAS and MYC oncogenes in vivo. These results support the continued development of U1 Adaptor technology as a strategy for therapeutic suppression of KRAS, MYC and possibly other oncogene targets in pancreatic cancer.

259. Targeted Gene Silencing by U1 Adaptor Oligonucleotides in Preclinical Models of Parkinson's Disease and Huntington's Disease

Many candidate genes are implicated in neurodegenerative disease, but to study potential therapeutic effects of modifying their expression in the central nervous system of animal models has been difficult, often requiring slow, expensive transgenic methods. Transient gene silencing with synthetic oligonucleotides can be a fast, inexpensive alternative to making new transgenic animal models, and a complimentary technique to extend the utility of existing ones. For genes with products that have been validated as therapeutic targets, but are not amenable to small molecule drugs, gene silencing may also be the therapeutic modality of choice.U1 Adaptors are a third generation of oligonucleotide-mediated gene silencing technology, mechanistically distinct from antisense or siRNA. U1 Adaptors act by selectively interfering with a key step in mRNA maturation: the addition of a 3’ polyadenosine (polyA) tail. Nearly all protein-coding mRNAs require a polyA tail, and failure to add one results in rapid degradation of the nascent mRNA inside the nucleus, preventing expression of a protein product. U1 Adaptor oligonucleotides are well suited to in vivo applications because they can accept extensive chemical modifications to improve nuclease resistance and the attachment of bulky groups, such as tags for imaging or ligands for receptor-mediated uptake by target cells, without loss of silencing activity.To explore the feasibility of U1 Adaptor technology for CNS targets, we designed panels of candidate U1 Adaptor oligos for mouse Scna (alpha-synuclein) and human HTT (Huntingtin), and screened them in cell culture. We identified U1 Adaptors that robustly suppress mouse Scna mRNA and reduce alpha-synuclein protein levels in mouse cells. Similarly, we identified U1 Adaptors that suppress the predominant, full length human HTT mRNA and reduce HTT protein levels in human cells. We also identified U1 Adaptors that suppress the HTT exon-1 truncation isoform recently implicated in HD pathogenesis. For in-vivo PK/PD studies, U1 Adaptors were delivered into the CNS of mice, by intracerebroventricular (ICV) injection or by direct stereotaxic injections into the striatum. To examine distribution, cellular uptake and persistence over time, fluorescently tagged U1 Adaptors were administered, then visualized by confocal microscopy in brain sections. ICV injection achieved broad distribution of fluorescent U1 Adaptors throughout the brain, with uptake visible in most cells. Subcellular distribution 24 hours after injection was diffuse in both cytoplasmic and nuclear compartments, but became more punctate and perinuclear by 48 hours. U1 Adaptor oligonucleotides were detected on northern blots of small RNA recovered from brain tissue specimens. Their levels in tissue were estimated by comparison to a standard loading curve, and correlated well with ICV-injected dose. After direct stereotaxic injection to the striatum, U1 Adaptors diffused rapidly and widely, and were taken up by all striatial cells, though preferentially by neurons. Adaptors persisted in tissue for at least five days (the last time point assayed) and reached the nuclei of striatial cells. We then did a series of studies with U1 Adaptors specific for mouse Scna mRNA. RNAScope was used to visualize relative levels of mRNA in situ. Injection of U1 Adaptors directly into the striatum resulted in clearly reduced expression of Scna mRNA and also of mRNA for synaptophysin, known to be down-regulated when α-synuclein expression is reduced.

694. Targeting KRAS in Pancreatic Cancer by Gene Silencing with U1 Adaptors

Activating mutations of the KRAS gene are key drivers of pancreatic cancer, but the KRAS protein has been refractory to small-molecule drugging. U1 Adaptors are oligonucleotides that enable the U1 small nuclear ribonucleoprotein complex to stably bind within the terminal exon of a specific pre-mRNA. This interferes with the obligatory polyadenylation step in mRNA maturation, causing selective destruction of the targeted mRNA in the nucleus. Unlike siRNA or antisense oligos, U1 Adaptors can be extensively modified for nuclease resistance or targeted delivery without loss of silencing activity, offering important advantages as therapeutic agents.We sought to translate U1 Adaptor technology to suppress KRAS in pancreatic cancer. A panel of U1 Adaptors targeting human KRAS (KRAS Adaptors) was screened in vitro using the human pancreatic cancer cell line MiaPaca-2 (KRAS G12D mutant). Candidate KRAS Adaptors reduced KRAS mRNA expression by up to 76%, as effectively as an siRNA control. Knockdown of KRAS protein expression was confirmed by western blot. Inhibition of cell growth in vitro was demonstrated for MiaPaca-2 and two additional pancreatic cancer cell lines, Panc1 (KRAS G12D mutant) and BXPC3 (KRAS wildtype).We evaluated KRAS Adaptors in mice bearing subcutaneous MiaPaca-2 xenograft tumors. For in-vivo delivery, the Adaptors were initially complexed with PAMAM dendrimers linked to a tumor-targeting, cyclic RGD peptide (cRGD), and administered by tail vein injection twice weekly for three weeks. Tumor growth was inhibited by as much as 68% compared to vehicle controls (p=0.0002). Excised tumors were analyzed by qPCR and IHC, which confirmed reductions of KRAS mRNA and protein.Although U1 Adaptors complexed with cRGD-dendrimers have been efficacious in this study and others, dendrimers have technical drawbacks and reported toxicities, and dendrimer-free formulations are preferable. As an alternative, we conjugated KRAS Adaptorsdirectly to cRGD peptide or to internalizing RGD (iRGD), which is a variant RGD peptide that triggers permeabilization of tumor endothelium and internalization by cells through secondary binding to neuropilin. The cRGD- and iRGD-conjugated KRAS Adaptors were tested for efficacy against subcutaneous MiaPaca-2 xenografts, and tumor growth was inhibited to equal or greater extent as with the original cRGD-dendrimer system. iRGD may be of particular benefit for pancreatic adenocarcinomas, which have a densely fibrotic stroma that impedes drug delivery. Enhanced delivery of small- and large-molecule therapeutics into primary pancreatic adenocarcinomas in KPC mice has been achieved previously by conjugating or coinjecting iRGD peptide.We have shown that U1 Adaptors can successfully target human KRAS both in vitro and in vivo. These results support the continued development of U1 Adaptor technology as a strategy for therapeutic suppression of KRAS in pancreatic cancer.

Abstract B52: Therapeutic targeting of human KRAS in pancreatic cancer using a novel method of gene-silencing: U1 adaptors

Background: While genetic knockdown of RAS in mouse tumor models has substantiated it as a therapeutic target, there is no effective means of targeting RAS currently available in the clinic today. Numerous RNA interference-based studies targeting RAS have demonstrated therapeutic effects, however, effective delivery has been a major obstacle that has impeded this approach. U1 Adaptors are a novel technology for oligonucleotide-mediated gene silencing that act by selectively interfering with polyadenylation of messenger RNA (mRNA) inside the cell nucleus. Polyadenosine (PolyA) tail addition is an obligatory step in mRNA maturation and function, and its failure results in rapid degradation of the nascent message by endogenous nucleases. The eukaryotic U1 small nuclear ribonucleoprotein complex (U1 snRNP) is best known for its role as a pre-mRNA splicing factor, but also acts naturally to silence some genes by suppressing polyadenylation. U1 Adaptors are synthetic oligonucleotides that enable the U1 snRNP complex to stably bind to the terminal exon of any chosen pre-mRNA target, thereby interfering with polyA tail addition and causing it to be selectively degraded in the nucleus. The silencing mechanism of U1 Adaptors is distinct from those of siRNA or antisense oligonucleotides and this distinction confers an important advantage for their use as therapeutic agents. We have validated this technology in vivo demonstrating an 85% tumor growth inhibition by targeting BCL-2 and GRM-1 in human melanoma xenografts. Our in vivo proof-of-concept study relied on delivery of the U1 Adaptors non-covalently complexed with a nanoparticle comprised of a positively charged dendrimer covalently linked to a cyclic penta-peptide containing Arginine-Glycine-Aspartate referred to as the cRGD peptide, a widely-used tumor-targeting moiety. Methods/Results: We sought to translate the U1 Adaptor technology to target human KRAS. We first designed a set of U1 Adaptors for screening purposes targeting human KRAS at eight different positions along the human KRAS pre-mRNA located at the junction of the terminal exon (position 632) and untranslated region (UTR). We have evaluated these adaptors in vitro using the human pancreatic cancer cell line MIA Paca-2 (KRASG12D). Screening of these eight U1 Adaptors reveals a range of KRAS gene silencing as measured by quantitative PCR. Notably, Adaptors 2 and 3 silenced KRAS down to 27 and 24% respectively, as effective as the siRNA control. We then evaluated Adaptors 2 and 3 in human MIA Paca-2 xenografts. These adaptors were coupled to the cRGD-nanoparticle complex and administered by tail vein injection twice weekly. We observed significant tumor growth inhibition (37.3% by KRAS Adaptor 2, p=0.025, and 68.3% by KRAS Adaptor 3, p=0.0002, as compared to the vehicle control by Day 34. We also observed significant tumor growth inhibition with a U1 Adaptor targeting BCL-2 albeit to a lesser extent than KRAS Adaptor 3. Conclusion: We have demonstrated that the U1 Adaptor method of gene silencing can be successfully applied to target human KRAS both in vitro and in vivo. These results support the continued investigation of U1 Adaptor technology as a strategy for therapeutic targeting of RAS oncogenes. Citation Format: Ashley T. Tsang, Xin Yu, Rafal Goraczniak, Mark Brenneman, Samuel Gunderson, Darren R. Carpizo. Therapeutic targeting of human KRAS in pancreatic cancer using a novel method of gene-silencing: U1 adaptors. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr B52. doi: 10.1158/1557-3125.RASONC14-B52