Anastasia Khvorova - US grants

Project Summary/Abstract: Highly inefficient transit of oligonucleotides from outside cells to the intracellular compartments where functional activity of oligonucleotides takes place is the most serious limitation to practical realization of a full potential of oligonucleotide-based therapies. Several classes of oligonucleotides therapeutics (ONT), including antisense oligonucleotides (ASO), hydrophobically modified siRNAs (hsiRNA), GalNAc conjugated siRNAs, and LNP formulated siRNAs have validated biological efficacy and are in clinic [1, 2]. In all cases, the fraction of oligonucleotides reaching the intended place of biological function is surprisingly low with the majority of molecules being trapped in wrong cellular compartments, resulting in low efficiency and clinically limiting toxicity. Concurrently, there is a significant and rapidly expanding body of evidence demonstrating natural small RNA trafficking between cells as a means of intracellular communication. Given the extremely high efficacy of oligonucleotide-mediated gene silencing (25-100 molecules of Ago2 loaded siRNAs per cell [3]), exploiting the natural pathways of intracellular small RNA trafficking opens a possibility to dramatically improve the efficacy of ONTs. We hypothesize that exploiting natural, evolutionarily conserved mechanisms and pathways for trafficking of small RNAs across cellular boundaries is the way to fundamentally improve ONTs efficiency. The goal of this proposal is to elucidate the pathways and mechanisms involved in natural small RNA and ONTs trafficking and leverage this knowledge to increase the efficacy of oligonucleotide therapies. We have developed a unique set of experimental tools and analytical methods which enable us to systematically map and study endogenous small RNA as well as ONTs trafficking pathways. We have developed (1) a novel class of hydrophobic siRNAs (hsiRNAs), which efficiently enter cells and tissues; (2) an approach for visualization of native RNA trafficking through use of hsiRNA loaded exosomes as a model, (3) an approach for dissection of productive and non-productive ONTs trafficking using a panel of small molecules dramatically enhancing hsiRNAs efficacy by altering their cellular distribution; (4) a panel of four major classes of ONTs, which utilize different uptake mechanisms and silence a clinically relevant target huntingtin (htt), (5) a panel of engineered cell lines (GFP-fusions of trafficking markers) to study internalization pathways. Using these novel experimental tools along with TESM intracellular imaging, high resolution mass spectrometry and functional and biochemical analyses, we will identify the productive and non-productive cellular uptake mechanisms for both natural and synthetic oligonucleotides Completion of this project will result in (1) significant progress in our understanding of the natural mechanisms of extracellular uptake of small regulatory RNAs, (2) mapping uptake pathways for different classes of ONTs (3) development of the conceptual framework for generation of novel RNA chemistries relying on native uptake mechanisms. These findings will lead to a new generation of ONTs with dramatically improved clinical efficacy.

DESCRIPTION (provided by applicant): The goal of this UH2 and UH3 is to study how exosomes can deliver siRNAs across the blood brain barrier to enter neurons and other brain cells. The immediate target is the mutant huntingtin mRNA. Huntington's disease (HD) is caused by an increase in the CAG trinucleotide repeats to ? 36 in series; it necessitates years in a high level nursing facility because of neurodegeneration first in striatum and cortex and then to other brain structures. HD patients have cognitive impairment, depression and aberrant movements. Most HD patient present by 30 to 40 years of age; a few have a juvenile onset. A rational treatment is to decrease expression of mutant huntingtin mRNA; this therapeutic can be accomplished in HD mouse models by siRNA, antisense oligonucleotides (ASO) and adeno-associated virus (AAV) with shRNAmir directed against huntingtin mRNA. However, delivery remains a pitfall to practical implementation of the therapeutics. siRNA and ASO require long-term infusion. In non-human primates, ASO administered to spinal fluid does not reach the striatum and spread of siRNA is limited in brain. Although promising, AAV-shRNA requires several injections into brain areas and the shRNAmir is unregulated. A gap in HD therapeutics can be filled by microvesicles normally extruded by cells, exosomes. Exosomes with rabies virus glycoprotein (RVG) on their surface can be injected into the blood, cross the blood brain barrier, and enter neurons and glia. RVG-exosomes can carry siRNA cargo. Delivered into the blood circulation, the exosomes deposit siRNA in neurons to engage in RNA interference. Our purpose is to develop exosomes as a therapeutic in HD. The UH2 examines the ability of RVG-exosomes carrying siRNA against huntingtin mRNA to cross the blood brain barrier to enter neurons. Localization in brain and RNAi dependent knock down will be studied. Hyper-functional siRNAs will be sought. Because exosomes are made from cytoplasm of cells, exosome mRNA, miRNA, and implaced siRNA will be identified by deep sequencing. Immune reactivity and immune-neutralization will be studied, since exosomes have potential antigens, like RVG, and will need to be administrated often. The UH3 further establishes exosome-based therapeutics, by study of reversal or prevention of neuropathology and aberrant movement in HD mouse models. Dosing of exosomes will be secured. A team of experts in HD pathogenesis, siRNA development, RNA identification and measurement, RNAi mechanisms and exosome production and brain delivery will carry out the studies. Harnessing exosomes for brain delivery is expected to form a viable therapeutic to reduce expression of mutant huntingtin in patients with HD. Patients with other genetically- based neurodegeneration will benefit.

? DESCRIPTION (provided by applicant): Oligonucleotide- based therapeutics and in vivo functional genomics is a cutting-edge area of research and development. The discovery of RNAi by University of Massachusetts Medical School Professor Craig Mello received the Nobel Prize in 2006. RNAi and Antisense-based drugs have proven clinical efficacy and are believed to become a novel major class of therapeutic modalities. At the University of Massachusetts Medical School (UMMS), there are multiple groups whose research has reached the stage at which functional analysis of novel and advanced oligonucleotides in animal models and preclinical development is necessary. These types of studies require access to mid-scale, uniquely chemically modified oligonucleotides that have limited commercial availability and are outside the reach of academic investigators. A Shared Instrumentation Grant will allow us to purchase the equipment to support mid scale synthesis, purification and quality control of chemically modified RNA that will enable ongoing and planned animal models and preclinical studies for research supported by 11 NIH grants and multiple grants from private foundations. Specifically, we are requesting funds to purchase a GE AKTA Pilot 10 RNA synthesizer, 218 Prep HPLC and 6530 QTOF LC- MS. The acquisition of this state-of-the-art RNA synthesis capability will propel our research and translational programs and enable the development of novel RNA-based therapies, specifically for treatment of Preeclampsia, ALS (amyotrophic lateral sclerosis), HSAN (hereditary sensory and autonomic neuropathy), Huntington, IBD (inflammatory bowel disease) and other diseases.

Complicating 5-8% of all pregnancies, preeclampsia (PE) is one of the three main causes of premature birth. Across the globe, PE and subsequent eclampsia are major contributors to maternal, fetal and neonatal morbidity and mortality. Although the root causes of PE have yet to be fully understood, it is now well established that the maternal signs and symptoms of hypertension, edema and proteinuria are caused by an excess of anti-angiogenic proteins in the mother's bloodstream. Chief among these are soluble fms-like tyrosine kinase 1 proteins (sFLT1s) produced by the placenta. sFLT1s are truncated forms of the membrane- bound vascular endothelial growth factor (VEGF) receptor FLT1 (aka, VEGFR1). When abnormally high in the mother's circulatory system, they can interfere with her body's ability to respond to VEGF. Selective elimination (filtration) of maternally circulating sFLT1 has been shown to be a successful strategy for treatment of PE, with a 30-40% reduction in circulating sFLT1 being sufficient to allow pregnancy extension. Our desire is to develop a simple and cost-effective PE therapeutic using RNA interference (RNAi) to limit excess placental expression of sFLT1 proteins. Based on RNA-Seq and PAS-Seq data, placental sFLT1 expression is dominated by three truncated mRNA isoforms generated by polyadenylation within Flt1 introns 13 and 14. Targeting these abnormally-expressed intronic regions with RNAi compounds enables selective silencing of the truncated mRNA variants that encode sFLT1 proteins without interfering with full-length FLT1 expression. We have developed a novel RNA chemistry that enables highly targeted delivery to trophoblasts in the placental labyrinth with up to 12% of injected dose accumulating in placentas (single SC or IV injection). Here, compound concentrations can achieve up to 100 µg/gram, over 1,000-fold above the dose required for effective silencing (100 ng/gram oligo). Most importantly, we have observed no detectable oligonucleotide transfer to the fetus, both by fluorescence microscopy and quantitative analysis. Using systematic screens, we have identified a pair of hyper-functional, fully-metabolically stabilized, hydrophobically modified siRNAs that selectively target the i13 (sFLT1-i13-2283) and i15a (sFLT1-i15a-2519) isoforms with EC50 <10 pM for RISC (RNA Induced Silencing Complex) entry and 40-80 nM for trophoblast delivery. Systemic administration of sFLT1-i13-2283 results in potent silencing of sFLT1-i13 mRNA in mouse placenta and liver/kidney endothelium and more than 40% reduction in circulating sFLT1 protein, with no observable adverse effects on fetal or maternal health. The goal of the current proposal is to generate sufficient data to move our currently identified lead candidate (sFLT-i13-2283/sFLT1-i15a-2519) toward formal GLP and IND-enabling studies for the development of a simple and cost-effective treatment of PE, a critically important unmet medical need.

ABSTRACT: Therapeutic oligonucleotides (e.g., small interfering RNAs (siRNAs) and antisense) hold promise as transformative drugs for the treatment of genetically defined neurodegenerative disorders, including Huntington's disease (HD) and amyotrophic lateral sclerosis (ALS). siRNAs silence disease-causing genes by targeting their cognate mRNAs for degradation, thereby preventing the expression of toxic gene products. Their inherent sequence specificity and prolonged activity provide a powerful therapeutic platform, as long as the disease is genetically defined and delivery to the relevant target tissue is achievable. However, siRNAs do not cross the blood?brain barrier and local CNS delivery by injection often results in poor retention, distribution or toxicity. Thus, efficient and non-toxic delivery represents a major hurdle in the development of RNAi-based drugs to treat neurodegenerative disorders. The goal of this proposal is to develop and characterize novel chemical scaffolds that promote simple, efficient, and non-toxic delivery of oligonucleotides and potent silencing of therapeutic targets in the central nervous system. We describe a class of fully chemically stabilized hydrophobic siRNAs (hsiRNAs) that elicit durable and potent silencing throughout the brain following bolus cerebrospinal fluid (CSF) infusion. Modifications include oligonucleotides structure, ribose, backbone and the addition of lipophilic conjugates? e.g., neuroactive steroids, endocannabinoid-like lipids, gangliosides. Extensive structure-activity relationship studies reveal that the type of conjugate defines the distribution, retention, efficacy, duration of effect, and toxicity of hsiRNA-conjugates in the central nervous system. Completion of this proposal will (i) define and characterize two novel chemical scaffolds that support potent, specific, and long-lasting silencing of target genes in the central nervous system, and (ii) validate this new platform in animal models of HD and ALS, establishing a path towards novel treatments for two neurodegenerative diseases. This proposal establishes a platform that allows direct targeting of any gene expressed in any region of the central nervous system in a rodent. Successful completion of this work will enable studies of gene function in the central nervous system and pave the way towards development of novel oligonucleotide-based therapies for genetically defined neurodegenerative diseases.

Project Summary Small interfering RNAs (siRNAs) are informational drugs that can be designed to treat genetically defined disorders and thereby reshape our approach to human medicine. The clinical utility of siRNAs depends on functional delivery to a tissue and cell type of interest, which is in turn defined by oligonucleotide chemistry. When a chemical architecture?i.e., oligonucleotide modification pattern?that provides functional and non- toxic delivery to a tissue is optimized, candidate drugs can be quickly developed to treat other diseases with the same tissue involvement. Currently, the clinical utility of siRNA is limited to liver, where conjugation of trivalent N-acetylgalactosamine (GalNAc) moiety enables efficient delivery to hepatocytes and therapeutic activity for a year after a single injection. To expand the utility of siRNAs to tissues beyond liver, we must (i) optimize chemical modification patterns that fully stabilize siRNAs and are non-toxic and compatible with the silencing machinery; (ii) understand the mechanisms that define siRNA pharmacokinetic and pharmacodynamic behavior; and (iii) identify and engineer novel ligands that enable targeted tissue delivery and sustained in vivo efficacy. We have the demonstrated expertise in organic chemistry, combinatorial chemistry, oligonucleotide chemistry, RISC biology, and siRNA pharmacology needed to solve these problems. To date, we have identified fully chemically stabilized siRNA scaffolds that exhibit minimal toxicity and immunogenicity; engineered novel conjugates that support functional delivery to liver, kidneys, heart, fat, muscle, and lung; defined chemical approaches to dynamically modulate siRNA clearance; and synthesized novel backbone modifications (phosphonate variants) that improve siRNA stability and, when placed in defined positions, enhance RISC efficacy and specificity. Building on these recent advances, we propose four principal research directions that seek to (i) chemically engineer siRNA scaffolds that enable complete stability and sustained efficacy of any RNA sequence in vivo; (ii) establish phosphonate variants as a new backbone for the modulation of therapeutic RNA properties; (iii) engineer and discover novel ligands that deliver siRNAs to tissues other than liver; and (iv) work with a network of expert collaborators to investigate the therapeutic potential of novel chemical configurations in models of diseases with unmet medical needs. The completion of these studies will establish siRNA chemical architectures that enable functional extrahepatic delivery of siRNAs and lead to the discovery of several compounds with the potential to transform therapeutic approaches for range of diseases.