Matthew R. Hassler

Exosome-mediated Delivery of Hydrophobically Modified siRNA for Huntingtin mRNA Silencing.

Delivery represents a significant barrier to the clinical advancement of oligonucleotide therapeutics for the treatment of neurological disorders, such as Huntingtons disease. Small, endogenous vesicles known as exosomes have the potential to act as oligonucleotide delivery vehicles, but robust and scalable methods for loading RNA therapeutic cargo into exosomes are lacking. Here, we show that hydrophobically modified small interfering RNAs (hsiRNAs) efficiently load into exosomes upon co-incubation, without altering vesicle size distribution or integrity. Exosomes loaded with hsiRNAs targeting Huntingtin mRNA were efficiently internalized by mouse primary cortical neurons and promoted dose-dependent silencing of Huntingtin mRNA and protein. Unilateral infusion of hsiRNA-loaded exosomes, but not hsiRNAs alone, into mouse striatum resulted in bilateral oligonucleotide distribution and statistically significant bilateral silencing of up to 35% of Huntingtin mRNA. The broad distribution and efficacy of hsiRNA-loaded exosomes delivered to brain is expected to advance the development of therapies for the treatment of Huntingtons disease and other neurodegenerative disorders.

Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain

Applications of RNA interference for neuroscience research have been limited by a lack of simple and efficient methods to deliver oligonucleotides to primary neurons in culture and to the brain. Here, we show that primary neurons rapidly internalize hydrophobically modified siRNAs (hsiRNAs) added directly to the culture medium without lipid formulation. We identify functional hsiRNAs targeting the mRNA of huntingtin, the mutation of which is responsible for Huntingtons disease, and show that direct uptake in neurons induces potent and specific silencing in vitro. Moreover, a single injection of unformulated hsiRNA into mouse brain silences Htt mRNA with minimal neuronal toxicity. Thus, hsiRNAs embody a class of therapeutic oligonucleotides that enable simple and straightforward functional studies of genes involved in neuronal biology and neurodegenerative disorders in a native biological context.

Docosahexaenoic Acid Conjugation Enhances Distribution and Safety of siRNA upon Local Administration in Mouse Brain

The use of siRNA-based therapies for the treatment of neurodegenerative disease requires efficient, nontoxic distribution to the affected brain parenchyma, notably the striatum and cortex. Here, we describe the synthesis and activity of a fully chemically modified siRNA that is directly conjugated to docosahexaenoic acid (DHA), the most abundant polyunsaturated fatty acid in the mammalian brain. DHA conjugation enables enhanced siRNA retention throughout both the ipsilateral striatum and cortex following a single, intrastriatal injection (ranging from 6-60 μg). Within these tissues, DHA conjugation promotes internalization by both neurons and astrocytes. We demonstrate efficient and specific silencing of Huntingtin mRNA expression in both the ipsilateral striatum (up to 73%) and cortex (up to 51%) after 1 week. Moreover, following a bilateral intrastriatal injection (60 μg), we achieve up to 80% silencing of a secondary target, Cyclophilin B, at both the mRNA and protein level. Importantly, DHA-hsiRNAs do not induce neural cell death or measurable innate immune activation following administration of concentrations over 20 times above the efficacious dose. Thus, DHA conjugation is a novel strategy for improving siRNA activity in mouse brain, with potential to act as a new therapeutic platform for the treatment of neurodegenerative disorders.The use of siRNA-based therapies for the treatment of neurodegenerative disease requires efficient, nontoxic distribution to the affected brain parenchyma, notably the striatum and cortex. Here, we describe the synthesis and activity of a fully chemically modified siRNA that is directly conjugated to docosahexaenoic acid (DHA), the most abundant polyunsaturated fatty acid in the mammalian brain. DHA conjugation enables enhanced siRNA retention throughout both the ipsilateral striatum and cortex following a single, intrastriatal injection (ranging from 6–60 μg). Within these tissues, DHA conjugation promotes internalization by both neurons and astrocytes. We demonstrate efficient and specific silencing of Huntingtin mRNA expression in both the ipsilateral striatum (up to 73%) and cortex (up to 51%) after 1 week. Moreover, following a bilateral intrastriatal injection (60 μg), we achieve up to 80% silencing of a secondary target, Cyclophilin B, at both the mRNA and protein level. Importantly, DHA-hsiRNAs do not induce neural cell death or measurable innate immune activation following administration of concentrations over 20 times above the efficacious dose. Thus, DHA conjugation is a novel strategy for improving siRNA activity in mouse brain, with potential to act as a new therapeutic platform for the treatment of neurodegenerative disorders.

Guanabenz (Wytensin™) selectively enhances uptake and efficacy of hydrophobically modified siRNAs

One of the major obstacles to the pharmaceutical success of oligonucleotide therapeutics (ONTs) is efficient delivery from the point of injection to the intracellular setting where functional gene silencing occurs. In particular, a significant fraction of internalized ONTs are nonproductively sequestered in endo-lysosomal compartments. Here, we describe a two-step, robust assay for high-throughput de novo detection of small bioactive molecules that enhance cellular uptake, endosomal escape, and efficacy of ONTs. Using this assay, we screened the LOPAC (Sigma–Aldrich) Library of Pharmacologically Active Compounds and discovered that Guanabenz acetate (Wytensin™), an FDA-approved drug formerly used as an antihypertensive agent, is capable of markedly increasing the cellular internalization and target mRNA silencing of hydrophobically modified siRNAs (hsiRNAs), yielding a ∼100-fold decrease in hsiRNA IC50 (from 132 nM to 2.4 nM). This is one of the first descriptions of a high-throughput small-molecule screen to identify novel chemistries that specifically enhance siRNA intracellular efficacy, and can be applied toward expansion of the chemical diversity of ONTs.

Taking charge of siRNA delivery

Delivery of siRNA into cells is achieved by neutralizing the negative charge of the phosphate backbone in a reversible manner.

Comparison of partially and fully chemically-modified siRNA in conjugate-mediated delivery in vivo

Abstract Small interfering RNA (siRNA)-based drugs require chemical modifications or formulation to promote stability, minimize innate immunity, and enable delivery to target tissues. Partially modified siRNAs (up to 70% of the nucleotides) provide significant stabilization in vitro and are commercially available; thus are commonly used to evaluate efficacy of bio-conjugates for in vivo delivery. In contrast, most clinically-advanced non-formulated compounds, using conjugation as a delivery strategy, are fully chemically modified (100% of nucleotides). Here, we compare partially and fully chemically modified siRNAs in conjugate mediated delivery. We show that fully modified siRNAs are retained at 100x greater levels in various tissues, independently of the nature of the conjugate or siRNA sequence, and support productive mRNA silencing. Thus, fully chemically stabilized siRNAs may provide a better platform to identify novel moieties (peptides, aptamers, small molecules) for targeted RNAi delivery.

Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways

Efficient delivery of therapeutic RNA is the fundamental obstacle preventing its clinical utility. Lipid conjugation improves plasma half-life, tissue accumulation, and cellular uptake of small interfering RNAs (siRNAs). However, the impact of conjugate structure and hydrophobicity on siRNA pharmacokinetics is unclear, impeding the design of clinically relevant lipid-siRNAs. Using a panel of biologically-occurring lipids, we show that lipid conjugation modulates siRNA hydrophobicity and governs spontaneous partitioning into distinct plasma lipoprotein classes in vivo. Lipoprotein binding influences siRNA distribution by delaying renal excretion and promoting uptake into lipoprotein receptor-enriched tissues. Lipid-siRNAs elicit mRNA silencing without causing toxicity in a tissue-specific manner. Lipid-siRNA internalization occurs independently of lipoprotein endocytosis, and is mediated by siRNA phosphorothioate modifications. Although biomimetic lipoprotein nanoparticles have been considered for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation.

Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing.

RNA-based drugs depend on chemical modifications to increase potency and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. Here, we explore chemical modifications at all positions of the crRNA guide and tracrRNA cofactor. We identify several heavily modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2′-OH groups) that are functional in human cells. These designs will contribute to Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes.Resistance of gRNA to ubiquitous ribonucleases is required for CRISPR-Cas9-based therapeutics. Here, the authors explore chemical modifications at all positions of the crRNA guide and tracrRNA cofactor, and identify modified versions that are more potent and stable than their unmodified counterparts in editing human cells.

Novel Cluster and Monomer-Based GalNAc Structures Induce Effective Uptake of siRNAs in Vitro and in Vivo

GalNAc conjugation is emerging as a dominant strategy for delivery of therapeutic oligonucleotides to hepatocytes. The structure and valency of the GalNAc ligand contributes to the potency of the conjugates. Here we present a panel of multivalent GalNAc variants using two different synthetic strategies. Specifically, we present a novel conjugate based on a support-bound trivalent GalNAc cluster, and four others using a GalNAc phosphoramidite monomer that was readily assembled into tri- or tetravalent designs during solid phase oligonucleotide synthesis. We compared these compounds to a clinically used trivalent GalNAc cluster both in vitro and in vivo. In vitro, cluster-based and phosphoramidite-based scaffolds show a similar rate of internalization in primary hepatocytes, with membrane binding observed as early as 5 min. All tested compounds provided potent, dose-dependent silencing, with 2-4% of injected dose recoverable from liver after 1 week. The two preassembled trivalent GalNAc clusters showed higher tissue accumulation and gene silencing relative to di-, tri-, or tetravalent GalNAc conjugates assembled via phosphoramidite chemistry.

Abstract PR12: Robust modulation of gene expression in aggressive glioblastoma mouse models: A new approach for in vivo target validation

Introduction: Glioblastoma (GBM) is an aggressive and highly malignant form of brain cancer with a devastatingly poor clinical prognosis. Despite years of research, there is a surprising lack of effective therapies and novel approaches for treatment. There exists a great, unmet need for strategies enabling efficient and targeted modulation of gene expression in established tumors. RNAi-mediated gene silencing is a promising strategy for straightforward modulation of gene expression, and siRNA-based therapies could result in more effective and less toxic treatment for GBM. The Khvorova laboratory has developed a fully chemically stabilized, self-delivering siRNA platform supporting efficient intratumoral uptake and target gene silencing in a GBM orthotopic mouse model. Methods: For all in vitro and in vivo studies, a patient-derived GBM8 cell line was used. Uptake in GBM8 cells was done by treating cells with 0.75 µM of fully chemically stabilized, Cy3-conjugated, hydrophobically-modified siRNA (hsiRNA) targeting the huntingtin mRNA (Cy3-FM-hsiRNAHTT). At different time points (0 to 5.5 hours) GBM8 cells were harvested by cytospin, fixed in 4% formaldehyde, and then imaged by confocal microscopy (40X oil). Next, levels of mRNA knockdown in GBM8 cells were determined by plating 50,000 cells/well in a 96-well plate and treating with increasing doses of FM-hsiRNAHTT (0.01 to 3 µM). After 72 hours, cells were lysed and mRNA was quantified using QuantiGene (Affymetrix) assay. In vivo distribution was done by unilaterally injecting Cy3-FM-hsiRNAHTT into the striatum (2 nmol) or the ventricle of nude mice (5 nmol), 4 weeks after GBM8 tumor implantation. 24 hours after injection, mice were sacrificed, perfused with 4% formaldehyde, and brains were sectioned into 4 µm sections. Knockout in nude mice following GBM8 tumor implantation was also conducted. Mice were treated with FM-hsiRNAHTT (5 nmoles, n=5), a non-targeting control hsiRNA (NTC, 5 nmoles, n=5), or vehicle (5 µL, n=5) and sacrificed after 5 days. 3 biopsies per animal were collected from the striatum of the tumor-bearing side. The levels of huntingtin mRNA expression were determined by QuantiGene® and normalized to a housekeeping gene and reported as percent of CSF. Results: We observed significant uptake in GBM8 cells treated with 0.75 µM Cy3-FM-hsiRNAHTT starting at 2 hours. In addition, huntingtin mRNA was reduced by 50% at 0.3 µM and was reduced by 80% when cells were treated with 1 µM. Following a direct intratumoral injection of Cy3-FM-hsiRNAHTT, we saw widespread distribution of Cy3-fluorescent co-localizing with the tumor. Intratumoral administration of 5 nmoles of FM-hsiRNAHTT resulted in 50% target silencing and was statistically significant when compared to the CSF or NTC treated groups. However, a single injection into the ventricle only resulted in 25% silencing and was only statistically significant relative to a non-targeting hsiRNA control. Summary/Conclusion: Here, we demonstrate that a single intratumoral injection of hydrophobically modified, metabolically stable siRNA (hsiRNA) results in robust and long-lasting silencing of targets in established tumors. Due to the high potency, long duration of effect, and predictable pharmacokinetics of hsiRNA, it is possible that medicine based on this technology can be tailored to a highly diverse patient population. As is, it is believed to be an ideal drug platform for treatment of cancer, as long as efficient delivery to the tumors and metastasis is achieved. Thus, this technology allows immediate modulation of expression in orthotropic tumor models and may contribute to our understating of tumor biology. In addition, this approach can, in the future, serve as a foundation for new therapeutic interventions for this devastating disease. This abstract is also presented as Poster B44. Citation Format: Andrew H. Coles, Maire F. Osborn, Diane Golebowski, Matthew Hassler, Miguel S. Esteves, Anastasia Khvorova. Robust modulation of gene expression in aggressive glioblastoma mouse models: A new approach for in vivo target validation. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr PR12.