Small interfering RNAs (siRNAs) are a promising class of drugs for treating genetically-defined diseases. Therapeutic siRNAs enable specific modulation of gene expression, but require chemical architecture that facilitates efficient in vivodelivery. siRNAs are informational drugs, therefore specificity for a target gene is defined by nucleotide sequence. Thus, developing a chemical scaffold that efficiently delivers siRNA to a particular tissue provides an opportunity to target any disease-associated gene in that tissue. The goal of this project was to develop a chemical scaffold that supports efficient siRNA delivery to the brain for the treatment of neurodegenerative diseases, specifically Huntington’s disease (HD). HD is an autosomal dominant neurodegenerative disorder that affects 3 out of every 100,000 people worldwide. This disorder is caused by an expansion of CAG repeats in the huntingtin gene that results in significant atrophy in the striatum and cortex of the brain. Silencing of the huntingtin gene is considered a viable treatment option for HD. This project: 1) identified a hyper-functional sequence for siRNA targeting the huntingtin gene, 2) developed a fully chemically modified architecture for the siRNA sequence, and 3) identified a new structure for siRNA central nervous system (CNS) delivery—Divalent-siRNA (Di-siRNA). Di-siRNAs, which are composed of two fully chemically-stabilized, phosphorothioate-containing siRNAs connected by a linker, support potent and sustained gene modulation in the CNS of mice and non-human primates. In mice, Di-siRNAs induced potent silencing of huntingtin mRNA and protein throughout the brain one month after a single intracerebroventricular injection. Silencing persisted for at least six months, with the degree of gene silencing correlating to guide strand tissue accumulation levels. In Cynomolgus macaques, a bolus injection exhibited significant distribution and robust silencing throughout the brain and spinal cord without detectable toxicity. This new siRNA scaffold opens the CNS for RNAi-based gene modulation, creating a path towards developing treatments for genetically-defined neurological disorders.

<p>Allosteric modulators are emerging as a new class of therapeutics for the treatment of complex disorders, including psychiatric illnesses such as schizophrenia. The disease is marked by hyperdopaminergic signaling in the striatum, which plays a role in the development of positive symptoms like delusions, hallucinations, and paranoia. Conventional antipsychotic drug therapy typically employs dopamine D2 receptor antagonists that compete with endogenous dopamine at the orthosteric, or dopamine-binding site, in an attempt to normalize these psychotic symptoms. However, they are often associated with adverse motor and metabolic side effects. Furthermore, only some antipsychotic drugs are able to treat the negative symptoms of schizophrenia, which include social withdrawal and anhedonia, and there is currently no treatment for the cognitive impairment associated with the disease.</p> <p>Allosteric modulators are safer alternatives to conventional orthosteric therapeutics as they interact with their receptor at a novel binding site and their mechanism involves modulation of endogenous signaling. Therefore, levels of endogenous ligand limit the activity of an allosteric modulator. Our lab has synthesized and evaluated over 185 compounds for their activity at the dopamine D2 receptor. Of these compounds, PAOPA is the most potent allosteric modulator, and has been shown to be effective in treating the MK-801 induced preclinical animal model of schizophrenia without causing the adverse effects induced by currently prescribed antipsychotic drugs. The objective of this study was to evaluate PAOPA’s ability to treat behavioural abnormalities in an amphetamine-sensitized model of schizophrenia.</p> <p>Four groups (n=10/group) of male Sprague Dawley rats received intraperitoneal injections three days per week on alternate days over three weeks. Group A received saline, group B received D-amphetamine (1mg/kg during week one, 2mg/kg during week two, 3mg/kg during week three), group C received PAOPA (1mg/kg), and group D received the same doses of amphetamine as group B with PAOPA (1mg/kg). Following a three-week withdrawal, each group was tested for prepulse inhibition, social interaction, and locomotor activity. Amphetamine-sensitized rats were subjected to the same tests following PAOPA administration (1mg/kg). To assess whether behavioural changes were associated with changes in brain chemistry, post-mortem dopamine levels were measured in the striatum, nucleus accumbens, and medial prefrontal cortex. Data were analyzed by one-way ANOVA or paired t test where appropriate.</p> <p>Amphetamine sensitization induced schizophrenic-like behavioural abnormalities, including deficits in prepulse inhibition and social interaction, as well as increased locomotor activity and sensitivity to amphetamine challenge. Concurrent amphetamine and PAOPA treatment prevented all amphetamine- induced behavioural abnormalities. Furthermore, amphetamine-induced deficits in prepulse inhibition and social interaction were reversed one hour following PAOPA treatment. PAOPA treatment alone had no effect on behaviour or post-mortem striatal dopamine. Behavioural changes in amphetamine-sensitized rats were accompanied by a reduction in post-mortem striatal dopamine levels. In correlation with behavioural results, PAOPA administration during amphetamine sensitization prevented this biochemical change.</p> <p>These results demonstrate that PAOPA can prevent and reverse behavioural and associated biochemical abnormalities in amphetamine-sensitized rats. PAOPA is a candidate for the development of treatments for schizophrenia.</p> / Master of Science (MSc)

Methyl group transfer from S-adenosyl-l-methionine (AdoMet) to various substrates including DNA, proteins, and natural products (NPs), is accomplished by methyltransferases (MTs). Analogs of AdoMet, bearing an alternative S-alkyl group can be exploited, in the context of an array of wild-type MT-catalyzed reactions, to differentially alkylate DNA, proteins, and NPs. This technology provides a means to elucidate MT targets by the MT-mediated installation of chemoselective handles from AdoMet analogs to biologically relevant molecules and affords researchers a fresh route to diversify NP scaffolds by permitting the differential alkylation of chemical sites vulnerable to NP MTs that are unreactive to traditional, synthetic organic chemistry alkylation protocols. The full potential of this technology is stifled by several impediments largely deriving from the AdoMet-based reagents, including the instability, membrane impermeability, poor synthetic yield and resulting diastereomeric mixtures. To circumvent these main liabilities, novel chemoenzymatic strategies that employ methionine adenosyltransferases (MATs) and methionine (Met) analogs to synthesize AdoMet analogs in vitro were advanced. Unstable AdoMet analogs are simultaneously utilized in a one-pot reaction by MTs for the alkylrandomization of NP scaffolds. As cell membranes are permeable to Met analogs, this also sets the stage for cell-based and, potentially, in vivo applications. In order to further address the instability of AdoMet and analogs thereof, MAT-catalyzed reactions utilizing Met and ATP isosteres generated highly stable AdoMet isosteres that were capable of downstream utilization by MTs. Finally, the development, use, and results of a high-throughput screen identified mutant-MAT/Met-analog pairs suitable for postliminary bioorthogonal applications.

Connor O’Hara July 29, 2019 Inhibition of Cancer Stem Cells by Glycosaminoglycan Mimetics In the United States cancer is the second leading cause of death, with colorectal cancer (CRC) being the third deadliest cancer and expected to cause over 51,000 fatalities in 2019 alone.1 The current standard of care for CRC depends largely on the staging, location, and presence of metastasis.2 As the tumor grows and invades nearby lymph tissue and blood vessels, CRC has the opportunity to invade not only nearby tissue but also metastasize into the liver and lung (most commonly).3 The 5-year survival rate for metastasized CRC is <15%, and standard of care chemotherapy regimens utilizing combination treatments only marginally improve survival.3-5 Additionally, patients who have gone into remission from late-stage CRC have a high risk of recurrence despite advances in treatment.6-7 The Cancer Stem-like Cell (CSC) paradigm has grown over the last 20 years to become a unifying hypothesis to support the growth and relapse of tumors previously regressed from chemotherapy (Figure 1).8 The paradigm emphasizes the heterogeneity of a tumor and its microenvironment, proposing that a small subset of cells in the tumor are the source of tumorigenesis with features akin to normal stem cells.9 The CSCs normally in a quiescent state survive this chemotherapy and “seed” tumor redevelopment.10 First observed in acute myeloid lymphoma models, CSCs have since been identified in various other cancers (to include CRC) by their cell surface antigens and unique properties characterizing them from normal cancer cells.11-12 These include tumor initiation, limitless self-renewal capacity to generate clonal daughter cells, as well as phenotypically diverse, mature, and highly differentiated progeny.13-14 Previously our lab has identified a novel molecule called G2.2 (Figure 2) from a unique library of sulfated compounds showing selective and potent inhibition of colorectal CSCs in-vitro.15 G2.2 is a mimetic of glycosaminoglycans (GAGs) and belongs to a class of molecules called non-saccharide GAG mimetics (NSGMs). Using a novel dual-screening platform, comparisons were made on the potency of G2.2 in bulk monolayer cells, primary 3D tumor spheroids of the same cell line, and subsequent generations of tumor spheroids. This work has shown in-vitro the fold-enhancement of CSCs when culturing as 3D tumor spheroids. Spheroid culture serves as a more accurate model for the physiological conditions of a tumor, as well as the functional importance of upregulating CSCs. Evaluation of G2.2 and other NSGMs was performed in only a few cell lines, developing a need to better understand the ability of G2.2 to inhibit spheroids from a more diverse panel of cancer cells to better understand G2.2’s mechanism. The last few decades have seen the advancement in fundamental biological and biochemical knowledge of tumor cell biology and genetics.16 CRC, in particular, has served as a useful preclinical model in recapitulating patient tumor heterogeneity in-vitro.17 Recent work has characterized the molecular phenotypes of CRC cell lines in a multi-omics analysis, stratifying them into 4 clinically robust and relevant consensus molecular subtypes (CMS).18-19 Our work was directed to screen a panel of cells from each of the molecular subtypes and characterize the action of G2.2 and 2nd generation lipid-modified analogs, synthesized to improve the pharmacokinetic properties of the parent compound. Four NSGMs, namely G2.2, G2C, G5C, and G8C (Figure 2) were studied for their ability to inhibit the growth of primary spheroids across a phenotypically diverse panel. Compound HT-29 IC50 (μM) Panel Average IC50 (μM) G2.2 28 ± 1 185 ± 55 G2C 5 ± 2 16 ± 15 G5C 8 ± 2 63 ± 19 G8C 0.7 ± 0.2 6 ± 3 Primary spheroid inhibition assays were performed comparing the potency of new NSGMs to G2.2. Fifteen cell lines were evaluated in a panel of colorectal adenocarcinoma cell lines with several cell lines representing each CMS. Primary spheroid inhibition assays revealed 3 distinct response with regard to G2.2’s ability to inhibit spheroid growth. Cells from CMS 3 and 4, which display poor clinical prognosis, metabolic dysregulation, and enhanced activation of CSC pathways, showed the most sensitivity to G2.2 (mean IC50 = 89 ± 55 μM). Mesenchymal CMS 4 cell lines were over 3-fold more sensitive to treatment with G2.2 when compared to CMS 1 cell lines. Resistant cell lines were composed entirely of CMS 1 and 2 (mean IC50 = 267 ± 105 μM). In contrast, all lipid-modified analogs showed greater potency than the parent NSGM in almost every CRC cell line. Of the three analogs, G8C showed the greatest potency with a mean IC50 of less than 15 μM. Of the CRC spheroids studied, HT-29 (CMS 3) was most sensitive to G8C (IC50 = 0.73 μM). To evaluate the selectivity of NSGMs for CSC spheroid inhibition, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) cytotoxicity assays were performed on monolayer cell culture, and the fold-selectivity of NSGM for spheroids was analyzed. Data shows that NSGMs preferentially target CSC-rich spheroids compared with monolayer cellular growth, with G2.2 having over 7-fold selectivity for spheroid conditions. This fold selectivity was enhanced in CMS 3/4, supporting the idea that G2.2 targets a mesenchymal and stem-like phenotype. To further validate this selectivity, limiting dilution assays were performed across the panel to determine the tumor-initiating capacity of each cell line. Cell lines which showed a sensitive response to G2.2 were over 2-fold more likely to develop into spheroids, validating the previous hypothesis. Further characterization was performed analyzing the changes G2.2 induced on CSC markers, as well as the basal expression of a unique pair of cancer cells. Western blots showed a reduction in self-renewal marker across all CMS after treatment with G2.2, and that cell lines sensitive to G2.2-treatment overexpress mesenchymal and stem-like markers. G2.2-resistant cell lines show an epithelial phenotype, lacking this expression. The positive results observed in these studies enhance the understanding of G2.2 and analogs, and further evaluation with additional cell lines of various tissues would improve the knowledge thus far gained. However, all experiments described take valuable time to perform and analyze. Thus, there became a need to develop a high-throughput screening (HTS) platform for our assays that standardized analysis and enhanced productivity. Initial development of the method for this assay are underway, and recent evidence from these evaluations of breast cancer spheroids suggests that G2.2 and analogs may be tissue-specific compounds for the treatment of cancer. Future work entails refining the application of this method for evaluation of the NCI-60 (National Cancer Institute) tumor cell panel. Overall, these results make several suggestions concerning the NSGMs evaluated against the panel. First, G2.2 selectively targets CSCs with limited toxicity to monolayer cells of the same cell line. Further, G2.2 has the greatest potency with CMS 3/4, whose mesenchymal phenotypes are associated with poor clinical prognosis and enrichment of CSCs. Supporting evidence include that sensitive cell lines are highly tumorigenic and show enhanced expression of mesenchymal/CSC markers compared to resistant cell lines. Lipid-modification of G2.2 enhances in-vitro potency against spheroid growth, with nM potency reached in the most sensitive cell lines. Evidence in the development of a HTS platform also suggests these NSGMs show tissue specificity to cancers of the intestine. Further work characterizing the mechanism of NSGMs in a broader multi-tissue panel will enhance our understanding of the compounds as a potential therapy to dramatically improve patient survival through specific targeting of tumorigenesis. References 1. Colorectal Cancer Facts & Figures 2017-2019. American Cancer Society 2017. 2. Compton, C. C.; Byrd, D. R.; Garcia-Aguilar, J.; Kurtzman, S. H.; Olawaiye, A.; Washington, M. K. Colon and rectum. In AJCC Cancer Staging Atlas, 2nd ed.; Ed. Springer Science: New York, 2012; pp 185–201. 3. Van Cutsem, E.; Cervantes, A.; Adam, R.; Sobrero, A.; Van Krieken, J. H.; Aderka, D.; Aranda Aguilar, E.; Bardelli, A.; Benson, A.; Bodoky, G.; et al. ESMO consensus guidelines for the management of patients with metastatic colorectal cancer. Ann. Oncol. 2016, 27, 1386–422. 4. Siegel, R. L.; Miller, K. D.; Fedewa, S. A.; Ahnen, D. J.; Meester, R. G. S.; Barzi, A.; Jemal, A. Colorectal cancer statistics, 2017. CA Cancer J. Clin. 2017, 67, 177–193. 5. Moriarity, A.; O'Sullivan, J.; Kennedy, J.; Mehigan, B.; McCormick, P. Current targeted therapies in the treatment of advanced colorectal cancer: a review. Ther. Adv. Med. Oncol. 2016, 8, 276–293. 6. Seidel, J.; Farber, E.; Baumbach, R.; Cordruwisch, W.; Bohmler, U.; Feyerabend, B.; Faiss, S. Complication and local recurrence rate after endoscopic resection of large high-risk colorectal adenomas of >/=3 cm in size. Int. J. Colorectal Dis. 2016, 31, 603–611. 7. Pugh, S. A.; Shinkins, B.; Fuller, A.; Mellor, J.; Mant, D.; Primrose, J. N. Site and stage of colorectal cancer influence the likelihood and distribution of disease recurrence and postrecurrence survival: data from the FACS randomized controlled trial. Ann. Surg. 2016, 263, 1143–1147. 8. Batlle, E.; Clevers, H. Cancer stem cells revisited. Nat. Med. 2017, 23, 1124–1134. 9. Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: the next generation. Cell 2011, 144, 646–674. 10. Tirino, V.; Desiderio, V.; Paino, F.; De Rosa, A.; Papaccio, F.; La Noce, M.; Laino, L.; De Francesco, F.; Papaccio, G. Cancer stem cells in solid tumors: an overview and new approaches for their isolation and characterization. FASEB J. 2013, 27, 13–24. 11. Bonnet, D.; Dick, J. E. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 1997, 3, 730–737. 12. Desai, A.; Yan, Y.; Gerson, S. L. Concise reviews: cancer stem cell targeted therapies: toward clinical success. Stem Cells Transl. Med. 2019, 8, 75–81. 13. Munro, M. J.; Wickremesekera, S. K.; Peng, L.; Tan, S. T.; Itinteang, T. Cancer stem cells in colorectal cancer: a review. J. Clin. Pathol. 2018, 71, 110–116. 14. Zhou, Y.; Xia, L.; Wang, H.; Oyang, L.; Su, M.; Liu, Q.; Lin, J.; Tan, S.; Tian, Y.; Liao, Q.; Cao, D. Cancer stem cells in progression of colorectal cancer. Oncotarget 2018, 9, 33403–33415. 15. Patel, N. J.; Karuturi, R.; Al-Horani, R. A.; Baranwal, S.; Patel, J.; Desai, U. R.; Patel, B. B. Synthetic, non-saccharide, glycosaminoglycan mimetics selectively target colon cancer stem cells. ACS Chem. Biol. 2014, 9, 1826–1833. 16. Punt, C. J.; Koopman, M.; Vermeulen, L. From tumour heterogeneity to advances in precision treatment of colorectal cancer. Nat. Rev. Clin. Oncol. 2017, 14, 235–246. 17. Mouradov, D.; Sloggett, C.; Jorissen, R. N.; Love, C. G.; Li, S.; Burgess, A. W.; Arango, D.; Strausberg, R. L.; Buchanan, D.; Wormald, S.; et al. Colorectal cancer cell lines are representative models of the main molecular subtypes of primary cancer. Cancer Res. 2014, 74, 3238–3247. 18. Guinney, J.; Dienstmann, R.; Wang, X.; de Reynies, A.; Schlicker, A.; Soneson, C.; Marisa, L.; Roepman, P.; Nyamundanda, G.; Angelino, P.; et al. The consensus molecular subtypes of colorectal cancer. Nat. Med. 2015, 21, 1350–1356. 19. Berg, K. C. G.; Eide, P. W.; Eilertsen, I. A.; Johannessen, B.; Bruun, J.; Danielsen, S. A.; Bjornslett, M.; Meza-Zepeda, L. A.; Eknaes, M.; Lind, G. E.; et al. Multi-omics of 34 colorectal cancer cell lines - a resource for biomedical studies. Mol. Cancer 2017, 16, 116–132.

Extracellular vesicles (EVs), exosomes and microvesicles, transfer endogenous RNAs between neurons over short and long distances. We have explored EVs for siRNA delivery to brain. (1) We optimized siRNA chemical modifications and siRNA conjugation to lipids for EV-mediated delivery. (2) We developed a GMP-compatible, scalable method to manufacture active EVs in bulk. (3) We characterized lipid and protein content of EVs in detail. (4) We established how protein and lipid composition relates to siRNA delivering activity of EVs, and we reverse engineered natural exosomes (small EVs) into artificial exosomes based on these data. We established that cholesterol-conjugated siRNAs passively associate to EV membrane and can be productively delivered to target neurons. We extensively characterized this loading process and optimized exosome-to-siRNA ratios for loading. We found that chemical stabilization of 5'-phosphate with 5'-E-vinylphosphonate and chemical stabilization of all nucleotides with 2'-O-methyl and 2'-fluoro increases the accumulation of siRNA and the level of mRNA silencing in target cells. Therefore, we recommend using fully modified siRNAs for lipid-mediated loading to EVs. Later, we identified that α-tocopherol-succinate (vitamin E) conjugation to siRNA increases productive loading to exosomes compared to originally described cholesterol. Low EV yield has been a rate-limiting factor in preclinical development of the EV technology. We developed a scalable EV manufacturing process based on three-dimensional, xenofree culture of mesenchymal stem cells and concentration of EVs from conditioned media using tangential flow filtration. This process yields exosomes more efficient at siRNA delivery than exosomes isolated via differential ultracentrifugation from two-dimensional cultures of the same cells. In-depth characterization of EV content is required for quality control of EV preparations as well as understanding composition–activity relationship of EVs. We have generated mass-spectrometry data on more than 3000 proteins and more than 2000 lipid species detected in exosomes (small EVs) and microvesicles (large EVs) isolated from five different producer cells: two cell lines (U87 and Huh7) and three mesenchymal stem cell types (derived from bone marrow, adipose tissue and umbilical cord Wharton’s jelly). These data represent an indispensable resource for the community. Furthermore, relating composition change to activity change of EVs isolated from cells upon serum deprivation allowed us to identify essential components of siRNA-delivering exosomes. Based on these data we reverse engineered natural exosomes into artificial exosomes consisting of dioleoyl-phosphatidylcholine, cholesterol, dilysocardiolipin, Rab7, AHSG and Desmoplakin. These artificial exosomes reproduced efficient siRNA delivery of natural exosomes both in vitro and in vivo. Artificial exosomes may facilitate manufacturing, quality control and cargo loading challenge that currently impede the therapeutic EV field.