A New Therapeutic Concept Based on Hyperstabilization of Sense-Antisense Duplexes.

We proposed that known helix-stabilizing, minor-groove binding agents such as CC-1065 and its analog, U-71,184, might be feasibly targeted to specific mRNA by tethering them to antisense oligonucleotides. The CC-1065-tethered antisense oligonucleotide might be hyperstabilized to the target mRNA and arrest the translation of mRNA. Our hypothesis is that these agents can bind and increase the stability of RNA-DNA duplexes which may occur between mRNA and antisense oligonucleotides. To verify our hypothesis, we evaluated the ability of CC-1065, U-71,184, and distamycin A to bind and hyperstabilize the 20-mer sense sequence (5\sp\prime-TTACTTCAGTTATGAGACCA-3\sp\prime) duplexed with complementary (antisense) oligonucleotide sequence made with phosphodiester (PO), phosphorothioate (PS), or methyl phosphonate (MP) linkages. Sense DNA sequences (20 mer), when duplexed with PO, PS, or MP DNA oligonucleotides, were saturated at 2, 2, and 1 CC-1065 molecules per duplex, respectively, or at 1 molecule of U-71,184 for each duplex. Each molecule of CC-1065 and U-71,184 was able to bind to poly(rA)-oligo(dT)\sb{20} at maximum binding. Similarly, a sense RNA sequence duplexed with either PO or PS structures was saturated at one CC-1065 molecule per duplex. It was apparent from the melting temperature study that CC-1065 was generally more effective than U-71,184 against DNA-DNA duplexes even if the antisense DNA strand has modified backbone structures. Only CC-1065 was able to hyperstabilize the RNA-DNA duplex as evidenced by a 29\sp\circC increase of the melting temperature. U-71,184 and distamycin A were able to increase the melting temperature of RNA-DNA duplex only about 2\sp\circC and 4\sp\circC, respectively. Translation (wheat germ extract) was inhibited in a dose-dependent manner at antisense:mRNA ratios ranging from 10 to 72. Concomitant incubations of the duplexes with CC-1065 caused significantly enhanced depression of translation at the higher doses. CC-1065 appears to induce hyperstabilization between the antisense oligonucleotide and the mRNA target and arrest translation. Further development of hyperstabilizing antisense oligonucleotide structures is amply justified

Screening, synthesis, and applications of "lipidoids", a novel class of molecules developed for the delivery of RNAi therapeutics

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2008.Vita.Includes bibliographical references.RNAi therapeutics represent a fundamentally new way of treating disease, permitting the selective abrogation of protein production at the genetic level. As nucleic acids do not readily enter cells, carriers are utilized to enhance uptake. While viruses are extremely efficient, concerns about their safety has led to the desire to engineer novel synthetic vectors. We have developed chemical methods using a combinatorial approach that has resulted in the synthesis of a large library of structurally-diverse compounds (1,200 in total), termed "lipidoids", without the need to use solvents, catalysts, or protection/deprotection steps. The members of this library were screened for the ability to transfect mammalian cells in vitro. The top-performing compounds were formulated for in vivo administration and resulted in durable, potent, and specific knockdown of reporter and therapeutically-relevant genes in four distinct species, including non-human primates. Local and systemic administration effectively silenced target genes in three different tissue types. In addition to reducing protein expression by directing cleavage of target mRNA through delivery of siRNA, lipidoids were shown to mediate upregulation of proteins by derpressing miRNA targets through delivery of anti-miR. Finally, inspection of the chemical functional groups common to the top-performing lipidoids from the first library informed the synthesis of a second-generation library. The dramatic increase in percentage of effective materials from this second library (52% as opposed to 3%) confirmed that a convergence of structure has been identified.by Michael Solomon Goldberg.Ph.D

Recent Progress Toward the Templated Synthesis and Directed Evolution of Sequence-Defined Synthetic Polymers

Biological polymers such as nucleic acids and proteins are ubiquitous in living systems, but their ability to address problems beyond those found in nature is constrained by factors such as chemical or biological instability, limited building-block functionality, bioavailability, and immunogenicity. In principle, sequence-defined synthetic polymers based on nonbiological monomers and backbones might overcome these constraints; however, identifying the sequence of a synthetic polymer that possesses a specific desired functional property remains a major challenge. Molecular evolution can rapidly generate functional polymers but requires a means of translating amplifiable templates such as nucleic acids into the polymer being evolved. This review covers recent advances in the enzymatic and nonenzymatic templated polymerization of nonnatural polymers and their potential applications in the directed evolution of sequence-defined synthetic polymers.Chemistry and Chemical Biolog

Branching into RNAi: Synthesis, Characterization and Biology of Branch and Hyperbranch siRNAs

The cancer epidemic continues to afflict millions of humans world-wide each year and despite a renewed hope with the development of new and improved forms of therapy, a cure for cancer remains an elusive goal. This is partly related to the rise of resilient forms of tumors that have evolved with resistance towards conventional chemotherapy and radiation treatments. Moreover, these non-specific therapeutic regimens are highly toxic, leading to severe immunosuppressive effects which poisons the body and compromises the road towards remission. In an effort to mitigate these limitations, cancer-targeting approaches are currently experiencing a renaissance in the translation of new medicines from pre-clinical to bedside use. Notably, gene therapy has recently gained widespread traction in cancer research in the advent of the first RNA interference (RNAi) application in humans. RNAi solicits the use of a double-stranded RNA substrate, aptly named short-interfering RNA (siRNA), which binds to and triggers the degradation of a targeted complementary mRNA strand within the catalytic site of the RNA-Induced Silencing Complex (RISC). In this manner, malignant mRNA expression is silenced, thereby inhibiting the translation of proteins that can lead to the production of pathological disorders such as cancer. In spite of their utility, several challenges still remain towards the development of a fruitful cancer-targeting gene therapy approach. Here, a new class of siRNA motifs is presented to increase substrate efficacy in the RNAi application. Our biological target is a member of the heat shock family of chaperone proteins, the Glucose Regulated Protein of 78 kilodaltons (GRP78) which signals tumor initiation, proliferation and resistance towards chemotherapy. Moreover, GRP78 is overexpressed and cell surface localized on a wide range of resilient tumor types but not on healthy cells, making it a viable bio-marker for the development of the proposed cancer-targeting gene therapy approach. Significantly, an efficient solid-phase synthesis method is described for the production of linear, V-shape, Y-branch and hyperbranch GRP78-silencing siRNAs. The novel V-shape, Y-branch and hyperbranch motifs were then studied by CD spectroscopy and thermal denaturation experiments. CD spectroscopy was used to characterize the requisite A-type double-stranded RNA helix for RNAi application; whereas thermal denaturation experiments were used to validate siRNA hybrid stabilities. With stable siRNA hybrids in hand, their biological activity was assessed in HepG2 hepatoblastoma cells, which constitutes a morbid form of pediatric liver cancer and a valid tumor model for studying our GRP78-targeting strategy. The GRP78 silencing activity of the putative branch and hyperbranch siRNAs is discussed and related to its underlying mechanisms for inducing apoptosis in cancer. Biological studies confirmed potent suppression of GRP78 expression (50-60%) while compromising cancer cell viability by ~20%. The development of an effective cancer-targeting gene therapy approach is highlighted by preliminary results that showcase the utility of a cancer-targeting peptide (CTP) to condense and deliver siRNA within cancer cells for therapeutic treatment. The latter forms the basis of our cancer-targeting gene therapy approach. Thus, branched and hyperbranched siRNAs may serve as potent siRNA candidates in cancer gene therapy applications

Covalent conjugates of therapeutic oligonucleotides for in vivo targeting

Over last few years, the development of oligonucleotide based therapeutics (antisense, siRNA, antagomirs) have received much interest as a novel class of drugs for the treatment of many diseases. Cell/organ specific targeting of oligonucleotides by covalent conjugation has become a promising approach for developing therapeutic RNAs. The major obstacle in the use of therapeutic RNAs is the cell/organ specific targeting and internalization of the large anionic oligonucleotides across the plasma membrane of the cells. This thesis focuses on the synthesis of different receptor specific ligand conjugates of oligonucleotides. The oligonucleotides are conjugated with different targeting ligands such as i) Galactose cluster, ii) Hyaluronic acid hexamer, and iii) Bisphosphonate derivative. Multi-galactose-conjugated 2’-O-methyl oligoribonucleotides showed remarkable galactose-dependent liver targeting of the conjugates monitored by in vivo positron emission tomography (PET) imaging in healthy rats. Hyaluronic acid hexasaccharide oligonucleotide were conjugated efficiently by using copper free click reaction (SPAAC conjugation approach) and also studied by in vivo PET imaging in myocardial infarction rat models. Bone targeting bisphosphonate oligonucleotide conjugates were prepared by SPAAC conjugation approach and in vivo PET imaging exhibited enriched radioactivity accumulation to bones in healthy rats. Additionally, a straightforward method was described for the synthesis of solid supported porphyrin biomolecule conjugates. The whole-body distribution of the conjugates in rats was monitored by PET. These oligonucleotide conjugates were efficiently labeled by complexing 68Ga, with a 3´-terminal 1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (NOTA) ligand. This allowed in vivo quantification of oligonucleotide pharmacokinetics and bio-distribution data in rats.Siirretty Doriast

Synthesis, characterizations and applications of C2'-modified oligonucleotide analogues

During the past two decades, oligonucleotide analogues have drawn considerable attention as potential therapeutic and diagnostic agents. Gene silencing through "RNA interference" (siRNA) or the more mature "antisense" technology (AONs) have proven to be powerful tools for studying gene functions. Chemical modifications of these compounds are generally required to improve their "drug-like" properties such as potency, selectivity and delivery, particularly in the development of oligonucleotide-based therapeutics. Aptamers are another emerging class of oligonucleotide therapeutics and diagnostics.This thesis focuses on oligonucleotides containing 1-(2-deoxy-2-alpha-C-hydroxymethyl-beta- D-ribofuranosyl)thymine (2'-alpha-hm-dT, abbreviated as "H") and 2'-deoxy-2'-fluoroarabinonucleotides (2'F-araN), and their applications. A major component of this work focused on the synthesis of 2'-alpha-hm-dT (H) and the first investigation of oligoribonucleotides containing this nucleoside analogue. Specifically, 2'-CH2O-phosphoramidite and 3'-O-phosphoramidite derivatives of H were synthesized and incorporated into both 2',5'-RNA and RNA chains. Incorporation of 3',5'-linked H units into a DNA, 2',5'-RNA or RNA strand led to significant destabilization of duplexes formed with unmodified RNA targets. 2',5'-Linked H units into 2',5'-RNA or RNA caused significantly less destabilization, and in fact, they were shown to stabilize the loop structure of some RNA hairpins. These results were rationalized in terms of the "compact" and "extended" conformations of nucleotides.A series of branched RNAs (Y-shaped) related to yeast pre-mRNA splicing intermediates were synthesized incorporating both natural (i.e., ribose) and nonnatural (i.e., H, and acyclic nucleoside) branch points in order to examine the effect of sugar conformation and phosphodiester configuration on yeast debranching enzyme (yDBR) hydrolytic efficiency. The results indicate that 2'-phosphodiester scission with yDBR occurs only with a ribose-phosphate backbone at the branch point, whereas some of the H-containing branched RNAs were found to competitively inhibit yDBR hydrolytic activity.This thesis also examines the stabilization of DNA guanine-quadruplexes (G-quadruplexes) by replacing the deoxyribose sugar by a 2-deoxy-2-fluoroarabinose. The effect of this substitution was assessed in the well-known thrombin-binding DNA aptamer d(G2T2G2TGTG2T 2G2), the telomeric DNA d(G4T4G 4) sequence and a phosphorothioate octanucleotide PS-d(T2G 4T2), all of which are known to fold into G-quadruplex structures. Stabilization of the G-quadruplexes was possible provided that the arabinose sugar was introduced at guanosine residues adopting an anti N-glycosidic bond conformation. Some of the arabinose modified thrombin-binding aptamers not only exhibited superior thermal stability and nuclease resistance, but also maintained high thrombin binding affinity.Finally, this thesis examines the ability of DNA polymerases to recognize and utilize 2'-deoxy-2'-fluoro-beta-D-arabinonucleoside 5'-triphosphates (2'F-araNTPs) as building blocks for the synthesis of 2'-deoxy-2'-fluoro-beta- D-arabinonucleic acids (2'F-ANA). The results obtained indicate that a few DNA polymerases can synthesize 2'F-ANA and 2'F-ANA-DNA chimeras on a DNA template. Conversely, certain enzymes were shown to catalyze 2'F-ANA template-directed DNA synthesis. While it was not possible to synthesize 2'F-ANA strands on a 2'F-ANA template, it is possible for some DNA polymerases to catalyze the formation of multiple 2'F-ANA:2'F-ANA base pairs within a DNA-FANA chimeric duplex. These results suggest that it should be possible to evolve FANA-modified aptamers via SELEX