Richard I. Hogrefe

Materializing the Potential of Small Interfering RNA via a Tumor-Targeting Nanodelivery System

The field of small interfering RNA (siRNA) as potent sequence-selective inhibitors of transcription is rapidly developing. However, until now, low transfection efficiency, poor tissue penetration, and nonspecific immune stimulation by in vivo administered siRNAs have delayed their therapeutic application. Their potential as anticancer therapeutics hinges on the availability of a vehicle that can be systemically administered, safely and repeatedly, and will deliver the siRNA specifically and efficiently to the tumor, both primary tumors and metastases. We have developed a nanosized immunoliposome-based delivery complex (scL) that, when systemically administered, will preferentially target and deliver molecules useful in gene medicine, including plasmid DNA and antisense oligonucleotides, to tumor cells wherever they occur in the body. This tumor-targeting nanoparticle delivery vehicle can also deliver siRNA to both primary and metastatic disease. We have also enhanced the efficiency of this complex by the inclusion of a pH-sensitive histidine-lysine peptide in the complex (scL-HoKC) and by delivery of a modified hybrid (DNA-RNA) anti-HER-2 siRNA molecule. Scanning probe microscopy confirms that this modified complex maintains its nanoscale size. More importantly, we show that this nanoimmunoliposome anti-HER-2 siRNA complex can sensitize human tumor cells to chemotherapeutics, silence the target gene and affect its downstream pathway components in vivo, and significantly inhibit tumor growth in a pancreatic cancer model. Thus, this complex has the potential to help translate the potent effects of siRNA into a clinically viable anticancer therapeutic.

Chemically modified short interfering hybrids (siHYBRIDS): nanoimmunoliposome delivery in vitro and in vivo for RNAi of HER-2.

A blunt-ended 19-mer short interfering hybrid (siHybrid) (H) comprised of sense-DNA/antisense-RNA targeting HER-2 mRNA was encapsulated in a liposomal nanoplex with anti-transferrin receptor single-chain antibody fragment (TfRscFv) as the targeting moiety for clinically relevant tumor-specific delivery. In vitro delivery to a human pancreatic cell line (PANC-1) was shown to exhibit sequence-specific inhibition of 48-h cell growth with an IC50 value of 37 nM. The inhibitory potency of this siHybrid was increased (IC50 value of 7.8 nM) using a homologous chemically modified siHybrid (mH) in which the 19-mer sense strand had the following pattern of 2 ′-deoxyinosine (dI) and 2 ′-O-methylribonucleotide (2 ′-OMe) residues: 5′-d(TITIT)-2′OMe(GCGGUGGUU)-d(GICIT). These modifications were intended to favor antisense strand-mediated RNAi while mitigating possible sense strand-mediated off-target effects and RNase H-mediated cleavage of the antisense RNA strand. The presently reported immunoliposomal delivery system was successfully used in vivo to inhibit HER-2 expression, and thus induce apoptosis in human breast carcinoma tumors (MDA-MB-435) in mice upon repeated i.v. treatment at a dose of 3 mg/kg of H or mH. The in vivo potency of modified siHybrid mH appeared to be qualitatively greater than that of H, as was the case in vitro.

Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phosphotriester (PTE) modification groups at 3′-terminal and 3′-penultimate internucleotide linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in one-step reverse transcription (RT)–PCR and in real-time PCR with SYBR Green I dye and Taqman® probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the specificity and efficiency of nucleic acid target amplification.

An improved method for the synthesis and deprotection of methylphosphonate oligonucleotides.

The methylphosphonate oligonucleotide synthesis methods described here give the desired products in good yield. Superior amounts of product are achieved by modifying both the DNA synthesis program and the reagent to compensate for the unstable methylphosphonite intermediate. Deprotection conditions have also been altered to maximize the recovery of oligonucleotide from DNA synthesis supports and to minimize the amount of base modification. Mass-spectrometry analysis of our oligonucleotides has verified their purity and confirmed the absence of modified bases. When compared to standard DNA synthesis methods, this procedure uses only about one-third the usual amount of monomer. Using these procedures, it should be possible to synthesize reliably methylphosphonate oligonucleotides at 1- and 15-mumol scales.

A convenient high yield synthesis of N4-isobutyryl-2'-O-methylcytidine and its monomer units for incorporation into oligonucleotides

Abstract We designed an efficient three step procedure for the synthesis of N4-isobutyryl-2′-O-methylcytidine. This protected nucleoside was then used to prepare a methylphosphonamidite monomer for incorporation into oligonucleotides. Transamination at the C4 position of cytidine using ethylenediamine, which has been reported for the N4-benzoyl cytidine, was not observed with N4-isobutyryl protected 2′-O-methylcytidine.

Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation.

For most sample types, the automation of RNA and DNA sample preparation workflows enables high throughput next-generation sequencing (NGS) library preparation. Greater adoption of small RNA (sRNA) sequencing has been hindered by high sample input requirements and inherent ligation side products formed during library preparation. These side products, known as adapter dimer, are very similar in size to the tagged library. Most sRNA library preparation strategies thus employ a gel purification step to isolate tagged library from adapter dimer contaminants. At very low sample inputs, adapter dimer side products dominate the reaction and limit the sensitivity of this technique. Here we address the need for improved specificity of sRNA library preparation workflows with a novel library preparation approach that uses modified adapters to suppress adapter dimer formation. This workflow allows for lower sample inputs and elimination of the gel purification step, which in turn allows for an automatable sRNA library preparation protocol.

Improved and reliable synthesis of 3'-azido-2',3'-dideoxyguanosine derivatives.

An improved synthesis of N2‐protected‐3′‐azido‐2′,3′‐dideoxyguanosine 20 and 23 is described. Deoxygenation of 2′‐O‐alkyl (and/or aryl) sulfonyl‐5′‐dimethoxytritylguanosine coupled with [1,2]‐hydride shift rearrangement gave protected 9‐(2‐deoxy‐threo‐pentofuranosyl)guanines ( 10 , 12 and 16 ). This rearrangement was accomplished in high yield with a high degree of stereoselectivity using lithium triisobutylborohydride (l‐Selectride®). Compounds 10 , 12 and 16 were transformed into 3′‐O‐mesylates ( 18 and 21 ), which can be used for 3′‐substitution. The 3′‐azido nucleosides were obtained by treatment of 18 and 21 with lithium azide. This procedure is reproducible with a good overall yield. †In honor and celebration of the 70th birthday of Professor Leroy B. Townsend.

The “Corey's Reagent,” 3,5-di-tert-butyl-1,2-Benzoquinone, as a Modifying Agent in the Synthesis of Fluorescent and Double-Headed Nucleosides

A new method for the synthesis of fluorescent nucleosides has been developed. It has been shown that a reaction of benzoquinone with aminopropenyl group at C-5-position of 2′-deoxyuridine or 2′-deoxycytidine and aminopropynyl group at the C-7-position of 8-aza-7-deazaadenosine under extremely mild conditions affords conjugated benzoxazole derivatives of nucleosides, which possess strong fluorescent properties. In a similar reaction 5′-amino-5′-deoxy-nucleosides form double-headed nucleoside derivatives with benzoxazole attached at C-4′-position.

Uridine Depletion and Chemical Modification Increase Cas9 mRNA Activity and Reduce Immunogenicity without HPLC Purification

The Cas9/guide RNA (Cas9/gRNA) system is commonly used for genome editing. mRNA expressing Cas9 can induce innate immune responses, reducing Cas9 expression. First-generation Cas9 mRNAs were modified with pseudouridine and 5-methylcytosine to reduce innate immune responses. We combined four approaches to produce more active, less immunogenic second-generation Cas9 mRNAs. First, we developed a novel co-transcriptional capping method yielding natural Cap 1. Second, we screened modified nucleotides in Cas9 mRNA to identify novel modifications that increase Cas9 activity. Third, we depleted the mRNA of uridines to improve mRNA activity. Lastly, we tested high-performance liquid chromatography (HPLC) purification to remove double-stranded RNAs. The activity of these mRNAs was tested in cell lines and primary human CD34+ cells. Cytokines were measured in whole blood and mice. These approaches yielded more active and less immunogenic mRNA. Uridine depletion (UD) most impacted insertion or deletion (indel) activity. Specifically, 5-methoxyuridine UD induced indel frequencies as high as 88% (average ± SD = 79% ± 11%) and elicited minimal immune responses without needing HPLC purification. Our work suggests that uridine-depleted Cas9 mRNA modified with 5-methoxyuridine (without HPLC purification) or pseudouridine may be optimal for the broad use of Cas9 both in vitro and in vivo.

651. Making the Optimal Messenger RNA for Gene Therapy Applications: Evaluation of Novel Nucleotide Modifications for Improved Activity

Recently, there has been significant interest in the use of messenger RNA (mRNA) based expression systems for gene therapy applications. Several groups have shown that mRNA is an attractive vehicle for therapeutic gene expression in mammals (Kormann et al. Nat. Biotechnol (2011) 29, 154; Kariko et al. Molecular Therapy (2012) 20, 948). mRNAs are expressed in the cytoplasm of cells which may improve expression in non-dividing cells, which are difficult to transfect. Additionally, Warren et al. demonstrated highly efficient induced pluripotent stem cell (iPS cells) generation by transfection of mRNAs encoding reprogramming factors (Warren et al. Cell Stem Cell (2010) 7, 618). The authors suggested that iPS cells generated in this manner should be safer than iPS cells derived by plasmid transfection or viral transduction since mRNA poses no risk of insertional mutagenesis and subsequent oncogenesis. In addition, transient expression from mRNA is desirable for applications such as genome editing using zinc-finger nucleases, TALENs and Cas9/CRISPR. Lastly, there is considerable interest in using mRNA for vaccines (Geall et al. Expert Rev Vaccines (2015) 14, 151).A key insight for the development of mRNA expression systems was the recognition that mRNA induces innate immune responses in transfected cells. Kariko et al. showed that substitution of uridine and cytidine residues with pseudouridine and 5-methylcytidine dramatically reduced innate immune recognition of mRNA (Kariko et al. Molecular Therapy (2008) 16, 1833). They also showed that pseudouridine modified RNA was translated more efficiently.These studies highlight the importance of the development of stable, non immunogenic mRNA. Activity and immunogenicity of mRNAs likely depends on the chemical modification pattern, the route of delivery and cell type or tissue transfected. To date, however, there have been few studies to assess novel chemically modifications of mRNAs. Here we synthesized a panel of numerous novel nucleotide triphosphates (NTPs). The panel consisted primarily of 5-position-modified pyrimidines but also included other pyrimidine and purine NTPs. A large number of combinations of the modifications were used to synthesize GFP and luciferase mRNAs in order to evaluate the ability of different combinations of modifications to support transcription by T7 RNA polymerase. The translation potential of the mRNAs was evaluated in rabbit reticulocyte lysates. The activity and toxicity profile of these mRNA was further evaluated in panels of different primary and immortalized cell lines. Comparison of in vitro translation with cell culture expression allows us uncouple translation from cytotoxicity, RNA stability and innate immune stimulation in cells. While trends were seen, cell type specific differences in expression were also observed. These studies greatly expand our knowledge of the optimal chemical modification of mRNA required to achieve maximal expression in different cell types.