Stefan Vonhoff

DNA vs. Mirror‐Image DNA: A Universal Approach to Tune the Absolute Configuration in DNA‐Based Asymmetric Catalysis

Mirror mirror on the wall: By taking advantage of the unique structural features of L-DNA, the first examples of left-helical enantioselective induction in the field of DNA-based asymmetric catalysis were realized. Most importantly, this approach is the only one that allows a reliable and predictable access to both enantiomers for any given reaction.

SDF-1 Inhibition Targets the Bone Marrow Niche for Cancer Therapy

Bone marrow (BM) metastasis remains one of the main causes of death associated with solid tumors as well as multiple myeloma (MM). Targeting the BM niche to prevent or modulate metastasis has not been successful to date. Here, we show that stromal cell-derived factor-1 (SDF-1/CXCL12) is highly expressed in active MM, as well as in BM sites of tumor metastasis and report on the discovery of the high-affinity anti-SDF-1 PEGylated mirror-image l-oligonucleotide (olaptesed-pegol). In vivo confocal imaging showed that SDF-1 levels are increased within MM cell-colonized BM areas. Using in vivo murine and xenograft mouse models, we document that in vivo SDF-1 neutralization within BM niches leads to a microenvironment that is less receptive for MM cells and reduces MM cell homing and growth, thereby inhibiting MM disease progression. Targeting of SDF-1 represents a valid strategy for preventing or disrupting colonization of the BM by MM cells.

Hematopoietic stem and progenitor cell mobilization in mice and humans by a first-in-class mirror-image oligonucleotide inhibitor of CXCL12.

NOX‐A12 is a PEGylated mirror‐image oligonucleotide (a so‐called Spiegelmer) that binds to CXCL12 (stromal cell–derived factor‐1, SDF‐1) with high affinity thereby inhibiting CXCL12 signaling on both its receptors, CXCR4 and CXCR7. In animals, NOX‐A12 mobilized white blood cells (WBCs) and hematopoietic stem and progenitor cells (HSCs) into peripheral blood (PB). In healthy volunteers, single doses of NOX‐A12 had a benign safety profile and also dose‐dependently mobilized WBCs and HSCs into PB. HSC peak mobilization reached a plateau at five times the baseline level at an i.v. dose of 5.4 mg/kg. In accordance with the plasma half‐life of 38 h, the duration of the WBC and HSC mobilization was long lasting and increased dose‐dependently to more than 4 days at the highest dose (10.8 mg/kg). In conclusion, NOX‐A12 may be appropriate for therapeutic use in and beyond mobilization of HSCs, e.g., in long‐lasting mobilization and chemosensitization of hematological cancer cells.

Polyetheylenimine-Polyplexes of Spiegelmer NOX-A50 Directed against Intracellular High Mobility Group Protein A1 (HMGA1) Reduce Tumor Growth in Vivo

High mobility group A1 (HMGA1) proteins belong to a group of architectural transcription factors that are overexpressed in a range of human malignancies, including pancreatic adenocarcinoma. They promote anchorage-independent growth and epithelial-mesenchymal transition and are therefore suggested as potential therapeutic targets. Employing in vitro selection techniques against a chosen fragment of HMGA1, we have generated biostable l-RNA oligonucleotides, so-called Spiegelmers, that specifically bind HMGA1b with low nanomolar affinity. We demonstrate that the best binding Spiegelmers, NOX-A50 and NOX-f33, compete HMGA1b from binding to its natural binding partner, AT-rich double-stranded DNA. We describe a formulation method based on polyplex formation with branched polyethylenimine for efficient delivery of polyethylene glycol-modified Spiegelmers and show improved tissue distribution and persistence in mice. In a xenograft mouse study using the pancreatic cancer cell line PSN-1, subcutaneous administration of 2 mg/kg per day NOX-A50 formulated in polyplexes showed an enhanced delivery of NOX-A50 to the tumor and a significant reduction of tumor volume. Our results demonstrate that intracellular targets can be successfully addressed with a Spiegelmer using polyethylenimine-based delivery and underline the importance of HMGA1 as a therapeutic target in pancreatic cancer.

RNA Aptamers and Spiegelmers: Synthesis, Purification, and Post‐Synthetic PEG Conjugation

This unit describes the solid‐phase synthesis and downstream processing for RNA oligonucleotides with a length of up to 40 to 50 nucleotides on a 1‐ to 4‐mmol scale with subsequent conjugation to PEG using the L‐RNA spiegelmer NOX‐E36 as an example. Following synthesis and two‐step deprotection, the crude oligonucleotide is purified by preparative reversed‐phase HPLC and desalted by tangential flow ultrafiltration. The resulting intermediate amino‐modified oligonucleotide is reacted with NHS‐ester‐activated PEG, and the oligonucleotide‐PEG conjugate is obtained after preparative AX‐HPLC purification, followed by ultrafiltration and lyophilization. Critical process parameters are described, as well as time considerations and examples for analytical methods used as in‐process and quality controls. Curr. Protoc. Nucleic Acid Chem. 46:4.46.1‐4.46.30.

Physicochemical Stability of NOX-E36, a 40mer L-RNA (Spiegelmer) for Therapeutic Applications

Spiegelmers are structured mirror-image oligonucleotides that are designed to bind and inhibit pharmacologically relevant target molecules. The synthesis and purification of mirror-image oligonucleotides is comparable to the manufacturing of standard oligonucleotides that consist of naturally configured nucleotides. Due to the use of the non-natural L-nucleotides in Spiegelmers, these oligonucleotides show an exceptional biostability. Further, they also display a high physicochemical stability in solution. These properties make them interesting substances for drug development.

Radiosynthesis of New [90Y]-DOTA-Based Maleimide Reagents Suitable for the Prelabeling of Thiol-Bearing l-Oligonucleotides and Peptides

We describe the radiosynthesis of two new [(90)Y]-DOTA-based maleimide reagents, suitable for the mild radiolabeling of L-RNAs and peptides modified with thiol-bearing linkers. The synthesis procedure of both maleimide-bearing (90)Y complexes, [{(2S)-2-[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)benzyl]-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl}tetraacetato][(90)Y]yttrate(1-)([(90)Y]3) and [{(2S)-2-(4-{[4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)butanoyl]amino}benzyl)-1,4,7,10-tetraaza-cyclododecane-1,4,7,10-tetrayl]tetraacetato}[(90)Y]yttrate(1-)([(90)Y]4), was optimized in terms of an easy purification method via solid-phase extraction (SPE). Application as well as reactivity of both maleimide reagents were initially evaluated by the prelabeling of glutathione (GSH) and a thiol-modified 12mer L-RNA as model substances. In comparison to the N-aryl maleimide-bearing complex [(90)Y]3, N-alkyl maleimide-bearing complex [(90)Y]4 showed an increased hydrolytic stability at pH > or = 7. A slightly higher reactivity was found for [(90)Y]3 by prelabeling of 0.1 and 1 microg glutathione, respectively, in phosphate buffer (pH 7.2) at room temperature. In terms of very high radiochemical yields, the direct radiolabeling of DOTA-L-RNA conjugate with [(90)Y]YCl(3) proved to be more suitable than the prelabeling of the thiol-modified 12mer L-RNA derivative with [(90)Y]4.

Methods for L-ribooligonucleotide sequence determination using LCMS

The ability to verify the sequence of a nucleic acid-based therapeutic is an essential step in the drug development process. The challenge associated with sequence identification increases with the length and nuclease resistance of the nucleic acid molecule, the latter being an important attribute of therapeutic oligonucleotides. We describe methods for the sequence determination of Spiegelmers, which are enantiomers of naturally occurring RNA with high resistance to enzymatic degradation. Spiegelmer sequencing is effected by affixing a label or hapten to the 5′-end of the oligonucleotide and chemically degrading the molecule in a controlled fashion to generate fragments that are then resolved and identified using liquid chromatography-mass spectrometry. The Spiegelmer sequence is then derived from these fragments. Examples are shown for two different Spiegelmers (NOX-E36 and NOX-A12), and the specificity of the method is shown using a NOX-E36 mismatch control.

A new class of Spiegelmers containing 2'-fluoro-nucleotides

Abstract Synthesis of 2′-fluoro-nucleosides from L-arabinose in order to perform the synthesis of 2′-fluoro-Spiegelmers binding to a neuropeptide.