Klaus Buchner

Development of an automated in vitro selection protocol to obtain RNA-based aptamers: identification of a biostable substance P antagonist

We have developed an automated SELEX (Systematic Evolution of Ligands by EXponential Enrichment) process that allows the execution of in vitro selection cycles without any direct manual intervention steps. The automated selection protocol is designed to provide for high flexibility and versatility in terms of choice of buffers and reagents as well as stringency of selection conditions. Employing the automated SELEX process, we have identified RNA aptamers to the mirror-image configuration (d-peptide) of substance P. The peptide substance P belongs to the tachykinin family and exerts various biologically important functions, such as peripheral vasodilation, smooth muscle contraction and pain transmission. The aptamer that was identified most frequently was truncated to the 44mer SUP-A-004. The mirror-image configuration of SUP-A-004, the so-called Spiegelmer, has been shown to bind to naturally occurring l-substance P displaying a Kd of 40 nM and to inhibit (IC50 of 45 nM) l-substance P-mediated Ca2+ release in a cell culture assay.

Late Onset of Ccl2 Blockade with the Spiegelmer mNOX-E36–3′PEG Prevents Glomerulosclerosis and Improves Glomerular Filtration Rate in db/db Mice

Diabetic kidney disease is associated with monocyte chemoattractant CC chemokine ligand 2 (CCL2)-dependent glomerular and interstitial macrophage recruitment. In addition, nephropathy is delayed in Ccl2 mutant diabetic mice. However, whether the late onset of therapeutic Ccl2 blockade modulates the progression of advanced diabetic nephropathy remains unknown. We addressed this question by antagonizing Ccl2 with mNOX-E36-3PEG, an anti-Ccl2 L-enantiomeric RNA aptamer (ie, a Spiegelmer), which binds murine Ccl2 and blocks the recruitment of ex vivo-labeled macrophages to the kidneys of db/db mice with type 2 diabetes. We injected mNOX-E36-3PEG subcutaneously at a dose of 50 mg/kg three times per week into uninephrectomized (1K) db/db mice with advanced glomerulopathy from 4 to 6 months of age. mNOX-E36-3PEG reduced the number of glomerular macrophages by 40% compared with nonfunctional (control) Spiegelmer-treated 1K db/db mice. This result was associated with protection from diffuse glomerulosclerosis and significantly improved the glomerular filtration rate. mNOX-E36-3PEG also reduced renal Ccl2 mRNA and protein expression compared with control Spiegelmer-treated 1K db/db mice of the same age. Together, the late onset of therapeutic Ccl2 blockade, eg, with specific Spiegelmers, offers protection from diffuse glomerulosclerosis in type 2 diabetic db/db mice and, thus, may represent a novel therapeutic strategy for advanced glomerulosclerosis.

The effects of the anti-hepcidin Spiegelmer NOX-H94 on inflammation-induced anemia in cynomolgus monkeys

Anemia of chronic inflammation is the most prevalent form of anemia in hospitalized patients. A hallmark of this disease is the intracellular sequestration of iron. This is a consequence of hepcidin-induced internalization and subsequent degradation of ferroportin, the hepcidin receptor and only known iron-export protein. This study describes the characterization of novel anti-hepcidin compound NOX-H94, a structured L-oligoribonucleotide that binds human hepcidin with high affinity (Kd = 0.65 ± 0.06 nmol/L). In J774A.1 macrophages, NOX-H94 blocked hepcidin-induced ferroportin degradation and ferritin expression (half maximal inhibitory concentration = 19.8 ± 4.6 nmol/L). In an acute cynomolgus monkey model of interleukin 6 (IL-6)-induced hypoferremia, NOX-H94 inhibited serum iron reduction completely. In a subchronic model of IL-6-induced anemia, NOX-H94 inhibited the decrease in hemoglobin concentration. We conclude that NOX-H94 protects ferroportin from hepcidin-induced degradation. Therefore, this pharmacologic approach may represent an interesting treatment option for patients suffering from anemia of chronic inflammation.

In vitro selection using a dual RNA library that allows primerless selection

High affinity target-binding aptamers are identified from random oligonucleotide libraries by an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since the SELEX process includes a PCR amplification step the randomized region of the oligonucleotide libraries need to be flanked by two fixed primer binding sequences. These primer binding sites are often difficult to truncate because they may be necessary to maintain the structure of the aptamer or may even be part of the target binding motif. We designed a novel type of RNA library that carries fixed sequences which constrain the oligonucleotides into a partly double-stranded structure, thereby minimizing the risk that the primer binding sequences become part of the target-binding motif. Moreover, the specific design of the library including the use of tandem RNA Polymerase promoters allows the selection of oligonucleotides without any primer binding sequences. The library was used to select aptamers to the mirror-image peptide of ghrelin. Ghrelin is a potent stimulator of growth-hormone release and food intake. After selection, the identified aptamer sequences were directly synthesized in their mirror-image configuration. The final 44 nt-Spiegelmer, named NOX-B11-3, blocks ghrelin action in a cell culture assay displaying an IC50 of 4.5 nM at 37°C.

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.

A novel C5a-neutralizing mirror-image (l-)aptamer prevents organ failure and improves survival in experimental sepsis.

Complement factor C5a is a potent proinflammatory mediator that contributes to the pathogenesis of numerous inflammatory diseases. Here, we describe the discovery of NOX-D20, a PEGylated biostable mirror-image mixed (l-)RNA/DNA aptamer (Spiegelmer) that binds to mouse and human C5a with picomolar affinity. In vitro, NOX-D20 inhibited C5a-induced chemotaxis of a CD88-expressing cell line and efficiently antagonized the activation of primary human polymorphonuclear leukocytes (PMN) by C5a. Binding of NOX-D20 to the C5a moiety of human C5 did not interfere with the formation of the terminal membrane attack complex (MAC). In sepsis, for which a specific interventional therapy is currently lacking, complement activation and elevated levels of C5a are suggested to contribute to multiorgan failure and mortality. In the model of polymicrobial sepsis induced by cecal ligation and puncture (CLP), NOX-D20 attenuated inflammation and organ damage, prevented the breakdown of the vascular endothelial barrier, and improved survival. Our study suggests NOX-D20 as a new therapeutic candidate for the treatment of sepsis.

A Mixed Mirror-image DNA/RNA Aptamer Inhibits Glucagon and Acutely Improves Glucose Tolerance in Models of Type 1 and Type 2 Diabetes

Background: An increased glucagon/insulin ratio is known to contribute to hyperglycemia in diabetes. Results: NOX-G15, a mirror-image mixed DNA/RNA glucagon-neutralizing aptamer, was identified. It improved glucose tolerance in models of type 1 and 2 diabetes. Conclusion: NOX-G15 may be useful for treatment of type 1 and 2 diabetes. Significance: The new therapeutic candidate may help to reduce insulin need in diabetes. Excessive secretion of glucagon, a functional insulin antagonist, significantly contributes to hyperglycemia in type 1 and type 2 diabetes. Accordingly, immunoneutralization of glucagon or genetic deletion of the glucagon receptor improved glucose homeostasis in animal models of diabetes. Despite this strong evidence, agents that selectively interfere with endogenous glucagon have not been implemented in clinical practice yet. We report the discovery of mirror-image DNA-aptamers (Spiegelmer®) that bind and inhibit glucagon. The affinity of the best binding DNA oligonucleotide was remarkably increased (>25-fold) by the introduction of oxygen atoms at selected 2′-positions through deoxyribo- to ribonucleotide exchanges resulting in a mixed DNA/RNA-Spiegelmer (NOX-G15) that binds glucagon with a Kd of 3 nm. NOX-G15 shows no cross-reactivity with related peptides such as glucagon-like peptide-1, glucagon-like peptide-2, gastric-inhibitory peptide, and prepro-vasoactive intestinal peptide. In vitro, NOX-G15 inhibits glucagon-stimulated cAMP production in CHO cells overexpressing the human glucagon receptor with an IC50 of 3.4 nm. A single injection of NOX-G15 ameliorated glucose excursions in intraperitoneal glucose tolerance tests in mice with streptozotocin-induced (type 1) diabetes and in a non-genetic mouse model of type 2 diabetes. In conclusion, the data suggest NOX-G15 as a therapeutic candidate with the potential to acutely attenuate hyperglycemia in type 1 and type 2 diabetes.

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 5.4u2009mg/kg. In accordance with the plasma half‐life of 38u2009h, the duration of the WBC and HSC mobilization was long lasting and increased dose‐dependently to more than 4 days at the highest dose (10.8u2009mg/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.

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.

Identification and characterization of a mirror-image oligonucleotide that binds and neutralizes sphingosine 1-phosphate, a central mediator of angiogenesis.

The sphingolipid S1P (sphingosine 1-phosphate) is known to be involved in a number of pathophysiological conditions such as cancer, autoimmune diseases and fibrosis. It acts extracellularly through a set of five G-protein-coupled receptors, but its intracellular actions are also well documented. Employing in vitro selection techniques, we identified an L-aptamer (Spiegelmer®) to S1P designated NOX-S93. The binding affinity of NOX-S93 to S1P had a Kd value of 4.3 nM. The Spiegelmer® shows equal binding to dihydro-S1P, but no cross-reactivity to the related lipids sphingosine, lysophosphatidic acid, ceramide, ceramide-1-phosphate or sphingosine phosphocholine. In stably transfected CHO (Chinese-hamster ovary) cell lines expressing the S1P receptors S1PR1 or S1PR3, NOX-S93 inhibits S1P-mediated β-arrestin recruitment and intracellular calcium release respectively, with IC50 values in the low nanomolar range. The pro-angiogenic activity of S1P, and of the growth factors VEGF-A (vascular endothelial growth factor-A), FGF-2 (fibroblast growth factor-2) and IGF-1 (insulin-like growth factor-1), was effectively blocked by NOX-S93 in a cellular angiogenesis assay employing primary human endothelial cells. These data provide further evidence for the relevance of extracellular S1P as a central mediator of angiogenesis, suggesting pharmacological S1P neutralization as a promising treatment alternative to current anti-angiogenesis approaches.