Diligent Design Enables Antibody-ASO Conjugates with Optimal Pharmacokinetic Properties.

Antisense-oligonucleotides (ASOs) are a promising drug modality for the treatment of neurological disorders, but the currently established route of administration via intrathecal delivery is a major limitation to its broader clinical application. An attractive alternative is the conjugation of the ASO to an antibody that facilitates access to the central nervous system (CNS) after peripheral application and target engagement at the blood-brain barrier, followed by transcytosis. Here, we show that the diligent conjugate design of Brainshuttle-ASO conjugates is the key to generating promising delivery vehicles and thereby establishing design principles to create optimized molecules with drug-like properties. An innovative site-specific transglutaminase-based conjugation technology was chosen and optimized in a stepwise process to identify the best-suited conjugation site, tags, reaction conditions, and linker design. The overall conjugation performance was found to be specifically governed by the choice of buffer conditions and the structure of the linker. The combination of the peptide tags YRYRQ and RYESK was chosen, showing high conjugation fidelity. Elaborate conjugate analysis revealed that one leading differentiating factor was hydrophobicity. The increase of hydrophobicity by the ASO payload could be mitigated by the appropriate choice of conjugation site and the heavy chain position 297 proved to be the most optimal. Evaluating the properties of the linker suggested a short bicyclo[6.1.0]nonyne (BCN) unit as best suited with regards to conjugation performance and potency. Promising in vitro activity and in vivo pharmacokinetic behavior of optimized Brainshuttle-ASO conjugates, based on a microtubule-associated protein tau (MAPT) targeting oligonucleotide, suggest that such designs have the potential to serve as a blueprint for peripherally delivered ASO-based drugs for the CNS in the future.

Glycoprotein 130 receptor signaling mediates α-cell dysfunction in a rodent model of type 2 diabetes.

Dysregulated glucagon secretion accompanies islet inflammation in type 2 diabetes. We recently discovered that interleukin (IL)-6 stimulates glucagon secretion from human and rodent islets. IL-6 family cytokines require the glycoprotein 130 (gp130) receptor to signal. In this study, we elucidated the effects of α-cell gp130 receptor signaling on glycemic control in type 2 diabetes. IL-6 family cytokines were elevated in islets in rodent models of this disease. gp130 receptor activation increased STAT3 phosphorylation in primary α-cells and stimulated glucagon secretion. Pancreatic α-cell gp130 knockout (αgp130KO) mice showed no differences in glycemic control, α-cell function, or α-cell mass. However, when subjected to streptozotocin plus high-fat diet to induce islet inflammation and pathophysiology modeling type 2 diabetes, αgp130KO mice had reduced fasting glycemia, improved glucose tolerance, reduced fasting insulin, and improved α-cell function. Hyperinsulinemic-euglycemic clamps revealed no differences in insulin sensitivity. We conclude that in a setting of islet inflammation and pathophysiology modeling type 2 diabetes, activation of α-cell gp130 receptor signaling has deleterious effects on α-cell function, promoting hyperglycemia. Antagonism of α-cell gp130 receptor signaling may be useful for the treatment of type 2 diabetes.

Increased expression of toll-like receptor 4 and inflammatory cytokines, interleukin-6 in particular, in islets from a mouse model of obesity and type 2 diabetes.

Toll-like receptor 4 (TLR4) has received much attention in the recent years due to its role in development of insulin resistance in type 2 diabetes mellitus. Its expression is elevated in fat and muscle from insulin-resistant mice. Several cells of the pancreatic islets, including ß-cells and resident macrophages, express TLR4. Our hypothesis is that expression of TLR4 and downstream signalling molecules in islets increases during progression of type 2 diabetes, thereby contributing to ß-cell damage. We investigated the hypothesis in the db/db mouse. Islets from male db/db (4, 8 and 15 weeks old) and control db/+ (4 and 15 weeks old) mice were examined for mRNA expression of TLR4 and selected cytokines using qPCR. In addition, cytokine secretion from islets was quantified. TLR4 is expressed in islets from lean and obese mice, displaying a 7.4-fold higher level in 15 weeks old db/db relative to age-matched control (p < 0.01). During progression of clinical type 2 diabetes manifested by hyperglycaemia, TLR4 expression increases 5.6-fold in islets from 15 weeks compared with 4 weeks old db/db mice (p < 0.01). Furthermore, both protein and mRNA levels of all cytokines examined increased. In particular, expression of IL-6 increased with 37 fold. Expression of TLR4 in db/db mouse islets increased in parallel with hyperglycaemia. A similar increase in expression and secretion of TNFα, IL-1 and IL-6 was observed. Our results demonstrate that, in addition to its contribution to insulin resistance, TLR4 might also play a role in ß-cell dysfunction in type 2 diabetes.

MicroRNA-29a is up-regulated in beta-cells by glucose and decreases glucose-stimulated insulin secretion.

Chronically elevated levels of glucose impair pancreatic beta-cell function while inducing beta-cell proliferation. MicroRNA-29a (miR-29a) levels are increased in several tissues in diabetic animals and mediate decreased insulin-stimulated glucose-transport of adipocytes. The aim was to investigate the impact of glucose on miR-29a levels in INS-1E beta-cells and in human islets of Langerhans and furthermore to evaluate the impact of miR-29a on beta-cell function and proliferation. Increased glucose levels up-regulated miR-29a in beta-cells and human and rat islets of Langerhans. Glucose-stimulated insulin-secretion (GSIS) of INS-1E beta-cells was decreased by forced expression of miR-29a, while depletion of endogenous miR-29a improved GSIS. Over-expression of miR-29a increased INS-1E proliferation. Thus, miR-29a up-regulation is involved in glucose-induced proliferation of beta-cells. Furthermore, as depletion of miR-29a improves beta-cell function, miR-29a is a mediator of glucose-induced beta-cell dysfunction. Glucose-induced up-regulation of miR-29a in beta-cells could be implicated in progression from impaired glucose tolerance to type 2 diabetes.