Cross-Reactive Polyclonal Antibodies Raised Against GalNAc-Conjugated siRNA Recognize Mostly the GalNAc Moiety.

Small interfering RNA (siRNA) is gaining momentum as a therapeutic modality with six approved products. Since siRNA has the potential to elicit undesired immune responses in patients, immunogenicity assessment is required during clinical development by regulatory authorities. In this study, anti-siRNA polyclonal antibodies were generated through animal immunization. These cross-reactive polyclonal antibodies recognized mostly the N-acetylgalactosamine (GalNAc) moiety with a small fraction against sequence-independent epitopes. We demonstrate that the polyclonal antibodies can be utilized as immunogenicity assay positive controls for the same class of GalNAc-conjugated siRNAs. In addition, anti-GalNAc mAbs showed desired sensitivity and drug tolerance, supporting their use as alternative surrogate positive controls. These findings can guide positive control selection and immunogenicity assay development for GalNAc-conjugated siRNAs and other oligonucleotide therapeutics.

The metabolic effects of GDF15 are mediated by the orphan receptor GFRAL.

Growth/differentiation factor 15 (GDF15), also known as MIC-1, is a distant member of the transforming growth factor-ß (TGF-ß) superfamily and has been implicated in various biological functions, including cancer cachexia, renal and heart failure, atherosclerosis and metabolism. A connection between GDF15 and body-weight regulation was initially suggested on the basis of an observation that increasing GDF15 levels in serum correlated with weight loss in individuals with advanced prostate cancer. In animal models, overexpression of GDF15 leads to a lean phenotype, hypophagia and other improvements in metabolic parameters, suggesting that recombinant GDF15 protein could potentially be used in the treatment of obesity and type 2 diabetes. However, the signaling and mechanism of action of GDF15 are poorly understood owing to the absence of a clearly identified cognate receptor. Here we report that GDNF-family receptor α-like (GFRAL), an orphan member of the GFR-α family, is a high-affinity receptor for GDF15. GFRAL binds to GDF15 in vitro and is required for the metabolic actions of GDF15 with respect to body weight and food intake in vivo in mice. Gfral-/- mice were refractory to the effects of recombinant human GDF15 on body-weight, food-intake and glucose parameters. Blocking the interaction between GDF15 and GFRAL with a monoclonal antibody prevented the metabolic effects of GDF15 in rats. Gfral mRNA is highly expressed in the area postrema of mouse, rat and monkey, in accordance with previous reports implicating this region of the brain in the metabolic actions of GDF15 (refs. 4,5,6). Together, our data demonstrate that GFRAL is a receptor for GDF15 that mediates the metabolic effects of GDF15.

Performance of a 3-bladed propeller pump to provide cavopulmonary assist in the failing Fontan circulation.

PURPOSE: We hypothesized that a propeller pump design would function optimally to provide cavopulmonary assist in a univentricular Fontan circulation. DESCRIPTION: The hydraulic and hemolysis performance of a rigid three-bladed propeller prototype (similar to a folding propeller design) was characterized. Pressure and flow measurements were taken for flow rates of 0.5 to 3 liters per minute (LPM) for 5,000 to 7,000 revolutions per minute (RPM) using a blood analog fluid. Hemolysis testing was performed using fresh bovine blood for 2 LPM at 6,000 RPM for a 6-hour duration. EVALUATION: The prototype performed well over the design operating range by producing a pressure rise of 5 to 50 mm Hg. Plasma free hemoglobin concentration remained less than 15 mg/dL. The normalized index of hemolysis peaked during the first hour, and then remained less than 10 mg/dL thereafter. CONCLUSIONS: A propeller pump has the pressure-flow characteristics and minimal risk of hemolysis and venous pathway obstruction which make it ideal for temporary cavopulmonary assist. This type of device has the potential to provide a new therapeutic option for patients with failing univentricular Fontan physiology as a bridge-to-recovery or transplantation.

Mechanical cavopulmonary assist for the univentricular Fontan circulation using a novel folding propeller blood pump.

A blood pump specifically designed to operate in the unique anatomic and physiologic conditions of a cavopulmonary connection has never been developed. Mechanical augmentation of cavopulmonary blood flow in a univentricular circulation would reduce systemic venous pressure, increase preload to the single ventricle, and temporarily reproduce a scenario analogous to the normal two-ventricle circulation. We hypothesize that a folding propeller blood pump would function optimally in this cavopulmonary circulation. The hydraulic performance of a two-bladed propeller prototype was characterized in an experimental flow loop using a blood analog fluid for 0.5-3.5 lpm at rotational speeds of 3,600-4,000 rpm. We also created five distinctive blood pump designs and evaluated their hydraulic performance using computational fluid dynamics (CFD). The two-bladed prototype performed well over the design range of 0.5-3.5 lpm, producing physiologic pressure rises of 5-18 mm Hg. Building upon this proof-of-concept testing, the CFD analysis of the five numerical models predicted a physiologic pressure range of 5-40 mm Hg over 0.5-4 lpm for rotational speeds of 3,000-7,000 rpm. These preliminary propeller designs and the two-bladed prototype achieved the expected hydraulic performance. Optimization of these configurations will reduce fluid stress levels, remove regions of recirculation, and improve the hydraulic performance of the folding propeller. This propeller design produces the physiologic pressures and flows that are in the ideal range to mechanically support the cavopulmonary circulation and represents an exciting new therapeutic option for the support of a univentricular Fontan circulation.