Application of a Quality-By-Design Approach to Optimise Lipid-Polymer Hybrid Nanoparticles Loaded with a Splice-Correction Antisense Oligonucleotide: Maximising Loading and Intracellular Delivery.

BACKGROUND: Antisense oligonucleotides (ASOs) are promising therapeutics for specific modulation of cellular RNA function. However, ASO efficacy is compromised by inefficient intracellular delivery. Lipid-polymer hybrid nanoparticles (LPNs) are attractive mediators of intracellular ASO delivery due to favorable colloidal stability and sustained release properties. METHODS: LPNs composed of cationic lipidoid 5 (L5) and poly(DL-lactic-co-glycolic acid) were studied for delivery of an ASO mediating splice correction of a luciferase gene transcript (Luc-ASO). Specific purposes were: (i) to increase the mechanistic understanding of factors determining the loading of ASO in LPNs, and (ii) to optimise the LPNs and customise them for Luc-ASO delivery in HeLa pLuc/705 cells containing an aberrant luciferase gene by using a quality-by-design approach. Critical formulation variables were linked to critical quality attributes (CQAs) using risk assessment and design of experiments, followed by delineation of an optimal operating space (OOS). RESULTS: A series of CQAs were identified based on the quality target product profile. The L5 content and L5:Luc-ASO ratio (w/w) were determined as critical formulation variables, which were optimised systematically. The optimised Luc-ASO-loaded LPNs, defined from the OOS, displayed high loading and mediated splice correction at well-tolerated, lower doses as compared to those required for reference L5-based lipoplexes, L5-modified stable nucleic acid lipid nanoparticles or LPNs modified with dioleoyltrimethylammonium propane (conventional cationic lipid). CONCLUSIONS: The optimal Luc-ASO-loaded LPNs represent a robust formulation that mediates efficient intracellular delivery of Luc-ASO. This opens new avenues for further development of LPNs as a broadly applicable technology platform for delivering nucleic acid cargos intracellularly.

[New-onset diabetes mellitus and liver transplantation, with special consideration of recurrent hepatitis C]. / De novo diabetes és májátültetés, különös tekintettel a hepatitis C-vírus kiújulására.

UNLABELLED: New-onset diabetes is a common complication after liver transplantation. AIM: We aimed to analyze the incidence and rate of known risk factors and the impact of new-onset diabetes mellitus on postoperative outcome. METHODS: We retrospectively evaluated the files of 310 patients who underwent liver transplantation between 1995 and 2009. Definition of new-onset diabetes included: repeated fasting serum glucose >6.8 mmol/l and/or sustained antidiabetic therapy that was present 3 months after transplantation. RESULTS: New-onset diabetes occurred in 63 patients (20%). Differences between the new-onset and the control group were the donor body mass index (24+/-3 vs. 22.4+/-3.6 kg/m 2 , p = 0.003), donor male gender (58% vs. 33%, p = 0.002), and recipient age (47.6+/-7.2 vs. 38.3+/-14.6 year, p<0.001), body mass index (26.7+/-3.8 vs. 23.3+/-5.6 kg/m 2 , p<0.001), male gender (60% vs. 44%, p = 0.031). The 66% of patients with new-onset diabetes were transplanted with cirrhosis caused by hepatitis C virus infection, while in the control group the rate was 23% (p<0.001). Cumulative patient survival rates at 1, 3, 5 and 8 year were 95%, 90.6%, 88% and 88% in the control group, and 87%, 79%, 79% and 64% in the de novo group, respectively (p = 0.011). Cumulative graft survival rates at 1, 3, 5 and 8 year in the control group were 92%, 87%, 86% and 79%, in the de novo diabetes group the rates were 87%, 79%, 79%, 65%, respectively (p = NS). In case of early recurrence (in 6 months), majority of patients developed new-onset diabetes (74% vs. control 26%, p = 0.03). More patients had more than 10 times higher increase of the postoperative virus titer correlate to the preoperative titer in the de novo diabetes group (53% vs. 20%, p = 0.028). Mean fibrosis score was higher in new-onset group one year after the beginning of antiviral therapy (2.05+/-1.53 vs. 1.00+/-1.08, p = 0.039). CONCLUSIONS: Risk factors for new-onset diabetes after transplantation are older age, obesity, male gender and cirrhosis due to hepatitis C infection. The early recurrence, viremia and more severe fibrosis after antiviral therapy have an impact on the occurrence of new-onset diabetes in hepatitis C positive patients.