Broadening the versatility of lentiviral vectors as a tool in nucleic acid research via genetic code expansion.

With the aim of broadening the versatility of lentiviral vectors as a tool in nucleic acid research, we expanded the genetic code in the propagation of lentiviral vectors for site-specific incorporation of chemical moieties with unique properties. Through systematic exploration of the structure-function relationship of lentiviral VSVg envelope by site-specific mutagenesis and incorporation of residues displaying azide- and diazirine-moieties, the modifiable sites on the vector surface were identified, with most at the PH domain that neither affects the expression of envelope protein nor propagation or infectivity of the progeny virus. Furthermore, via the incorporation of such chemical moieties, a variety of fluorescence probes, ligands, PEG and other functional molecules are conjugated, orthogonally and stoichiometrically, to the lentiviral vector. Using this methodology, a facile platform is established that is useful for tracking virus movement, targeting gene delivery and detecting virus-host interactions. This study may provide a new direction for rational design of lentiviral vectors, with significant impact on both basic research and therapeutic applications.

Maternal Serum Polyols and Its Link to Gestational Diabetes Mellitus: A Population-Based Nested Case-Control Study.

CONTEXT: Sugar alcohols (also called polyols) are regarded as a "healthy" sugar substitute. One of the possible reasons for their safe use in pregnant women is their natural origin and the presence of polyols in maternal and fetal samples during normal human gestation. But little is known about the association between circulating sugar alcohols levels and maternal metabolic disorders during pregnancy. OBJECTIVE: We aimed to detect the concentration of the polyols in participants with and without gestational diabetes mellitus (GDM), and to investigate the association between maternal serum levels of polyols and GDM, as well as newborn outcomes. METHODS: A nested population-based case-control study was conducted in 109 women with and without GDM. Maternal concentrations of serum erythritol, sorbitol, and xylitol in the fasting state were quantified using a time of flight mass spectrometry system. RESULT: In women with GDM, serum concentrations of erythritol and sorbitol were higher, but serum concentrations of xylitol were lower than those in women without GDM. Per 1-SD increment of Box-Cox-transformed concentrations of erythritol and sorbitol were associated with the increased odds of GDM by 43% and 155% (95% CI 1.07-1.92 and 95% CI 1.77-3.69), while decreased odds were found for xylitol by 25% (95% CI 0.57-1.00). Additionally, per 1-SD increase of Box-Cox-transformed concentrations of serum sorbitol was associated with a 52% increased odds of large for gestational age newborns controlling for possible confounders (95% CI 1.00-2.30). CONCLUSION: Maternal circulating sugar alcohols levels during pregnancy were significantly associated with GDM. These findings provide the potential roles of polyols on maternal metabolic health during pregnancy.

Leave Nothing Behind: Treatment of Intracranial Atherosclerotic Disease with Drug-Coated Balloon Angioplasty.

PURPOSE: Intracranial atherosclerosis disease (ICAD) is an essential cause of stroke. The characteristics of effective treatment include low periprocedural risk and a sustained treatment effect. Angioplasty with a conventional balloon for ICAD is safe but has a dauntingly high restenosis rate. Drug-coated balloon (DCB) angioplasty might reduce the risk of restenosis while maintaining the overall safety of the procedure. METHODS: This study included symptomatic ICAD patients with more than 70% stenosis. Intermediate catheters were placed distally, and the lesions were predilated with a conventional balloon, followed by a DCB (SeQuent Please, B Braun, Melsungen, Germany). The primary endpoint was any stroke or death within 30 days or ipsilateral ischemic stroke thereafter. The secondary endpoint was arterial restenosis of more than 50% during follow-up. RESULTS: A total of 39 sessions of DCB angioplasty were performed for 39 lesions in 35 patients between October 2015 and April 2018 in a single center. All of the DCBs could be navigated to the lesions. Major periprocedural complications were noted in two patients (5.7%, 2/35), and minor periprocedural complications were also noted in two patients (5.7%, 2/35). The average percentages of stenosis of the lesions were 76.6% ± 7% before treatment, 32.4% ± 11.2% after DCB angioplasty, and 25% ± 16% at follow-up. Stenosis over 50% was present in 3 lesions during the follow-up period (8.3%, 3/36). CONCLUSION: In this study, the application of DCBs to treat ICAD patients was feasible and safe. A larger scale clinical trial is warranted to further evaluate the safety and efficacy of this treatment.

Precise and combinatorial PEGylation generates a low-immunogenic and stable form of human growth hormone.

In this study, we aimed to develop a safe and stable form of human growth hormone (hGH) and to refine PEGylation methods for therapeutic proteins via genetic code expansion. Through this precise approach, a series of polyethylene glycol (PEG) moieties and sites were combined in various ways. Additionally, the effects of combinatorial PEGylation on the biological, pharmacological, and immunogenic properties of hGH in vitro and vivo were analyzed. Our results showed that combinatorial PEGylation at Y35, G131, and K145 significantly reduced immunogenicity and improved pharmacokinetic (PK) profiles compared with mono-PEGylation, while retaining biological activity. Upon re-examination of the pharmacodynamics in hypophysectomized rats, multi-PEGylated hGH was found to be much more stable than mono-PEGylated hGH. Thus, this method for combinatorial, precise PEGylation may facilitate the development of next-generation, long-acting hGH with low immunogenicity.

Development of next generation of therapeutic IFN-α2b via genetic code expansion.

With the aim to overcome the heterogeneity associated with marketed IFN-α2b PEGylates and optimize the size of the PEG moiety and the site of PEGylation, we develop a viable and facile platform through genetic code expansion for PEGylation of IFN-α2b at any chosen site(s). This approach includes site-specific incorporation of an azide-bearing amino acid into IFN-α2b followed by orthogonal and stoichiometric conjugation of a variety of PEGs via a copper-free click reaction. By this approach, only the chosen site(s) within IFN-α2b is consistently PEGylated under mild conditions, leading to a single and homogenous conjugate. Furthermore, it makes the structure-activity relationship study of IFN-α2b possible by which the opposite effects of PEGylation on the biological and pharmacological properties are optimized. Upon re-examination of the PEGylated IFN-α2b isomers carrying different sizes of PEG at different sites, we find mono-PEGylates at H34, A74 and E107 with a 20-, 10- and 10-kDa PEG moiety, respectively, have both higher biological activities and better PK profiles than others. These might represent the direction for development of the next generation of PEGylated IFN-α2b.