Glycosylation of recombinant adeno-associated virus serotype 6.

Glycosylation of biopharmaceuticals can affect their safety and efficacy. Glycans can occur on recombinant adeno-associated viruses (rAAVs) that are used for gene therapy; however, the types of glycans that attach to rAAVs are controversial. Here, we conducted lectin microarray analyses on six rAAV serotype 6 (rAAV6) preparations that were produced differently. We demonstrate that O-glycans considered to be attached to rAAV6 were recognized by Agaricus bisporus agglutinin (ABA) and that N-glycans were detected in rAAV6 purified without affinity chromatography. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis showed that the N-glycans detected in rAAV6 were derived from host cell proteins. A combination of ABA-based fractionation and LC-MS/MS revealed that rAAV6 was O-glycosylated with the mucin-type glycans, O-GalNAc (Tn antigen), and mono- and di-sialylated Galß1-3GalNAc (T antigen) at S156, T162, T194, and T201 in viral protein (VP) 2 and with O-GlcNAc at T242 in VP3. The mucin-type O-glycosylated rAAV6 particles were 0.1%-1% of total particles. Further physicochemical and biological analyses revealed that mucin-type O-glycosylated rAAV6 had a lower ratio of VP1 to VP2/VP3, resulting in a lower transduction efficiency both in vitro and in vivo compared with rAAV6 without mucin-type O-glycans. This report details conclusive evidence of rAAV glycosylation and its impact on rAAV-based therapeutics.

Differential Glycoform Analysis of MUC1 Derived from Biological Specimens Using an Antibody-Overlay Lectin Microarray.

The association between altered glycosylation of MUC1 and various disease events has sparked significant interest. However, analytical technologies to investigate the disease-related glycoforms of endogenous MUC1 in blood and tissue specimens are limited. Therefore, we devised a reliable technique for differential analysis of endogenous MUC1 glycoforms based on an antibody-assisted lectin microarray. Its highly sensitive detection aids in analyzing soluble MUC1 from relatively small amounts of serum via a simple enrichment process. Micro-/macro-dissection of the MUC1-positive region is combined with glycoform analysis of the membrane-tethered MUC1. Thus, we have optimized the protocol for sample qualification using immunohistochemistry, sample pretreatment for tissue sections, protein extraction, purification via immunoprecipitation, and the antibody-overlay lectin microarray, which are sequentially essential for differential glycoform analysis of endogenous MUC1.

Studies on the alpha-(1-->4)- and alpha-(1-->8)-fucosylation of sialic acid for the total assembly of the glycan portions of complex HPG-series gangliosides.

A synthetic study on alpha-(1-->4) and alpha-(1-->8)-fucosylation of sialic acid is reported, with the ultimate aim being the total assembly of the glycan portion of HPG-series gangliosides. In both types of fucosylations, the combination of a phenylthio fucosyl donor and a 1,5-lactamized acceptor provided high-yielding glycosylations to afford alpha-fucosyl-sialic acid sequences. The obtained alpha-Fucp-(1-->8)-NeupNAc glycan having a 1,5-lactam bridge has been successfully transformed into the corresponding glycosyl donor.

Schizophrenia and narcolepsy: a review with a case report.

Several reports emphasize the importance of differentiating between psychosis in schizophrenia and the psychotic form of narcolepsy. The failure to identify narcolepsy leads to the labeling of patients as refractory to standard treatments for schizophrenia and retards consideration of intervention for narcolepsy in which psychosis can improve with psychostimulant treatment. Psychosis in patients with narcolepsy can occur in three ways: (i) as the psychotic form of narcolepsy with hypnagogic and hypnopompic hallucinations; (ii) as a result of psychostimulant use in a patient with narcolepsy; and (iii) as the concurrent psychosis of schizophrenia in a patient with narcolepsy. The present case report describes a difficult-to-treat patient who likely had concurrent schizophrenia and narcolepsy. It then summarizes the literature related to the treatment of the three types of patients with psychosis associated with narcolepsy.