Moss-Derived Human Recombinant GAA Provides an Optimized Enzyme Uptake in Differentiated Human Muscle Cells of Pompe Disease.

Pompe disease is an autosomal recessive lysosomal storage disorder (LSD) caused by deficiency of lysosomal acid alpha-glucosidase (GAA). The result of the GAA deficiency is a ubiquitous lysosomal and non-lysosomal accumulation of glycogen. The most affected tissues are heart, skeletal muscle, liver, and the nervous system. Replacement therapy with the currently approved enzyme relies on M6P-mediated endocytosis. However, therapeutic outcomes still leave room for improvement, especially with regard to skeletal muscles. We tested the uptake, activity, and effect on glucose metabolism of a non-phosphorylated recombinant human GAA produced in moss (moss-GAA). Three variants of moss-GAA differing in glycosylation pattern have been analyzed: two with terminal mannose residues in a paucimannosidic (Man3) or high-mannose (Man 5) configuration and one with terminal N-acetylglucosamine residues (GnGn). Compared to alglucosidase alfa the moss-GAA GnGn variant showed increased uptake in differentiated myotubes. Moreover, incubation of immortalized muscle cells of Gaa-/- mice with moss-GAA GnGn led to similarly efficient clearance of accumulated glycogen as with alglucosidase alfa. These initial data suggest that M6P-residues might not always be necessary for the cellular uptake in enzyme replacement therapy (ERT) and indicate the potential of moss-GAA GnGn as novel alternative drug for targeting skeletal muscle in Pompe patients.

Host Cell Proteome of Physcomitrella patens Harbors Proteases and Protease Inhibitors under Bioproduction Conditions.

Host cell proteins are inevitable contaminants of biopharmaceuticals. Here, we performed detailed analyses of the host cell proteome of moss ( Physcomitrella patens) bioreactor supernatants using mass spectrometry and subsequent bioinformatics analysis. Distinguishing between the apparent secretome and intracellular contaminants, a complex extracellular proteolytic network including subtilisin-like proteases, metallo-proteases, and aspartic proteases was identified. Knockout of a subtilisin-like protease affected the overall extracellular proteolytic activity. Besides proteases, also secreted protease-inhibiting proteins such as serpins were identified. Further, we confirmed predicted cleavage sites of 40 endogenous signal peptides employing an N-terminomics approach. The present data provide novel aspects to optimize both product stability of recombinant biopharmaceuticals as well as their maturation along the secretory pathway. Data are available via ProteomeXchange with identifier PXD009517.

A novel mitochondrial MAVS/Caspase-8 platform links RNA virus-induced innate antiviral signaling to Bax/Bak-independent apoptosis.

Semliki Forest virus (SFV) requires RNA replication and Bax/Bak for efficient apoptosis induction. However, cells lacking Bax/Bak continue to die in a caspase-dependent manner. In this study, we show in both mouse and human cells that this Bax/Bak-independent pathway involves dsRNA-induced innate immune signaling via mitochondrial antiviral signaling (MAVS) and caspase-8. Bax/Bak-deficient or Bcl-2- or Bcl-xL-overexpressing cells lacking MAVS or caspase-8 expression are resistant to SFV-induced apoptosis. The signaling pathway triggered by SFV does neither involve death receptors nor the classical MAVS effectors TNFR-associated factor-2, IRF-3/7, or IFN-ß but the physical interaction of MAVS with caspase-8 on mitochondria in a FADD-independent manner. Consistently, caspase-8 and -3 activation are reduced in MAVS-deficient cells. Thus, after RNA virus infection MAVS does not only elicit a type I antiviral response but also recruits caspase-8 to mitochondria to mediate caspase-3 activation and apoptosis in a Bax/Bak-independent manner.

Uptake of moss-derived human recombinant GAA in Gaa -/- mice.

Pompe disease, an autosomal recessive lysosomal storage disorder, is caused by deficiency of lysosomal acid alpha-glucosidase (GAA). On cellular level, there is lysosomal-bound and free accumulation of glycogen and subsequent damage of organelles and organs. The most severe affected tissues are skeletal muscles and heart. The only available treatment to date is an enzyme replacement therapy (ERT) with alglucosidase alfa, a recombinant human GAA (rhGAA) modified with mannose-6-phosphate (M6P), which is internalized via M6P-mediated endocytosis. There is an unmet need to improve this type of therapy, especially in regard to skeletal muscle. Using different tissue culture models, we recently provided evidence that a moss-derived nonphosphorylated rhGAA (moss-GAA), carrying a glycosylation with terminal N-acetylglucosamine residues (GnGn), might have the potential to improve targeting of skeletal muscle. Now, we present a pilot treatment of Gaa -/- mice with moss-GAA. We investigated general effects as well as the uptake into different organs following short-term treatment. Our results do confirm that moss-GAA reaches the target disease organs and thus might have the potential to be an alternative or complementary ERT to the existing one.

A Survey of Anti-Depressant Prescribing Practice and the Provision of Psychological Therapies in a South London CAMHS from 2003-2006.

A survey was conducted in a South London CAMHS before and after the publication and implementation of NICE guidelines of 2005 for the treatment of depression in children and adolescents. The results for 2006 indicate that 28% of cases were receiving medication without psychological therapy. Of those prescribed medication, 96% were receiving a prescription for fluoxetine. Of those receiving psychological therapy most received cognitive behaviour therapy whilst none received interpersonal therapy. Although 72% of cases were receiving medication with psychological therapy this falls short of the 100% expectation of the NICE guidelines. Other services are encouraged to survey young people receiving ant-depressant medication against the NICE guidelines.

A fast and flexible PEG-mediated transient expression system in plants for high level expression of secreted recombinant proteins.

Plant expression systems offer a valuable alternative to traditional systems for the production of recombinant biopharmaceuticals. A highly efficient polyethyleneglycol (PEG)-mediated transient expression system for secreted recombinant proteins in plants has been developed. The human vascular endothelial growth factor 121 (rhVEGF) has been successfully expressed and efficiently secreted into the culture medium by transiently transformed moss protoplasts. In order to obtain secretion efficiency data, different expressed signal peptides were analysed and time course studies were performed with expression constructs containing different promoters. The transformation procedure was optimised for high level expression (up to 10 microg/ml) and successfully performed even with a transgenic glyco-engineered strain lacking plant-specific immunogenic sugar residues in N-glycans. The amount of rhVEGF was produced in such quantity that it allowed for the analysis of biological activity, silver-staining and Western blotting, revealing the correct formation and processing of the homodimer. This fast and flexible transient expression system enables feasibility studies and construct optimisation to be concluded within a few days, thus avoiding the time consuming step of having to generate stably transformed lines.

Use of Psychotropic Medication in an Outpatient CAMHS Over a 5-Year Period.

BACKGROUND: Psychotropic medication is being prescribed with increasing frequency for children and adolescents but there are very few prescription reviews on the extent of use in the UK. METHOD: A retrospective prescriptions' survey was carried out in an outpatient CAMHS in three time-bands between 1997 and 2002. RESULTS: We found a five-fold increase in the number of prescriptions, a ten-fold increase in the number of young people prescribed medications, and a dramatic increase in the range of psychotropics prescribed. CONCLUSIONS: There is a need to further evaluate prescribing for children and adolescents in the UK. Clearer guidelines should be developed to ensure safe practices.