Packages

Work Package 2

Long-term studies in immune-tolerant alpha-Mannosidosis mice

Involves Partners CAU, ZYMX, K.U. Leuven and DTI

Objectives:
The aim of this WP is to obtain detailed knowledge about the effect of chronic pre-clinical enzyme replacement studies on the neuropathology and underlying neurological impairments in immune-tolerant alpha-Mannosidosis mice using purified recombinant human LAMAN as the therapeutic agent. In addition, in vitro and in vivo studies will be performed to study the uptake mechanism of rhLAMAN into the brain.

Description of work:
The first goal within WP2 is to find the minimum effective dose in the CNS. After the minimum effective dose has been established in mice, serious long-term treatment studies using the minimum effective dose in immune-tolerant alpha-Mannosidosis mice will be performed. The aim of this study is to evaluate the effect of chronic dosing on the neuropathology and underlying behavioural deficits by detailed morphological, biochemical and behavioural analyses, that will be performed by partners 1, 4 and 9 in collaboration. ERT and subsequent analyses includes the following steps: Prior to injection, blood is taken from the retroorbital plexus to follow antibody titers against the injected enzyme. Mice are then injected into the tail vein with either enzyme solution or PBS (control). Prior to analysis, mice are perfused with PBS and organs dissected out. Organs can then be frozen at either -80°C for biochemical use or postfixed in 4% paraformaldehyde for histological analyses. For electron microscopy, mice have to be perfused with 6% glutaraldehyde. The biochemical analyses include: Sugar extraction and subsequent thin layer chromatography or HPLC, tissue extraction, Western Blot analysis, enzyme activity measurements in tissue extracts and serum, ELISA to determine IgG levels against rhLAMAN in serum, immunoprecipation, pull down assays and tissue mRNA analysis. A brief workplan about implementation of WP2 is outlined below:

Task 1: Minimum effective dose for the CNS: 8-12 weeks old mice will be injected over a period of 3 months with different doses and intervals. After injection mice will be analysed for their sugar storage in brain.
Task 2: Chronic effect of the minimum effective dose on neuropathology: 8-12 weeks old mice will be injected over a period of 3, 6, 12 and 24 months with the minimum effective dose. Injected mice will then be analysed for their brain storage and neuropathology by detailed morphological, biochemical and behavioural analyses.
Task 3: Reversibility of neuropathology by chronic ERT: Old mice (up to 12 months) will be injected over a period of 3, 6 and 12 months with the minimum effective dose. For analyses see Task 2.
Task 4: Prevention of a neuropathology by chronic ERT: Young mice (3-4 weeks) will be injected over a period of 3, 6 and 12 months with the minimum effective dose. For analyses see Task 2.
Task 5: Uptake mechanims of rhLAMAN into the brain: Adult mice will be injected and brain lysates used for pull down assays and proteomic analyses to look for putative in vivo interactions partners. In vitro pull down experiments with immobilized rhLAMAN and brain lysates will be performed in addition.