Progress report from the research team
Principal Investigator, Prof. Alan Mackay-Sim provided this report:
Non-SPAST HSP project
Fan quantified proteins in the non-SPAST HSP cells, comparing them with SPAST HSP cells and healthy controls (beta-tubulin, acetylated alpha tyrosinated alpha-tubulin, spastin, stathmin, etc) using western blot analysis of purified proteins as well as cellular imaging using the Operetta system. He also analysed the distributions of cellular components that depend on intracellular trafficking (peroxisomes, mitochondria and endoplasmic reticulum). He also quantified oxidative stress in the three cell types using the 4-HNE expression assay developed by Gautam for SPAST HSP cells. Results of these studies are not yet available.
Cultures of mice (heterozygous, homozygous and wild type) cortical neurons have now been successfully established.
This is a major milestone in the study and a very pleasing outcome. These cells will now be investigated and analysed in vitro for:
- qualification of their morphology
- the growth rate of axons
- expression of proteins of interest including SPG4/SPAST, SPG3A, STMN, acetylated alpha-tubulin.
Now that this project is well underway, forward planning estimates can be established with some confidence. Given the delays in receiving the mice with travel and quarantine restrictions in the UK, together with issues described in the last progress report with initial attempts at breeding and technological problems with imaging equipment, the new projection for completion is the end of June 2016, 6 months behind schedule. Original project funding terminates as of year-end, but the HSP Research Foundation has agreed to provide the $76,000 funding required to complete the project and fulfil the need for supporting data for an application to the TGA for clinical trials.
Induced pluripotent stem cell project
A major breakthrough has been achieved in this project over the past 3 months.
The first batch of induced pluripotent stem cells (iPSCs) generated from an HSPer with a SPAST mutation and from two people without HSP now passes the rigourous quality control process, meaning that the cells have been successfully reprogrammed and are suitable and reliable for further experimentation.
This is the end point of a repetitive and tedious phase of culturing and genetically engineering the stem cells, constantly evaluating them on measures of gene expression such as chromosome stability, differentiation potential and pluripotency status, making further adjustments in engineering them, re-evaluating and so on in multiple experimental cycles.
The next step is to differentiate the cells into corticospinal neurons, which are specialised nerve cells in the brain that have a motor function, and are the particular types of cells that are damaged and degenerated in people with SPAST HSP. It is in these cells where the impairment associated with SPAST HSP mutations shows up.
This differentiation process is largely experimental, elaborate, complex, variable and lengthy. The estimated time required for the process is 60 days and cells from the 1st batch that have passed the quality control tests are presently undergoing this process.
Batch 2 iPSC reprogramming
There are plans to generate and reprogram a 2nd batch of iPSCs. This batch will be based on samples taken from two HSPers with SPAST mutations, two HSPers with SPG7 mutations and one from a person without HSP. Cultures of stem cell lines and other genetic material required for the genetic reprogramming process are ready. The reprogramming process for the second batch will begin before the end of the year.
Despite the advances and progress made in this project, creating the material on which to test HSP candidate drugs is not achievable within the original project timeframe of the end of 2015. A further 6 months and approximately $60,000 is required to complete this project with the aim of providing supporting data for an application to the TGA for clinical trials. The HSP Research Foundation has agreed in principle to provide this additional funding subject to submission and consideration of detailed plans for extension.