Research
CURRENT RESEARCH IN CDKL5 Funded by IFCR
Your research dollars at work!
Mouse Model (Knock-In)
Researcher: Zhaolan (Joe) Zhou, Ph.D., Asst. Professor Department of Genetics
Laboratory: University of Pennsylvania School of Medicine, Philadelphia, PA
Project started July 2011
Project: IFCR has just begun a second mouse model with Dr. Zhaolan (Joe) Zhou at the University of Pennsylvania. He is developing a germline transmitted knock-in mouse which will have a nonsense mutation R59X. It is imperative that different types of mutated mice are developed to be able to further develop studies to determine what the CDKL5 protein really does and to see how new drugs affect different types of mutations. This particular nonsense mutation was selected for a few reasons. It appears to be a more common mutation for CDKL5 with 5 reported cases in the literature, it is at a strategic site in the gene and we have correlative samples from both a male and female affected with CDKL5 in which iPS cells are also being developed.
Biotech companies are working to develop read-through compounds which will allow the production of RNA and protein despite having a nonsense mutation. Many of the CDKL5 affected children have this type of mutation. These studies will help to correlate activities in mouse and human cells and will help identify a reproducible cellular phenotype that would allow us to test the read-through compounds that are already available. With these correlations it will be much easier to take these compounds into clinical trials.
In Progress
- These mice will still need to be characterized to assure the presence of the CDKL5 nonsense mutation (anticipate August 2012)
- Continued breeding
Future Studies required
- Phenotype characterization (Anticipate 2013)
- Attempt to reverse the phenotype (symptoms of CDKL5) in mice with currently available “read-through” drugs.
- Need 2-3 other mice created with different “human” mutations to allow better phenotype characteization and allow for best model prediction of drug discovery and modifier genes
- Correlation of mouse neuronal development with iPS neuronal development
Development of iPS cells and Differentiation into Neurons
Researcher: Alysson Muotri, PhD
Laboratories: University of California San Diego
Started: April 2011
Project:
Create induded pluripotent stem cells from skin fibroblasts of patients with CDKL5 and differentiate into neuronal cells. Update
Currently have multiple cell lines in growth from males with CDKL5, and corresponding their controls. In addition, we have one cell line from a female with a nonsense mutation R59X, and one from a male with the same mutation. In Progress
Growth and differentiation of cell lines
Characterization of cell lines
Future Studies Required
- May need more females
- Need to develop lines with variety of mutations
- Mouse correlates (see above)
- Use for drug screening and molecular profiling
Mouse Model (Knock out)
Researchers: Cornelius Gross, PhD and Elena Amendola, PhD- post doctoral fellow
European Molecular Biology Laboratory, Monterotondo, Italy
Project started September 2010
Project
Create CDKL5 Knockout mouse model (Generate a mouse that lacks the Cdkl5 gene). Other labs have said that CDKL5 KO mice were embryonic lethal so this lab is using a specilaized Cre-lox technique to create mice derived entirely from the knockout ES cells, while in the traditional procedure only part of the offspring are derived form the knockout ES cells. Update
Currently have heterozygote mice which have been bred and expecting knockout offspring to be born end of July, 2011.
In Progress
- These mice will still need to be characterized to assure absence of CDKL5 (anticipate November 2011)
- Continued breeding
- Phenotype characterization (Anticipate May 2012)
Future Studies required
- May need more full characterization of phenotype (Psychogenics)
- Need 2-3 other mice created with different “human” mutations to allow better phenotype characteization and allow for best model prediction of drug discovery and modifier genes
- Correlation of mouse neuronal development with iPS neuronal development
CDKL5 Antibody Development
Researchers: Cornelius Gross, PhD and Elena Amendola, PhD- post doctoral fellow
European Molecular Biology Laboratory, Monterotondo, Italy
Project started September 2010
Project
- Create 2 different antibodies against the CDKL5 protein to be used for future studies utilizing Immunflourescence for Enzyme Linked ImmunoSorbent Assays (ELISA)
- There are 2 other antibodies that have been previously developed but neither are specific enough and do not work well in ELISA’s
Update
Two protein fragments of the mouse Cdkl5 protein were produced in vitro and have been injected into mice to induce the production of antibodies. These antibodies are currently being tested and appropriate blood cells have been taken for the production of cell lines (hybridomas). In Progress
Hybridomas need to be selected for high level expression and eventually production, purification, and testing of the antibodies. Estimated to be purified and tested by February 2012 Future Studies to consider
Development of 2 more antibodies
Determination of CDKL5 Function
Researchers/Laboratories:
- Elena Amendola, PhD- EMBL, Monterotondo, Italy
- Alysson Muotri, PhD , University of California, San Diego
Project
Variety of approaches using mice, iPS, and cell cultures to determine CDKL5 function
In- Progress
- Awaiting final success with both knock-out and knock-in mice creation, as well as final differentiation of iPS cell lines.
Future Studies
- Correlations between mouse and iPS
Other CDKL5 projects currently in progress:
iPS Cell Lines
Researchers and Laboratories:
Alessandra Renieri, MD-PhD, University of Siena, Siena Italy
John Christodoulou, MD PhD, Childrens Hospital of Westmead, Australia
Project
Create induded pluripotent stem cells from skin fibroblasts of patients with CDKL5 and differentiate into neuronal cells.
Update
Currently 2 iPS cells lines have been developed (1 male, 1 female) in Raneiri lab Future Studies
- Growth and differentiation
- Characterization
- May need more females
- Need to develop lines with variety of mutations
- Mouse correlates (see above)
- Use for drug screening and molecular profiling
Determination of CDKL5 Function
Researchers/Laboratories:
- Nicoletta Landsberger, PhD, Univ of Insurbia, Busto Arsizio, Italy
- Thierry Bienvenu, MD Institut Cochin, Universite´ Paris Descartes, CNRS (UMR 8104), Paris, France
- Alessandra Renieri, MD-PhD, University of Siena, Siena Italy
- John Christodoulou , MD PhD, Childrens Hospital of Westmead, Australia Project Variety of approaches using mice, iPS, and cell cultures to determine CDKL5 function
- Landsberger- CDKL5 phophorylates MeCP2, autophosphorylation
- Kameshita, I., Sekiguchi, M., Hamasaki, D., Sugiyama, Y., Hatano, N., Suetake, I. et al. Cyclin-dependent kinase-like 5 binds and phosphorylates DNA methyltransferase 1. Biochem. Biophys. Res. Commun. 377, 1162–1167 (2008).
- Christodoulou- Microtubule Function (Personal comm)v
- Bienvenu- Significant differences from MeCP2 (personal comm)
Progress
Very little data. All in progress.
Drs Helen Leonard and John Christodoulou have teamed up with clinical geneticist, Dr. Meredith Wilson, for the following CDKL5 project: