A gene therapy approach to childhood parkinsonism

Lead Research Organisation: University College London
Department Name: Institute of Child Health

Abstract

Infantile parkinsonism is a severe progressive disease that is incurable and children affected die in adolescence. It begins in infancy with features similar to adult Parkinsons disease with severe difficulties with movement and unsteadiness. The faulty gene that causes this condition has been identified and its function understood. This makes it a potential condition that could be treated by gene therapy as no other treatments have worked or helped so far.

The faulty gene is responsible for producing proteins involved in transporting a chemical in the brain that controls movements. In this condition the protein does not work effectively leading the severe movement difficulties and death in adolescence.

I will research ways to deliver a normal form of the gene to a mouse model with the disease. An animal study is required as it is not safe to study these techniques directly in humans. There is a mouse model that simulates the human from of infantile parkinsonism condition well. This mouse does not produce any of form of the protein and shows all the signs with movement difficulties and reduced longevity seen in the human form of the condition.

We would aim to give the mouse the normal gene through an injection into the blood stream. We would assess for improvements in movements, weight gain and lifespan to assess whether the gene treatment has helped. We would assess carefully for side effects and ability for the gene treatment to reach the brain in the mouse, the area where we want the treatment to work.

We would study the mice to see if the normal function of the gene is restored and would see this with improvements in movements and longevity of the mice. We would assess the mice to ensure the gene treatment is safe and effective and assess the best age to give the treatment and ideal dosage. Further studies of the effects of the gene treatment will be performed by assessing tissue samples from the mice. Successful gene therapy study in mice will help us to proceed to establish future studies in humans. In the future these techniques may also be applicable to other incurable childhood brain disorders.

Technical Summary

Dopamine transporter deficiency syndrome (DTDS) is a newly recognised autosomal recessive neurotransmitter disorder clinically characterised by progressive infantile parkinsonism dystonia. It is caused by loss-of-function mutations in the SLC6A3 gene that encodes for the dopamine transporter (DAT).This progressive condition does not respond to medical or surgical treatments leading to early death.

Gene therapy is increasingly being considered as a realistic treatment option for human disorders including a number of successful clinical gene therapy trials. Indeed there has been a recent clinical gene therapy trial showing positive results in children with AADC deficiency. This is another devastating genetic neurotransmitter disorder lacking in medical treatment. This study demonstrated improvement in oculogyric crisis and motor abilities in all children treated and provides impetus to study potential gene therapy in DTDS.

A proof of concept study would be required to describe expression of transgene delivery and restoration of DAT expression in the first instance. We aim to develop a gene therapy strategy in the knockout mouse model of DTDS. We hypothesise that by inserting human DAT through gene transfer to the DAT-KO mouse model will restore gene function and DAT expression and function.

The key aims of the study are to:

1. Establish a DAT-KO mouse colony at UCL
2. Develop a safe and efficient vector for transgene delivery to the central nervous system in DAT- KO mouse model
3. Demonstrate efficacy through restoration or augmentation of DAT expression through locomotor phenotyping and tissue histological study.
4. Establish optimal time of administration of gene transfer
5. Establish optimal dosage and correlate dosage effects

In the longer term this study would underpin future translation to clinical trials of gene therapy in DTDS. The expertise gained may also be applicable to other untreatable childhood neurological diseases.

Planned Impact

Through this study we would aim that gene vector design, safety and efficacy, optimal method, dosing and timing of gene transfer therapy will have been established in the DAT KO mouse. We would hope that successful gene therapy in a murine model may lead to further translational study.

With Dr Kurian's complimentary work in cell line model and Professor Thrasher's expertise in translating animal study to clinical trial we would hope to use the data from this project to underpin future clinical study. The long term goal of this research may lead to the development of suitable vector delivery systems to be used in DTDS patients trial in patients within a 10 years time-line.

The findings of this study and future application are especially beneficial and relevant to children with DTDS. It will be of relevance to other children with neurotransmitter disorders, movement disorders. In addition, techniques developed from this study may be applicable more widely for research in to gene transfer therapeutic options for other related movement disorders and other untreatable childhood neurological disorders.

Dr Kurian is an established expert clinician in childhood movement disorders and has access to recruit future children with DTDS for study. As Great Ormond Street houses the National Neurotransmitter laboratory in the UK, all children found to have a characteristic neurotransmitter profile associated with DTDS will be easily identified for consideration for clinical study and recruitment for further study through Dr Kurian's established collaboration with Professor Simon Heales, Clinical Chemistry unit, Institute Child Health and Great Ormond Street Hospital.

The findings of this research will be disseminated through relevant scientific meetings and publication in high impact factor journals that would lead to further national and international awareness of this condition and awareness of our collaborative's long term aims of translational research towards future clinical trials. This will lead to further collaboration with other groups which is often required in rare diseases and gene therapy trials.

In addition the members of this collaborative group are already sit on the scientific boards of relevant international family and patientsgroups for children with Neurotransmitter disorders (AADC Trust and Pediatric Neurotransmitter Disorder) who would also be informed of the findings of this work. This again will enhance all future collaboration at an international level to bringing this work towards clinical trial and ensuring ths research of the highest standard is undertaken.

Publications

10 25 50
 
Description 5th International Pediatric Movement Disorders Symposium Travel grant
Amount € 500 (EUR)
Organisation International Parkinson and Movement Disorder Society (MDS) 
Sector Charity/Non Profit
Country United States
Start 03/2017 
End 04/2017
 
Description Development of novel synthetic promoters for neurological gene therapy and childhood parkinsonism
Amount £155,000 (GBP)
Funding ID 550099 
Organisation Synpromics 
Sector Private
Country United Kingdom
Start 01/2019 
End 12/2022
 
Description GOSHCC Starter Grant
Amount £87,000 (GBP)
Funding ID 1PAAE 
Organisation Great Ormond Street Hospital Children's Charity (GOSHCC) 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2013 
End 09/2016
 
Description Gene therapy for Childhood Parkinsonism: Dopamine transporter deficiency syndrome
Amount £495,190 (GBP)
Funding ID MR/R015325/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 06/2018 
End 12/2020
 
Description MRC Developmental Pathway funding scheme
Amount £591,951 (GBP)
Funding ID MR/R015325/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start  
 
Description Ono Pharmaceuticals Rising Star
Amount £65,000 (GBP)
Organisation Ono Pharmaceutical 
Sector Private
Country Japan
Start 07/2017 
End 12/2017
 
Description Project Grant
Amount £32,500 (GBP)
Funding ID Project M576 or A1204 UC 
Organisation Rosetrees Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 10/2016 
End 06/2017
 
Description Project grant
Amount £30,000 (GBP)
Organisation Gracious Heart Charity Foundation 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2014 
End 09/2017
 
Description Project grant
Amount $150,000 (USD)
Organisation NBIA Disorders Association 
Sector Public
Country United States
Start 09/2014 
End 09/2017
 
Description Rosetrees Trust Continuation funding
Amount £102,742 (GBP)
Funding ID A1793 
Organisation Rosetrees Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2018 
End 03/2021
 
Description Synpromics
Amount £122,800 (GBP)
Organisation Synpromics 
Sector Private
Country United Kingdom
Start 01/2018 
End 12/2018
 
Title Adeno associated virus preparation 
Description Establishing making of adenoassociated virus production and purification. 
Type Of Material Cell line 
Provided To Others? No  
Impact Enables AAV production within our research group and reduces reliance on other groups 
 
Title Behavioural studies of DAT-KO mice 
Description Motor behavioural analysis in transgenic mouse colony 
Type Of Material Physiological assessment or outcome measure 
Provided To Others? No  
Impact Enables standardised outcome measure for future gene therapy efficacy 
 
Title Cloning therapeutic vectors 
Description Cloning of transgenes into viral vectors to develop gene therapy vectors. 
Type Of Material Biological samples 
Provided To Others? No  
Impact Vital for rescue of mouse model of disease for this proof of concept gene therapy model 
 
Title Electrophysiology 
Description Electrophysiology of neurons in the striatum of mice were assessed to look at the effects of gene therapy. Mice were collected and brain slices taken for patch-clamp electrophysiology. There were clear differences in firing rates of DAT knockout and wildtype animals. In DAT knockout animals treated with gene therapy the firing rates was restored back to wildtype animal patters. 
Type Of Material Physiological assessment or outcome measure 
Provided To Others? No  
Impact This is another readout to assess the effect of gene therapy 
 
Title Elisa 
Description Quantification method for protein expression delivered by gene therapy 
Type Of Material Biological samples 
Provided To Others? No  
Impact Quantification of protein expression in target organs and off target transduction 
 
Title Establishment of DAT-KO mouse colony 
Description Establish colony at UCL as model of childhood dopamine transporter deficiency syndrome for proof of concept study of gene therapy. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Provided To Others? No  
Impact Establish colony at UCL as model of childhood dopamine transporter deficiency syndrome for proof of concept study of gene therapy. 
 
Title Immunoblotting 
Description CSF analysis of dopaminergic system related proteins in patient CSF. 
Type Of Material Biological samples 
Provided To Others? No  
Impact These observations may demonstrate effects down and upstream of loss of function of dopamine transporter May be a future method to understand disease mechanism in patients 
 
Title Immunocytochemistry 
Description Staining of neuronal cultures 
Type Of Material Cell line 
Provided To Others? No  
Impact To demonstrate location of gene therapy expression protein 
 
Title Immunohistochemistry 
Description Immunohistochemistry used to assess marker gene studies in mouse brain transduction. Immunohistochemistry also used to delineate neurohistopathology in transgenic mouse models of neurological disease 
Type Of Material Antibody 
Year Produced 2014 
Provided To Others? Yes  
Impact Enable marker gene studies for other viral vectors and neurohistopathology in other mouse models. 
 
Title MACS cell seperation for Neuronal isolation 
Description Isolation of neurons using magnetically labelled cells and antibodies using MACS brain dissociation and seperation kits. 
Type Of Material Biological samples 
Provided To Others? No  
Impact Able to isolate cell lines and perform invitro studies from in vivo tissues that have been treated with gene therapy 
 
Title Neurotransmitter analysis 
Description HPLC analysis of neurotransmitter metabolites in mouse brain homogenates 
Type Of Material Technology assay or reagent 
Provided To Others? No  
Impact Reliable biochemical assay for future outcome measure of gene therapy efficacy 
 
Title Stereotactic injection 
Description We have now established stereotactic delivery of AAV vectors to adult mice as a clinically applicable delivery method for translational studies to evaluate gene therapy. 
Type Of Material Model of mechanisms or symptoms - mammalian in vivo 
Year Produced 2018 
Provided To Others? No  
Impact We were awarded MRC DPFS grant and Rosetrees Trust project grant funding to develop this clinically applicable delivery method. This will be considered the delivery method taken towards clinical translation of an clinically applicable gene therapy vector. 
 
Title Stereotactic injection of mice to model human delivery 
Description We have modelled gene therapy delivery in mice that would be applied for DAT deficiency patients in a clinical trial. We demonstrated gene transfer and function with restricted targetting to the area of interest in patients. 
Type Of Material Model of mechanisms or symptoms - human 
Year Produced 2019 
Provided To Others? No  
Impact Having developed this methodology we are now moving to translating the gene therapy in clinical trial with MRC follow on DPFS application for first in human study 
 
Title Database of undiagnosed childhood movement disorders 
Description Children referred to Dr Kurian Childhood movement disorder clinic with undiagnosed movement disorders. Database established to enable grouping of phenotypes to facilitate analysis of whole exome sequencing data. 
Type Of Material Database/Collection of data 
Provided To Others? No  
Impact Database established to enable grouping of phenotypes to facilitate analysis of whole exome sequencing data. 
 
Description Dr Gabriele Lignani 
Organisation University College London
Department Department of Clinical and Experimental Epilepsy
Country United Kingdom 
Sector Academic/University 
PI Contribution Further analysis of the gene therapy in DAT knock mouse model was possible using ex vivo electrophysiology. DAT knockout mice treated with gene therapy and their litter mate controls (untreated knockouts and wildtype mice) were collected for analysis. Brain slices were prepared for patch clamp electrophysiology by Dr Lignani.
Collaborator Contribution Dr Llignany performed patch clamp electrophysiology and showed differences between wildtype and DAT knockout animals in neuronal firing rates. The DAT knockout animals treated with gene therapy showed the neuronal firing rate was restored to wildtype firing patters.
Impact These data will be compiled for publication for gene therapy approach for DAT deficiency.
Start Year 2016
 
Description Gene therapy for cerebrovascular diseases 
Organisation University of North Carolina at Chapel Hill
Department Gene Therapy Center
Country United States 
Sector Academic/University 
PI Contribution Professor Jude Sumulski has provided modified AAV capsid to enable evaluation of viral vectors to develop gene therapy for cerebrovascular disease.
Collaborator Contribution Provision of novel capsid
Impact Materials provided for new project
Start Year 2018
 
Description Gene therapy for neurological disease Dr Ahad Rahim 
Organisation University College London
Department School of Pharmacy
Country United Kingdom 
Sector Academic/University 
PI Contribution Joint experience in gene therapy approaches to neurological diseases
Collaborator Contribution Refinement of neuronal gene therapy tranduction approaches for both neurological and multisystem neurological diseases
Impact Joint grant applications leading towards preclinical gene therapy study of brain iron disorders
Start Year 2013
 
Description Gene therapy infantile epileptic encephalopathy and intellectual disability 
Organisation University of Aberdeen
Department Translational Neuroscience
Country United Kingdom 
Sector Academic/University 
PI Contribution Project to develop gene therapy for childhood epilepsy disorder. We supported establishment of new transgenic colony and in vivo validation of neonatal gene therapy approach for Professor Schorge's research group. Previously her group has focussed on adult epilepsy models.
Collaborator Contribution Establish collaboration with Professor Abbott at University of Edinburgh who runs the patient registry and established other transgenic models of disease.
Impact MTA for transgenic animals, future publications planned. Further longer term project grant application with Ono Pharmaceutics, outcome awaited
Start Year 2017
 
Description In vitro modelling of Dopamine Transporter 
Organisation Oregon Health and Science University
Country United States 
Sector Academic/University 
PI Contribution We have identified novel missense mutations in SLC6A3 for modelling.
Collaborator Contribution Professor Aaron Janowsky research group and undertaking in vitro modelling of Dopamine transporter missense mutations identified in new patient cohort
Impact Manuscript in preparation
Start Year 2018
 
Description In vivo modelling of Dopamine transporter 
Organisation Professor John Glover Memorial Fund
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution We have identified novel missense mutations in DTDS patients
Collaborator Contribution Professor De Schiavi's research group have modelled and characterised c. elegans models of 2 novel missense mutations in DTDS
Impact Manuscript in preparation
Start Year 2017
 
Description In vivo modelling of SLC6A3 mutations 
Organisation Italian National Research Council
Department Institute of Biosciences and Bioresources
Country Italy 
Sector Public 
PI Contribution Professor Elia Di Schiavi's research group have previously modelled disease causing SCL6A3 mutations identified by our research team. We have established a collaboration to model 4 novel missense mutations identified in a cohort of 7 new Dopamine transporter deficiency patients.
Collaborator Contribution Professor Di Schiavi's group have modelled 2 of the 4 novel missense mutations in c elegans model supporting predicted loss of function.
Impact Future publication in progress of new cohort of DTDS patients.
Start Year 2017
 
Description Maarten Reith 
Organisation New York University
Department School of Medicine
Country United States 
Sector Academic/University 
PI Contribution We identify novel genotypes in DAT deficiency syndrome for further function study.
Collaborator Contribution Maarten Reith's group provide expertise and support in functional modelling of novel genotypes in the dopamine transporter. His group provided the plasmids initially used in cloning of gene therapy constructs.
Impact Original publication in Brain 2014: 137; 1107-1119 Dopamine transporter deficiency syndrome: phenotypic spectrum from infancy to adulthood
Start Year 2013
 
Description Marc Caron 
Organisation Duke University
Country United States 
Sector Academic/University 
PI Contribution We have delineated the clinical phenotypes and genotypes of dopamine transporter deficiency leading to greater understanding of the animal model developed prior to the clinical disease identification.
Collaborator Contribution Marc Caron developed the DAT-KO mouse and delivered a personal seminar on this model to our group. He provides expertise in this model to support this project.
Impact The expertise from Marc Caron and his laboratory enabled the rapid and successful establishment of the DAT-KO transgenic colony at UCL
Start Year 2013
 
Description Raul Gainetdinov 
Organisation Italian Institute of Technology (Istituto Italiano di Tecnologia IIT)
Country Italy 
Sector Academic/University 
PI Contribution Professor Gainetdinov kindly provided the DAT-KO mouse to enable a colony to be established at UCL for this fellowship.
Collaborator Contribution Establish colony at UCL as model of childhood dopamine transporter deficiency syndrome for proof of concept study of gene therapy.
Impact DAT-KO colony established at UCL
Start Year 2013
 
Description Simon Heales 
Organisation University College London
Department Institute of Child Health
Country United Kingdom 
Sector Academic/University 
PI Contribution Clinical input into the neurotransmitter analysis of patients referred to Professor Heales Neurotransmitter laboratory
Collaborator Contribution Able to link CSF neurotransmitter patients and DAT=KO mouse model of DTDS deficiency.
Impact Collaboration between clinicians, clinical biochemists and gene therapy groups for biomarker analysis in murine models of human disease.
Start Year 2013
 
Description Synpromics Ltd 
Organisation Synpromics
Country United Kingdom 
Sector Private 
PI Contribution Collaboration with Synpromics Ltd, leaders in gene control to develop novel promoters for dopamine transporter deficiency gene therapy
Collaborator Contribution We will be collaborating to develop novel synthetic promoters that are selective for dopaminergic neurons. This aim is to increase efficiency and specificity of gene expression in AAV gene therapy vectors for Parkinsonian disorders and may allow non invasive delivery for clinical translation.
Impact New project to develop novel promoters commenced in January 2018. Subsequent to this project we have been awarded UCL Synpromics Impact studentship
Start Year 2018
 
Description AADC deficiency research trust 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Patients, carers and/or patient groups
Results and Impact 50 patients and family members is childhood neurotransmitter disease attended conference with clinicians and researchers working in the neurotransmitter field. There was opportunity to engage with families to discuss their treatment experiences, suggestions for future research, participation in current research.
Year(s) Of Engagement Activity 2016
URL http://www.aadcresearch.org/
 
Description AADC deficiency research trust conference 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research and patient groups
Results and Impact The conference combines patients and families with AADC deficiency and experts in neurotransmitter disease and gene therapy.
The conference focussed on the launch for gene therapy clinical trial for this disease and also patients describing their main clinical difficulties.

After this conference further efforts for fundraising and applications for a European site for gene therapy clinical trial were further discussed
Year(s) Of Engagement Activity 2014
 
Description Patient group dialogue 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Patients, carers and/or patient groups
Results and Impact Engagement with patients with 20 Parkinson's disease to discuss gene therapy, the delivery methods and reasons for and against gene therapy trials. Patients reported they would now be more interested in receiving gene therapy.
Year(s) Of Engagement Activity 2018
 
Description School work experience placement 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact School work experience for sixth form students. Exposure to role of animal work in novel therapy development.
Year(s) Of Engagement Activity 2018
 
Description Spring Meeting for Clinician Scientists in training 
Form Of Engagement Activity A broadcast e.g. TV/radio/film/podcast (other than news/press)
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Dr Ng was selected to be interviewed for podcast for the Academy of Medical Sciences Spring meeting for Clinician Scientists in training. In this podcast she was able to discuss the success of the gene therapy developed from this award.
Year(s) Of Engagement Activity 2016
URL https://soundcloud.com/acmedsci/joanne-ng/s-BA6yt?in=acmedsci/sets/spring-meeting-2016/s-5kJCb
 
Description Video for MRC insight 
Form Of Engagement Activity Engagement focused website, blog or social media channel
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Professional Practitioners
Results and Impact Dr Ng was invited to video a mock MRC clinical training fellowship interview for training purposes. The video is a guide to give insight into MRC fellowship application process.
Year(s) Of Engagement Activity 2016
URL https://www.insight.mrc.ac.uk/2016/09/27/interview-day-whats-it-like-to-face-a-fellowship-panel/
 
Description Visit Gracious Heart Foundation 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Supporters
Results and Impact The talk lead to further questions from the public and supporters of the charity. Also lead to other medical research charities contacting our group with further funding proposals to support our studies.

The talk raised awareness of alternative diagnosis for cerebral palsy. Many practitioners involved in conductive education for children with cerebral palsy were present and keen to be aware of alternative diagnoses and treatments.
Year(s) Of Engagement Activity 2014
URL http://www.graciousheart.co.uk