Final Report Summary - NEUGENE (Advanced gene therapy tools for treatment of CNS-specific disorders)
Executive summary:
Neurodegenerative disorders like Alzheimer's and Parkinson's disease are affecting substantial cohorts of patients, with incidences dramatically increasing in the elderly societies of Western Europe, as ageing is the major risk factor for these otherwise idiopathic disorders. No curative therapies are currently available despite decades of intense research, and even symptomatic treatments are effective only temporally.
Gene therapy is an approach to restore function of diseases organs, e.g. the brain, by means of transferring genetic information specifically into the affected cells and tissues. This genetic information may encode proteins which can restore or protect functionality, e.g. neurotrophic factors which support neurons to function properly and to survive critical situations, or enzymes which help neurons to synthesise essential neurotransmitters needed to maintain critical communication pathways. In its current setting gene therapy is an irreversible process, as the transferred genetic information cannot be withdrawn in case of unforeseen problems, as it is possible with pharmaceutical or physical therapies. This issue can be regarded as a two edged sword: on the one hand gene therapy ensures that a potentially curative molecule can act in the diseased cells and tissues for a long time, but on the other hand this irreversibility may eventually turn into severe problems in case that unwanted side-effects occur.
The major tools for gene therapy are engineered viruses, as it can be regarded the raison d'être of viruses to efficiently transport genetic information into their target cells. During the last two decades these recombinant viruses (viral vectors) have been developed into safe and efficient tools for both, basic research and for some therapeutic applications. However, substantial technical and ethical hurdles still need to be overcome in order to let gene therapy fulfil its promises in larger cohorts of patients.
The NEUGENE consortium was founded to overcome currently existing limitations of gene transfer vectors for safe and efficient gene therapy of major neurodegenerative disorders. The consortium used Parkinson's disease as a model, but the advanced tools developed are thought to be of paramount importance for development of gene therapeutic strategies for other brain diseases, as well. The major objectives were:
1) the targeting of viral vectors to specific cell types of the brain, in order to be able to restrict expression of therapeutic molecules to either neurons or glia (which actively supports neuronal maintenance and thus may be a novel target for forthcoming therapies), or even to specific subtypes of neurons;
2) the regulation of transgene expression, i.e. the implementation of switches which can turn on or off the therapeutic molecule;
3) the safety of the advanced viral vectors; and
4) the efficacy of the advanced tools in standardised rodent models of Parkinson's disease.
Most (but not all) of our attempts have resulted in successful development of viral vectors with characteristics superior over previously existing material. Even though the NEUGENE consortium was mainly dedicated to basic research and not to the direct clinical application of advanced viral vector tools, the substantial success in preclinical evaluation has already allowed to enter the first recombinant viruses aiming to restore neurotransmitter synthesis into a clinical development programme for treatment of Parkinson's patients.
Project context and objectives:
According to the consensus document on European brain research? (Olesen et al, J Neurol Neurosurg Psychiatry 77, 2006) the socioeconomic burden of diseases affecting the human brain is estimated to constitute 35 % of all European Union (EU) disease burden. Demographic changes in ageing societies of the EU will increase this rate considerably and this will represent a crucial challenge to forthcoming generations. Fortunately, enormous progress has been made during the last decade in understanding mechanisms and principles of both normal brain function and CNS disorders. This increase in knowledge makes new treatments for nervous system diseases realistic. Unfortunately, current pharmacological treatment strategies for neurodegenerative diseases are at best partially effective. Typically, they offer only some symptomatic relief. Generally, they are not able to affect disease progression. Furthermore, pharmacotherapies are limited by restrictions set by the blood-brain-barrier and by their relatively untargeted mode of action. As a consequence, they often provoke side effects, especially following long-term treatment. Thus, the targeted delivery of protective, or even curative genes to specific populations of disease-affected brain cells is an exciting future alternative. This approach may offer significant relief or even cure to presently untreatable or only symptomatically treatable brain diseases. Several proteins and regulatory Ribonucleic acid (RNAs) involved in normal brain function and disease progression have been identified or will be identified in the near future. Targeting these molecules directly rather than developing drugs that modulate them may drastically reduce cost and time efforts in developing medications as compared to classical pharmacology. Ultimately, targeted and tightly controlled delivery of genetic medicines may open up venues for treatment strategies taking into account specific personal requirements of individual patients.
The objectives of the NEUGENE consortium are thus to develop and to validate the tools necessary for safe, efficient, durable, targeted and regulated expression of therapeutic molecules within the Central nervous system (CNS).
These objectives are reflected by the 4 scientific Work packages (WPs):
WP1: Targeting, aimed at directing or restricting expression of therapeutic proteins or short hairpin RNAs (shRNAs) to specific populations of CNS neurons, as characterised by specific neurotransmitters or susceptibility to disease conditions. Another major objective was to achieve specific targeting of astroglial cells, as these actively support neurons and can open novel venues for CNS gene therapy, e.g. as sources for neurotrophic factors.
WP2: Regulation, aimed at development of tightly regulated vector systems capable to control and turn on and off transgene expression. These systems were developed for two different scenarios, i.e. controlled expression of neurotrophic factors and controlled expression of neurotransmitter synthesising enzymes.
WP3: Safety, addressed immune responses to LV vectors, to novel types of AAV vectors, the influence of inflammatory lesions, and other safety issues.
WP4: Functional validation: aimed at verifying technological advances such as regulated and targeted AAV and LV vectors in a standardised rodent model of Parkinson's disease, in order to be able to compare efficacy of the different approaches against each other and to be able to identify the most promising strategy for forthcoming gene therapy approaches to treat Parkinson patients.
Project results:
Targeting of AAV and LV vectors
Glial cells and especially astrocytes serve essential roles in maintenance of the CNS. Thus, making brain astrocytes available for CNS gene transfer strategies was one major focus of NEUGENE, which has been fully achieved. Using different strategies for AAV and LV vectors the consortium was successful in developing astrocytes-specific vectors, which are already used for functional studies including gene therapy in animal models. We also optimised gene transfer into the main target cell population affected by Parkinson's disease, nigral dopaminergic neurons. Neuronal subtype-specific promoters allowed for targeting of transgene expression to neuronal subtypes or specific disease-affected neurons.
Regulation of transgene expression
We followed two different strategies for development of systems capable of controlling transgene expression levels in the brain: regulatory protein-based and RNA aptamer-based. Work on the latter is on-going, but for the time being we have not met this objective. In contrast, two different regulatory protein-based strategies have been tested successfully in vivo. Both regulated systems under development towards clinical applicability have demonstrated excellent manageability. Both systems needed a large body of work (and indeed more work than expected) for fine-tuning of their respective components to achieve optimal results in pre-clinical animal models. As outlined below, both systems have been functionally tested in relevant pre-clinical animal models of PD.
Safety / Immunology
In order to mimic the human situation as closely as possible, we developed an immunisation protocol for rats making use of the antigen which humans encounter, i.e. the wild-type AAV-2 virus. Preimmunisation with wt AAV-2 significantly decreased transgene expression in the brain following intracerebral administration of AAV-2 vector encoding EGFP. These results clearly indicate that preexisting immunity to AAV-2 may compromise the efficiency of AAV-2-based vector systems even in immune privileged organs such as brain. Additional induction of a lesion which causes up-regulation of inflammatory markers did not further reduce transduction efficacy, indicating that it was only the specific anti-capsid directed immune reaction precluding successful transgene expression.
Furthermore, we investigated immune issues related to Equine infectious anemia virus (EIAV) lentiviral vectors, to which humans are not naturally immunocompetent. Our results demonstrated that CNS application of these vectors is not substantially hindered by peripheral immunity, arguing for a superior safety profile.
During the course of our studies, novel safety aspects arose and were investigated. Unexpected persistence of AAV capsids and genomes in CNS neurons however did not introduce uncontrollable safety risks.
Functional validation
Expression of optimised levels of DOPA synthesising TH / GCH1 successfully restored motor performance in a model of late PD. This tool entered a clinical development programme. An AAV vector was generated to constitutively express Glial cell line-derived neurotrophic factor (GDNF) in astrocytes, thereby preventing off-target delivery of the neurotrophin without any restriction in therapeutic efficacy. This tool offers novel delivery options for neurotrophic factor-based gene therapy. An AAV vector system was generated for short-term intermitted induction of GDNF expression, providing substantial improvements in motor performance and neuroprotection in a model of early PD. This tool is currently developed further towards clinical applicability. A non-integrating LV vector expressing GDNF was generated and provided the same level of neuroprotection as conventional integrating LV vectors. These tools will provide added safety for forthcoming therapeutic strategies using LV vectors.
Neuronal subpopulation-specific and glia-specific AAV and LV vectors were generated, which are currently exploited to generated advanced animal models of neurodegenerative diseases.
Potential impact:
The majority of projects conducted by the NEUGENE consortium were of a basic research nature, and thus direct economic or wider societal impacts were not intended. Clearly, it will be the scientific community (neuroscientists, gene therapists etc.) who will have the major benefit from the work presented so far and from further work resulting from current projects.
None the less, the most advanced vector tool developed through our studies is patented and has entered into a clinical development program for DOPA restoration gene therapy of PD. Regulated neurotrophin-expressing vectors have attracted the interest of a leading SEM in the field of gene therapy, and will be developed towards clinical applicability in due course. As stated in the initial plan for dissemination of foreground these further studies will comprise the use of non-human primates to prove safety in a more advanced model, and thus initiation of clinical trials can be envisaged no earlier than 2015.
Several other advanced vector tools like cell type-specific standard and non-integrating lentiviral vectors are about to be patented, and will be made available for the scientific community as soon as possible.
Much of the work progress of the NEUGENE consortium is freely available to the scientific community. Our results have been presented at 19 conference contributions and in 14 peer-reviewed publications, mostly in high-ranked neuroscience or gene therapy journals. The number of high ranked publications will further increase in the next months, as several manuscripts are in revision, submitted, or in preparation. Actually, several highly interesting studies resulting from work in the WP4 ('Functional validation') are just about to be prepared for publication.
List of websites: www.neugene.eu
Neurodegenerative disorders like Alzheimer's and Parkinson's disease are affecting substantial cohorts of patients, with incidences dramatically increasing in the elderly societies of Western Europe, as ageing is the major risk factor for these otherwise idiopathic disorders. No curative therapies are currently available despite decades of intense research, and even symptomatic treatments are effective only temporally.
Gene therapy is an approach to restore function of diseases organs, e.g. the brain, by means of transferring genetic information specifically into the affected cells and tissues. This genetic information may encode proteins which can restore or protect functionality, e.g. neurotrophic factors which support neurons to function properly and to survive critical situations, or enzymes which help neurons to synthesise essential neurotransmitters needed to maintain critical communication pathways. In its current setting gene therapy is an irreversible process, as the transferred genetic information cannot be withdrawn in case of unforeseen problems, as it is possible with pharmaceutical or physical therapies. This issue can be regarded as a two edged sword: on the one hand gene therapy ensures that a potentially curative molecule can act in the diseased cells and tissues for a long time, but on the other hand this irreversibility may eventually turn into severe problems in case that unwanted side-effects occur.
The major tools for gene therapy are engineered viruses, as it can be regarded the raison d'être of viruses to efficiently transport genetic information into their target cells. During the last two decades these recombinant viruses (viral vectors) have been developed into safe and efficient tools for both, basic research and for some therapeutic applications. However, substantial technical and ethical hurdles still need to be overcome in order to let gene therapy fulfil its promises in larger cohorts of patients.
The NEUGENE consortium was founded to overcome currently existing limitations of gene transfer vectors for safe and efficient gene therapy of major neurodegenerative disorders. The consortium used Parkinson's disease as a model, but the advanced tools developed are thought to be of paramount importance for development of gene therapeutic strategies for other brain diseases, as well. The major objectives were:
1) the targeting of viral vectors to specific cell types of the brain, in order to be able to restrict expression of therapeutic molecules to either neurons or glia (which actively supports neuronal maintenance and thus may be a novel target for forthcoming therapies), or even to specific subtypes of neurons;
2) the regulation of transgene expression, i.e. the implementation of switches which can turn on or off the therapeutic molecule;
3) the safety of the advanced viral vectors; and
4) the efficacy of the advanced tools in standardised rodent models of Parkinson's disease.
Most (but not all) of our attempts have resulted in successful development of viral vectors with characteristics superior over previously existing material. Even though the NEUGENE consortium was mainly dedicated to basic research and not to the direct clinical application of advanced viral vector tools, the substantial success in preclinical evaluation has already allowed to enter the first recombinant viruses aiming to restore neurotransmitter synthesis into a clinical development programme for treatment of Parkinson's patients.
Project context and objectives:
According to the consensus document on European brain research? (Olesen et al, J Neurol Neurosurg Psychiatry 77, 2006) the socioeconomic burden of diseases affecting the human brain is estimated to constitute 35 % of all European Union (EU) disease burden. Demographic changes in ageing societies of the EU will increase this rate considerably and this will represent a crucial challenge to forthcoming generations. Fortunately, enormous progress has been made during the last decade in understanding mechanisms and principles of both normal brain function and CNS disorders. This increase in knowledge makes new treatments for nervous system diseases realistic. Unfortunately, current pharmacological treatment strategies for neurodegenerative diseases are at best partially effective. Typically, they offer only some symptomatic relief. Generally, they are not able to affect disease progression. Furthermore, pharmacotherapies are limited by restrictions set by the blood-brain-barrier and by their relatively untargeted mode of action. As a consequence, they often provoke side effects, especially following long-term treatment. Thus, the targeted delivery of protective, or even curative genes to specific populations of disease-affected brain cells is an exciting future alternative. This approach may offer significant relief or even cure to presently untreatable or only symptomatically treatable brain diseases. Several proteins and regulatory Ribonucleic acid (RNAs) involved in normal brain function and disease progression have been identified or will be identified in the near future. Targeting these molecules directly rather than developing drugs that modulate them may drastically reduce cost and time efforts in developing medications as compared to classical pharmacology. Ultimately, targeted and tightly controlled delivery of genetic medicines may open up venues for treatment strategies taking into account specific personal requirements of individual patients.
The objectives of the NEUGENE consortium are thus to develop and to validate the tools necessary for safe, efficient, durable, targeted and regulated expression of therapeutic molecules within the Central nervous system (CNS).
These objectives are reflected by the 4 scientific Work packages (WPs):
WP1: Targeting, aimed at directing or restricting expression of therapeutic proteins or short hairpin RNAs (shRNAs) to specific populations of CNS neurons, as characterised by specific neurotransmitters or susceptibility to disease conditions. Another major objective was to achieve specific targeting of astroglial cells, as these actively support neurons and can open novel venues for CNS gene therapy, e.g. as sources for neurotrophic factors.
WP2: Regulation, aimed at development of tightly regulated vector systems capable to control and turn on and off transgene expression. These systems were developed for two different scenarios, i.e. controlled expression of neurotrophic factors and controlled expression of neurotransmitter synthesising enzymes.
WP3: Safety, addressed immune responses to LV vectors, to novel types of AAV vectors, the influence of inflammatory lesions, and other safety issues.
WP4: Functional validation: aimed at verifying technological advances such as regulated and targeted AAV and LV vectors in a standardised rodent model of Parkinson's disease, in order to be able to compare efficacy of the different approaches against each other and to be able to identify the most promising strategy for forthcoming gene therapy approaches to treat Parkinson patients.
Project results:
Targeting of AAV and LV vectors
Glial cells and especially astrocytes serve essential roles in maintenance of the CNS. Thus, making brain astrocytes available for CNS gene transfer strategies was one major focus of NEUGENE, which has been fully achieved. Using different strategies for AAV and LV vectors the consortium was successful in developing astrocytes-specific vectors, which are already used for functional studies including gene therapy in animal models. We also optimised gene transfer into the main target cell population affected by Parkinson's disease, nigral dopaminergic neurons. Neuronal subtype-specific promoters allowed for targeting of transgene expression to neuronal subtypes or specific disease-affected neurons.
Regulation of transgene expression
We followed two different strategies for development of systems capable of controlling transgene expression levels in the brain: regulatory protein-based and RNA aptamer-based. Work on the latter is on-going, but for the time being we have not met this objective. In contrast, two different regulatory protein-based strategies have been tested successfully in vivo. Both regulated systems under development towards clinical applicability have demonstrated excellent manageability. Both systems needed a large body of work (and indeed more work than expected) for fine-tuning of their respective components to achieve optimal results in pre-clinical animal models. As outlined below, both systems have been functionally tested in relevant pre-clinical animal models of PD.
Safety / Immunology
In order to mimic the human situation as closely as possible, we developed an immunisation protocol for rats making use of the antigen which humans encounter, i.e. the wild-type AAV-2 virus. Preimmunisation with wt AAV-2 significantly decreased transgene expression in the brain following intracerebral administration of AAV-2 vector encoding EGFP. These results clearly indicate that preexisting immunity to AAV-2 may compromise the efficiency of AAV-2-based vector systems even in immune privileged organs such as brain. Additional induction of a lesion which causes up-regulation of inflammatory markers did not further reduce transduction efficacy, indicating that it was only the specific anti-capsid directed immune reaction precluding successful transgene expression.
Furthermore, we investigated immune issues related to Equine infectious anemia virus (EIAV) lentiviral vectors, to which humans are not naturally immunocompetent. Our results demonstrated that CNS application of these vectors is not substantially hindered by peripheral immunity, arguing for a superior safety profile.
During the course of our studies, novel safety aspects arose and were investigated. Unexpected persistence of AAV capsids and genomes in CNS neurons however did not introduce uncontrollable safety risks.
Functional validation
Expression of optimised levels of DOPA synthesising TH / GCH1 successfully restored motor performance in a model of late PD. This tool entered a clinical development programme. An AAV vector was generated to constitutively express Glial cell line-derived neurotrophic factor (GDNF) in astrocytes, thereby preventing off-target delivery of the neurotrophin without any restriction in therapeutic efficacy. This tool offers novel delivery options for neurotrophic factor-based gene therapy. An AAV vector system was generated for short-term intermitted induction of GDNF expression, providing substantial improvements in motor performance and neuroprotection in a model of early PD. This tool is currently developed further towards clinical applicability. A non-integrating LV vector expressing GDNF was generated and provided the same level of neuroprotection as conventional integrating LV vectors. These tools will provide added safety for forthcoming therapeutic strategies using LV vectors.
Neuronal subpopulation-specific and glia-specific AAV and LV vectors were generated, which are currently exploited to generated advanced animal models of neurodegenerative diseases.
Potential impact:
The majority of projects conducted by the NEUGENE consortium were of a basic research nature, and thus direct economic or wider societal impacts were not intended. Clearly, it will be the scientific community (neuroscientists, gene therapists etc.) who will have the major benefit from the work presented so far and from further work resulting from current projects.
None the less, the most advanced vector tool developed through our studies is patented and has entered into a clinical development program for DOPA restoration gene therapy of PD. Regulated neurotrophin-expressing vectors have attracted the interest of a leading SEM in the field of gene therapy, and will be developed towards clinical applicability in due course. As stated in the initial plan for dissemination of foreground these further studies will comprise the use of non-human primates to prove safety in a more advanced model, and thus initiation of clinical trials can be envisaged no earlier than 2015.
Several other advanced vector tools like cell type-specific standard and non-integrating lentiviral vectors are about to be patented, and will be made available for the scientific community as soon as possible.
Much of the work progress of the NEUGENE consortium is freely available to the scientific community. Our results have been presented at 19 conference contributions and in 14 peer-reviewed publications, mostly in high-ranked neuroscience or gene therapy journals. The number of high ranked publications will further increase in the next months, as several manuscripts are in revision, submitted, or in preparation. Actually, several highly interesting studies resulting from work in the WP4 ('Functional validation') are just about to be prepared for publication.
List of websites: www.neugene.eu