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Kügler lab

Research in our laboratory is based on two major pillars:

  • understanding the pathophysiological mechanisms causing neurodegenerative disorders like Parkinson´s disease. Special attention is paid to proteins of the synuclein family, since a-synuclein is a major component of aggregate structures that form a hallmark of this disease. Blood-borne immunoglobulins against synucleins may invade the brain through an impaired blood-brain-barrier, thereby linking heart and brain science.
  • development of adeno-associated viral vector (AAV) technology, enabling us to express any kind of protein or regulatory RNA in cells refractory to conventional gene transfer methods both in vitro and in vivo. AAV vectors are used for basic science as well as for establishment of pre-clinical gene therapy protocols. While we focus on CNS disorders, collaborative projects address diseases of the heart and the muscular system.

Vector tech and general applications

We develop and use AAV viral vectors for a wide variety of applications, i.e. for expression of Parkinson´s disease related proteins and genetically encoded sensors (e.g. for quantification of levels of calcium, ATP, ROS, cAMP etc.), both in cultured neurons andin the rodent brain. Expression of proteins or regulatory RNAs can be targeted to specific cell types or to specific sub-cellular organelles. The AAV toolkit offers extraordinary efficacy, safety and flexibility, in basic science but also in development of gene therapy.

For many internal and external collaborations, we have manufactured a large number and variety of AAV vectors (some of which reported here:  ADDIN EN.CITE.DATA  ADDIN EN.CITE.DATA (1-36)), while lentiviral (LV) vectors are  rarely used (37) and adenoviral (AD) and Semliki-forest-virus based vectors (SFV) have meanwhile been fully discontinued.

Fig 1: Examples of AAV vector applications routinely used in the lab

A) Concomitant expression of cytoplasmic Ca2+ sensor GCaMP3.5 (green) and mitochondrial Ca2+ sensor RCaMP1e (red) in cultured neurons. Both sensors expressed by AAV-6 vectors under hSyn promoter. B) Mutually exclusive transduction of neurons (AAV-6-hSyn-Cherry, red) and astrocytes (AAV-5-GFAP-EGFP) in mouse cortical layer V. C) Neurodegeneration of nigral dopaminergic neurons after transduction with AAV-2 vectors expressing synuclein proteins (TH immunohistochemistry). D) Imaging of living neurons in mouse brain by 2-photon microscopy through a cranial window.

Pre-clinical gene therapy protocols

Molybdenum Cofactor Deficiency

Fig 2: Molybdenum Cofactor (MoCo) gene therapy A) Biosynthesis of MoCo; the MOCS1A/1B genes are deficient in most patients. B) Control and MOCS1 -/- mice at 7 and 66 days after application of AAV-EGFP or AAV-MOCS1. AAV-EGFP injected animals die at 8-10 days after birth, while AAV-MOCS1 treated animals grow up with a normal phenotype. C) Dilution of AAV-vector genomes after intrahepatic injection as visualized by EGFP expression; DAPI nuclear counterstain in blue.

Molybdenum cofactor (MoCo) deficiency is an ultra-rare disease causing early neonatal death. MoCo is synthesized in the liver and functions as an essential co-factor for enzymes detoxifying sulphur components. During pregnancy maternal supply of MoCo prevents lesions, but very soon after birth highly toxic sulphur and xanthine deposits in the brain cause irreversible damages. MOCS1A/1B deficient mice were generated by Jochen Reiss (Dept. of Human Genetics, University Medicine Göttingen), and in a collaborative project we developed an AAV-based gene therapy protocol resulting in healthy animals after liver gene transfer (38). At times of development of this protocol, ultra-rare diseases were out of focus of pharmaceutical industry, and thus no clinical application was initiated. This situation has changed meanwhile (see below), and thus clinical applicability of MoCo gene transfer may still be within reach.

Dual-AAV for large transgenes: treatment of deafness

Fig 3: Dual AAV gene therapy in deaf Otoferlin -/- mice A) The two approaches used (trans-splicing genomes and hybrid genomes) to generate the full length Otoferlin construct are schematically depicted. B) A nice representation of the study by EMBO´s artists.

AAV are very small viruses, and thus can package only a quite limited transgene capacity of about 5000 base pairs, of which about 500-1000 bp are necessary for transcriptional control elements like promoter, enhancer and polyadenylation sites. This is unfortunate as several promising gene therapy candidates in sensory systems like retina or inner ear exceed this size by far. Sensory organs are probably the best suited targets for successful gene therapy approaches in the near future, due to their relatively small size, good accessibility and partial protection from immune responses towards the vectors. The Otoferlin protein is defective in a fairly large percentage of people suffering from hereditary hearing loss. Otoferlin is crucially involved into the auditory signal transmission process from inner hair cells to spiral ganglion neurons of the inner ear. As Otoferlin´s cDNA comprises about 6000 bp, it is not suitable to be packaged into a single AAV vector. In addition, we and other groups have demonstrated that vectors with enhanced transgene capacity such as lentiviral or adenoviral vectors are not suitable for inner hair cell transduction.

Thus, in a collaboration with Ellen Reisinger (Dept. of Otorhinolaryngology, University Medicine Göttingen) we exploited a dual AAV vector system in Otoferlin -/- mice, where the 5´-part of Otoferlin was incorporated into one AAV vector, and the 3´-part of Otoferlin was incorporated into a second AAA vector. Appropriately positioned splice sites allowed for reconstitution of full length Otoferlin cDNA in transduced inner hair cells, and enabled a partial but still impressive recovery of auditory function (39). Dual AAV-Otoferlin vectors were out-licensed and are now in highly successful clinical use in the US (with license) and in China (without license J).

Pharmacological control of therapeutic transgene expression

Fig 4: Pharmacological control of neurotrophic factor expression A) The principle of regulated GDNF expression is shown to demonstrate that different levels of Mifepristone can induce GDNF levels over two orders of magnitude. B) Basic principle of “GeneSwitch”-regulated expression. Both transcription units fit into a single AAV vector genome, and need a certain spatial configuration in order to allow for high-level but background-free GDNF expression. The “GeneSwitch” fusion protein consists primarily of human components, except for about 70 amino acids of the yeast DNA binding domain. Therefore, immunological studies in immunocompetent organs of non-human primates must now prove that this peptide will not be immunogenic in humans.

Major neurodegenerative disorders like Alzheimer´s or Parkinson´s disease are characterized by a multifaceted etiology and disease progression. Thus, it is unlikely that a single target will be identified, modification of which might halt disease progression or even revert symptoms in the majority of idiopathic patients. To circumvent this issue, neurotrophic factors (NTFs), that are able to stimulate multiple survival-promoting pathways are considered a valuable option to prevent further loss of neurons and even to restore neuronal functionality. However, the enormous potency of these molecules provokes side effects as well. As NTFs cannot cross the blood-brain-barrier, their expression within the CNS by means of gene therapy is currently considered the optimal route of delivery. As a drawback, gene therapy in its current layout is an irreversible process, meaning that in case of excess supply of NTFs, expression cannot be stopped, nor can the expression level be regulated according to individual patient´s needs.

In order to provide an alternative, we have developed and further optimized an AAV vector system that takes advantage of pharmacological control over transgene expression. The FDA-approved human drug Mifepriston (Mfp), a synthetic steroid, controls expression of the neurotrophic factor GDNF through the so-called “GeneSwitch”, enabling us to tightly control the level and duration of GDNF expression in the brain. The system has proven to work favorably in rat models of Parkinson´s disease, providing robust recovery from motor impairments (40-43).

A regulatory transgene expression system approved for human use will enable a wide portfolio of products, as it allows to address “high risk – high benefit” gene therapy targets, e.g. intracranially expressed single chain antibodies to deplete aggregates in PD and AD, gene editing tools based on Crispr/Cas to directly modify genomic sites, or temporal expression of Yamanaka factors to re-juvenile the aged CNS.  

In general, a regulated gene therapy system would enable a highly personalized concept of gene therapy, as it would be no longer an irreversible process.

Safety studies in periphery and CNS of non-human primates are currently underway to prove clinical applicability of this system, so far with promising results.

Synuclein research: a link between blood and brain?

The synuclein protein family consist of three closely related proteins, a-, b-, and g-Synuclein. They all show very little secondary or tertiary structure in solution, and are thus members of the ever-growing family of “unfolded” proteins. a-Synuclein can form protein aggregates under certain conditions, similar to other proteins linked to “aggregopathies” like Tau, Aß or Huntingtin. b-Synuclein was long thought to be an anti-aggregating counterpart to a-Synuclein, but we have recently demonstrated that it forms proteinase K-resistant aggregates in the rodent brain very similar to a-Synuclein (44). a-Synuclein is intimately linked to the etiology of Parkinson´s disease (PD), as mutants and gene multiplications of a-Synuclein are directly causative for parkinsonian symptoms, and aggregations of a-Synuclein are found in all idiopathic PD patients. Throughout the last years we have conducted a series of investigations addressing pathological and physiological functions of the synucleins (2, 6, 7, 18, 22, 45-52). Current research is prompted by the unexpected presence of auto-antibodies against the synucleins in blood of almost every individual. While we have shown that these IgGs cannot serve as reliable biomarkers for Parkinson´s disease (53), recent research has demonstrated that they can induce prominent neurodegeneration when binding to synuclein associated with NMDA receptors on excitatory neurons (54). Given the impaired blood-brain-barrier of many individuals suffering from ageing-associated neurodegenerative diseases, infiltration of blood-borne IgGs into the CNS is likely. Thus, we are about to investigate how relevant this mechanism may be for Parkinsonian patients, by exploiting animal models of IgG transfer into the CNS.

References

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  21. Mironov SL, Skorova EY, Kügler S. Epac-mediated cAMP-signalling in the mouse model of Rett Syndrome. Neuropharmacology. 2011;60(6):869-77.
  22. Garrido M, Tereshchenko Y, Zhevtsova Z, Taschenberger G, Bähr M, Kügler S. Glutathione depletion and overproduction both initiate degeneration of nigral dopaminergic neurons. Acta Neuropathol. 2011;121(4):475-85.
  23. Kurimoto T, Yin Y, Omura K, Gilbert HY, Kim D, Cen LP, et al. Long-distance axon regeneration in the mature optic nerve: contributions of oncomodulin, cAMP, and pten gene deletion. J Neurosci. 2010;30(46):15654-63.
  24. Jaworski T, Kügler S, Van Leuven F. Modeling of tau-mediated synaptic and neuronal degeneration in Alzheimer's disease. Int J Alzheimers Dis. 2010;2010.
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  29. Mironov SL, Skorova E, Hartelt N, Mironova LA, Hasan MT, Kügler S. Remodelling of the respiratory network in a mouse model of Rett syndrome depends on brain-derived neurotrophic factor regulated slow calcium buffering. J Physiol. 2009;587(Pt 11):2473-85.
  30. Wallace DJ, Meyer zum Alten Borgloh S, Astori S, Yang Y, Bausen M, Kügler S, et al. Single-spike detection in vitro and in vivo with a genetic Ca2+ sensor. Nat Methods. 2008;5(9):797-804.
  31. Hartelt N, Skorova E, Manzke T, Suhr M, Mironova L, Kügler S, et al. Imaging of respiratory network topology in living brainstem slices. Mol Cell Neurosci. 2008;37(3):425-31.
  32. Tomita H, Sugano E, Yawo H, Ishizuka T, Isago H, Narikawa S, et al. Restoration of visual response in aged dystrophic RCS rats using AAV-mediated channelopsin-2 gene transfer. Invest Ophthalmol Vis Sci. 2007;48(8):3821-6.
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  34. Malik JM, Shevtsova Z, Bähr M, Kügler S. Long-term in vivo inhibition of CNS neurodegeneration by Bcl-XL gene transfer. Mol Ther. 2005;11(3):373-81.
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  39. Al-Moyed H, Cepeda AP, Jung S, Moser T, Kügler S, Reisinger E. A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock-out mice. EMBO Mol Med. 2019;11(1).
  40. Maddalena A, Tereshchenko J, Bähr M, Kügler S. Adeno-associated Virus-mediated, Mifepristone-regulated Transgene Expression in the Brain. Mol Ther Nucleic Acids. 2013;2:e106.
  41. Cheng S, van Gaalen MM, Bähr M, Garea-Rodriguez E, Kügler S. Optimized pharmacological control over the AAV-Gene-Switch vector for regulable gene therapy. Mol Ther Methods Clin Dev. 2021;23:1-10.
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  46. Psol M, Darvas SG, Leite K, Mahajani SU, Bähr M, Kügler S. Dementia with Lewy bodies-associated ss-synuclein mutations V70M and P123H cause mutation-specific neuropathological lesions. Hum Mol Genet. 2021;30(3-4):247-64.
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