Luis Quintino

Visualization and genetic modification of resident brain microglia using lentiviral vectors regulated by microRNA-9.

Functional studies of resident microglia require molecular tools for their genetic manipulation. Here we show that microRNA-9-regulated lentiviral vectors can be used for the targeted genetic modification of resident microglia in the rodent brain. Using transgenic reporter mice, we demonstrate that murine microglia lack microRNA-9 activity, whereas most other cells in the brain express microRNA-9. Injection of microRNA-9-regulated vectors into the adult rat brain induces transgene expression specifically in cells with morphological features typical of ramified microglia. The majority of transgene-expressing cells colabels with the microglia marker Iba1. We use this approach to visualize and isolate activated resident microglia without affecting circulating and infiltrating monocytes or macrophages in an excitotoxic lesion model in rat striatum. The microRNA-9-regulated vectors described here are a straightforward and powerful tool that facilitates functional studies of resident microglia.

Destabilizing domains mediate reversible transgene expression in the brain.

Regulating transgene expression in vivo by delivering oral drugs has been a long-time goal for the gene therapy field. A novel gene regulating system based on targeted proteasomal degradation has been recently developed. The system is based on a destabilizing domain (DD) of the Escherichia coli dihydrofolate reductase (DHFR) that directs fused proteins to proteasomal destruction. Creating YFP proteins fused to destabilizing domains enabled TMP based induction of YFP expression in the brain, whereas omission of TMP resulted in loss of YFP expression. Moreover, induction of YFP expression was dose dependent and at higher TMP dosages, induced YFP reached levels comparable to expression of unregulated transgene., Transgene expression could be reversibly regulated using the DD system. Importantly, no adverse effects of TMP treatment or expression of DD-fusion proteins in the brain were observed. To show proof of concept that destabilizing domains derived from DHFR could be used with a biologically active molecule, DD were fused to GDNF, which is a potent neurotrophic factor of dopamine neurons. N-terminal placement of the DD resulted in TMP-regulated release of biologically active GDNF. Our findings suggest that TMP-regulated destabilizing domains can afford transgene regulation in the brain. The fact that GDNF could be regulated is very promising for developing future gene therapies (e.g. for Parkinsons disease) and should be further investigated.

Functional neuroprotection and efficient regulation of GDNF using destabilizing domains in a rodent model of Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) has great potential to treat Parkinsons disease (PD). However, constitutive expression of GDNF can over time lead to side effects. Therefore, it would be useful to regulate GDNF expression. Recently, a new gene inducible system using destabilizing domains (DD) from E. coli dihydrofolate reductase (DHFR) has been developed and characterized. The advantage of this novel DD is that it is regulated by trimethoprim (TMP), a well-characterized drug that crosses the blood-brain barrier and can therefore be used to regulate gene expression in the brain. We have adapted this system to regulate expression of GDNF. A C-terminal fusion of GDNF and a DD with an additional furin cleavage site was able to be efficiently regulated in vitro, properly processed and was able to bind to canonical GDNF receptors, inducing a signaling cascade response in target cells. In vivo characterization of the protein showed that it could be efficiently induced by TMP and it was only functional when gene expression was turned on. Further characterization in a rodent model of PD showed that the regulated GDNF protected neurons, improved motor behavior of animals and was efficiently regulated in a pathological setting.

A Model of GDNF Gene Therapy in Mice with 6-Hydroxydopamine Lesions: Time Course of Neurorestorative Effects and ERK1/2 Activation

BACKGROUND Glial cell line-derived neurotrophic factor (GDNF) is the most promising neurotrophin for restorative treatments in Parkinsons disease, but its biological effects are not completely understood. OBJECTIVE To define a model of GDNF gene therapy in the mouse, we studied the long-term effects of lentiviral GDNF delivery in mice with striatal 6-hydroxydopamine (6-OHDA) lesions. METHODS Lentiviral vectors coding for GDNF or green fluorescent protein (GFP) were injected unilaterally in the striatum two weeks prior to the 6-OHDA lesion. Mice were monitored on tests of spontaneous activity and amphetamine-induced rotation at 1, 4, 10 and 35 weeks post-lesion. Brains were processed immunohistochemically for tyrosine hydroxylase (TH) and markers of extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation at the same time points. RESULTS Lentiviral GDNF significantly inhibited both spontaneous and amphetamine-induced rotation. Compared to the control vector, lentiviral GDNF resulted in a partial protection of TH-positive cells in the substantia nigra, and in a nearly total restoration of striatal TH immunostaining by 35 weeks. A progressive sprouting of TH-positive neurites occurred in both the globus pallidus and the substantia nigra, reaching a 4-5 fold increase above controls by 35 weeks. This effect was paralleled by a long-term supranormal activation of ERK1/2 and its downstream target, phospho-Ser31 TH. CONCLUSIONS Lentiviral GDNF delivery produced robust long-term signaling responses and neurorestoration. This experimental model of GDNF gene therapy will be particularly suitable to study the molecular mechanisms of dopaminergic fiber sprouting, a long-term response to GDNF delivery that also occurs in Parkinsons disease patients.

Novel disease-specific promoters for use in gene therapy for Parkinson's disease

Gene therapy is a promising therapeutic tool for Parkinsons disease (PD), but there is a lack of evaluated cell specific promoters that are relevant for the disease. We have chosen PD relevant promoter candidates for gene therapy vectors based on either previous studies; Drd1a, Drd2 and pDyn, or from a microarray study on parkinsonian patients; ACE, DNAJC3, GALNS, MAP1a and RNF25. These candidates have been evaluated in rat striatum to determine their suitability for use in cell specific vectors. The promoters had a neuronal specificity of 91-100%. The efficiency of the promoters was variable, but RNF25, DNAJC3 and MAP1a were comparable to widely used ubiquitous promoters. MAP1a was also affected by dopamine depletion.

FACS binding assay for analysing GDNF interactions.

Glial cell-line derived neurotrophic factor (GDNF) is a secreted protein with great therapeutic potential. However, in order to analyse the interactions between GDNF and its receptors, researchers have been mostly dependent of radioactive binding assays. We developed a FACS-based binding assay for GDNF as an alternative to current methods. We demonstrated that the FACS-based assay using TGW cells allowed readily detection of GDNF binding and displacement to endogenous receptors. The dissociation constant and half maximal inhibitory concentration obtained were comparable to other studies using standard binding assays. Overall, this FACS-based, simple to perform and adaptable to high throughput setup, provides a safer and reliable alternative to radioactive methods.

Rational design of a compact CRISPR-Cas9 activator for AAV- mediated delivery

Akin to Zinc Finger and Transcription Activator Like Effector based transcriptional modulators, nuclease-null CRISPR-Cas9 provides a groundbreaking programmable DNA binding platform, begetting an arsenal of targetable regulators for transcriptional and epigenetic perturbation, by either directly tethering, or recruiting, transcription enhancing effectors to either component of the Cas9/guide RNA complex. Application of these programmable regulators is now gaining traction for the modulation of disease-causing genes or activation of therapeutic genes, in vivo. Adeno-Associated Virus (AAV) is an optimal delivery vehicle for in vivo delivery of such regulators to adult somatic tissue, due to the efficacy of viral delivery with minimal concerns about immunogenicity or integration. However, present Cas9 activator systems are notably beyond the packaging capacity of a single AAV delivery vector capsid. Here, we engineer a compact CRISPR-Cas9 activator for convenient AAV-mediated delivery. We validate efficacy of the CRISPR-Cas9 transcriptional activation using AAV delivery in several cell lines.

Molecular barcoding of viral vectors enables mapping and optimization of mRNA trans-splicing

Genome editing has proven to be highly potent in the generation of functional gene knockouts in dividing cells. In the CNS however, efficient technologies to repair sequences are yet to materialize. Reprogramming on the mRNA level is an attractive alternative as it provides means to perform in situ editing of coding sequences without nuclease dependency. Furthermore, de novo sequences can be inserted without the requirement of homologous recombination. Such reprogramming would enable efficient editing in quiescent cells (e.g., neurons) with an attractive safety profile for translational therapies. In this study, we applied a novel molecular-barcoded screening assay to investigate RNA trans-splicing in mammalian neurons. Through three alternative screening systems in cell culture and in vivo, we demonstrate that factors determining trans-splicing are reproducible regardless of the screening system. With this screening, we have located the most permissive trans-splicing sequences targeting an intron in the Synapsin I gene. Using viral vectors, we were able to splice full-length fluorophores into the mRNA while retaining very low off-target expression. Furthermore, this approach also showed evidence of functionality in the mouse striatum. However, in its current form, the trans-splicing events are stochastic and the overall activity lower than would be required for therapies targeting loss-of-function mutations. Nevertheless, the herein described barcode-based screening assay provides a unique possibility to screen and map large libraries in single animals or cell assays with very high precision.

Regulated Gene Therapy

Gene therapy represents a promising approach for the treatment of monogenic and multifactorial neurological disorders. It can be used to replace a missing gene and mutated gene or downregulate a causal gene. Despite the versatility of gene therapy, one of the main limitations lies in the irreversibility of the process: once delivered to target cells, the gene of interest is constitutively expressed and cannot be removed. Therefore, efficient, safe and long-term gene modification requires a system allowing fine control of transgene expression.Different systems have been developed over the past decades to regulate transgene expression after in vivo delivery, either at transcriptional or post-translational levels. The purpose of this chapter is to give an overview on current regulatory system used in the context of gene therapy for neurological disorders. Systems using external regulation of transgenes using antibiotics are commonly used to control either gene expression using tetracycline-controlled transcription or protein levels using destabilizing domain technology. Alternatively, specific promoters of genes that are regulated by disease mechanisms, increasing expression as the disease progresses or decreasing expression as disease regresses, are also examined. Overall, this chapter discusses advantages and drawbacks of current molecular methods for regulated gene therapy in the central nervous system.

Striatonigral neurons divide into two distinct morphological-physiological phenotypes after chronic L-DOPA treatment in parkinsonian rats

Dendritic regression of striatal spiny projection neurons (SPNs) is a pathological hallmark of Parkinson’s disease (PD). Here we investigate how chronic dopamine denervation and dopamine replacement with L-DOPA affect the morphology and physiology of direct pathway SPNs (dSPNS) in the rat striatum. We used a lentiviral vector optimized for retrograde labeling (FuG-B-GFP) to identify dSPNs in rats with 6-hydroxydopamine (6-OHDA) lesions. Changes in morphology and physiology of dSPNs were assessed through a combination of patch-clamp recordings and two photon microscopy. The 6-OHDA lesion caused a significant reduction in dSPN dendritic complexity. Following chronic L-DOPA treatment, dSPNs segregated into two equal-sized clusters. One group (here called “cluster-1”), showed sustained dendritic atrophy and a partially normalized electrophysiological phenotype. The other one (“cluster-2”) exhibited dendritic regrowth and a strong reduction of intrinsic excitability. Interestingly, FosB/∆FosB induction by L-DOPA treatment occurred preferentially in cluster-2 dSPNs. Our study demonstrates the feasibility of retrograde FuG-B-GFP labeling to study dSPNs in the rat and reveals, for the first time, that a subgroup of dSPNs shows dendritic sprouting in response to chronic L-DOPA treatment. Investigating the mechanisms and significance of this response will greatly improve our understanding of the adaptations induced by dopamine replacement therapy in PD.