Patrick Aldrin-Kirk

DREADD Modulation of Transplanted DA Neurons Reveals a Novel Parkinsonian Dyskinesia Mechanism Mediated by the Serotonin 5-HT6 Receptor

Summary Transplantation of DA neurons is actively pursued as a restorative therapy in Parkinson’s disease (PD). Pioneering clinical trials using transplants of fetal DA neuroblasts have given promising results, although a number of patients have developed graft-induced dyskinesias (GIDs), and the mechanism underlying this troublesome side effect is still unknown. Here we have used a new model where the activity of the transplanted DA neurons can be selectively modulated using a bimodal chemogenetic (DREADD) approach, allowing either enhancement or reduction of the therapeutic effect. We show that exclusive activation of a cAMP-linked (Gs-coupled) DREADD or serotonin 5-HT6 receptor, located on the grafted DA neurons, is sufficient to induce GIDs. These findings establish a mechanistic link between the 5-HT6 receptor, intracellular cAMP, and GIDs in transplanted PD patients. This effect is thought to be mediated through counteraction of the D2 autoreceptor feedback inhibition, resulting in a dysplastic DA release from the transplant.

Novel AAV-based rat model of forebrain synucleinopathy shows extensive pathologies and progressive loss of cholinergic interneurons.

Synucleinopathies, characterized by intracellular aggregation of α-synuclein protein, share a number of features in pathology and disease progression. However, the vulnerable cell population differs significantly between the disorders, despite being caused by the same protein. While the vulnerability of dopamine cells in the substantia nigra to α-synuclein over-expression, and its link to Parkinsons disease, is well studied, animal models recapitulating the cortical degeneration in dementia with Lewy-bodies (DLB) are much less mature. The aim of this study was to develop a first rat model of widespread progressive synucleinopathy throughout the forebrain using adeno-associated viral (AAV) vector mediated gene delivery. Through bilateral injection of an AAV6 vector expressing human wild-type α-synuclein into the forebrain of neonatal rats, we were able to achieve widespread, robust α-synuclein expression with preferential expression in the frontal cortex. These animals displayed a progressive emergence of hyper-locomotion and dysregulated response to the dopaminergic agonist apomorphine. The animals receiving the α-synuclein vector displayed significant α-synuclein pathology including intra-cellular inclusion bodies, axonal pathology and elevated levels of phosphorylated α-synuclein, accompanied by significant loss of cortical neurons and a progressive reduction in both cortical and striatal ChAT positive interneurons. Furthermore, we found evidence of α-synuclein sequestered by IBA-1 positive microglia, which was coupled with a distinct change in morphology. In areas of most prominent pathology, the total α-synuclein levels were increased to, on average, two-fold, which is similar to the levels observed in patients with SNCA gene triplication, associated with cortical Lewy body pathology. This study provides a novel rat model of progressive cortical synucleinopathy, showing for the first time that cholinergic interneurons are vulnerable to α-synuclein over-expression. This animal model provides a powerful new tool for studies of neuronal degeneration in conditions of widespread cortical α-synuclein pathology, such as DLB, as well an attractive model for the exploration of novel biomarkers.

Chemogenetic modulation of cholinergic interneurons reveals their regulating role on the direct and indirect output pathways from the striatum

The intricate balance between dopaminergic and cholinergic neurotransmission in the striatum has been thoroughly difficult to characterize. It was initially described as a seesaw with a competing function of dopamine versus acetylcholine. Recent technical advances however, have brought this view into question suggesting that the two systems work rather in concert with the cholinergic interneurons (ChIs) driving dopamine release. In this study, we have utilized two transgenic Cre-driver rat lines, a choline acetyl transferase ChAT-Cre transgenic rat and a novel double-transgenic tyrosine hydroxylase TH-Cre/ChAT-Cre rat to further elucidate the role of striatal ChIs in normal motor function and in Parkinsons disease. Here we show that selective and reversible activation of ChIs using chemogenetic (DREADD) receptors increases locomotor function in intact rats and potentiate the therapeutic effect of L-DOPA in the rats with lesions of the nigral dopamine system. However, the potentiation of the L-DOPA effect is accompanied by an aggravation of L-DOPA induced dyskinesias (LIDs). These LIDs appear to be driven primarily through the indirect striato-pallidal pathway since the same effect can be induced by the D2 agonist Quinpirole. Taken together, the results highlight the intricate regulation of balance between the two output pathways from the striatum orchestrated by the ChIs.

A novel adeno-associated virus capsid with enhanced neurotropism corrects a lysosomal transmembrane enzyme deficiency

Tordo et al. present a novel AAV gene therapy vector, AAV-TT, which exceeds the current benchmark neurotropic serotypes AAV9 and AAVrh10 and enables unprecedented correction of a lysosomal transmembrane enzyme deficiency. AAV-TT based gene therapies may thus be suitable for the treatment of human neurological diseases characterised by global neuropathology.

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.

Barcoded Rational AAV Vector Evolution enables systematic in vivo mapping of peptide binding motifs

Engineering of Adeno-associated viral (AAV) vector capsids through directed evolution has been used to generate novel capsids with altered tropism and function1-9. This approach, however, involves a selection process that requires multiple generations of screenings to identify real functional capsids2-4. Due to the random nature of this process, it is also inherently unreproducible, and the resulting capsid variants provide little mechanistic insights into the molecular targets engaged. To overcome this, we have developed a novel method for rational capsid evolution named Barcoded Rational AAV Vector Evolution (BRAVE). The key to this method is a novel viral production approach where each virus particle displays a protein-derived peptide on the surface which is linked to a unique barcode in the packaged genome10. Through hidden Markov model-based clustering11, we were able to identify novel consensus motifs for cell-type specific retrograde transport in neurons in vivo in the brain. The BRAVE approach enables the selection of novel capsid structures using only a single-generation screening. Furthermore, it can be used to map, with high resolution, the putative binding sequences of large protein libraries.

256. A Novel Rationally Designed AAV Capsid Yields a Potent Neurotropic Gene Therapy Vector

Recent clinical trials have demonstrated safety and efficacy of adeno-associated virus (AAV)-mediated gene therapy, and precipitated the approval of the first gene therapy product for commercial use. Although AAV is the most commonly used vector, there are limitations due to its restricted biological activity. This can be partially overcome by aligning capsid choice and target tissue. Tailoring of AAV capsids has traditionally been accomplished by simply selecting the most suitable available serotype or by further optimization of available serotypes by peptide insertion or capsid reshuffling and in vivo selection in mouse or humanised mouse models. A novel approach based on in silico reconstruction of ancestral viruses recently yielded a promising vector for gene therapy of diseases that affect liver, muscle or retina. Here we report an alternative evolutionary approach to AAV capsid design based on the introduction of amino acids that have been found at the other end of the evolutionary spectrum. Specifically, we have designed a novel AAV2 capsid by including amino acids that are conserved in natural AAV isolates found in human pediatric tissues. We have determined the structural and immunological characteristics of this novel AAV2-based capsid and performed biodistribution studies. When the bioactivity of the vector was evaluated in various in vivo contexts the data revealed extraordinary transduction characteristics in eye and brain tissues. Intracranial injections in mice and rats revealed a significantly increased spread and enhanced transduction of brain tissues as compared to AAV2, with evidence for retrograde transport. Additionally, intra-ocular injections in adult mice revealed a marked enhancement of transduction of photoreceptor cells by the novel vector when compared to AAV2. Peripheral injections also demonstrated strong affinity for the brain and showed little evidence for transduction of non-neuronal tissues. Importantly, we were able to demonstrate superior performance in a disease model that affects the central nervous system when compared to other serotypes that have a strong affinity for neuronal tissues. In summary, we have engineered a novel capsid to include key residues found in natural variants of AAV2, which yields a unique gene therapy vector for the treatment of diseases that affect the central nervous system.

542. Novel Barcode-Based In Vivo Screening Method for Generating De Novo AAV Serotypes for CNS-Directed Gene Therapy

Capsid modification is a useful strategy to create adeno-associated virus (AAV) vectors with subtype specific neuronal targeting and enhanced retrograde transport. Incorporating known cell-specific binding ligands is a rational method, but the creation of vectors without prior knowledge has the potential to reveal novel targets. Directed evolution and phage display are broadly utilized high-throughput methods, but are inefficient due to displaying random peptides wherein the vast majority will be non-functional.Here, we have developed a novel AAV library in which each virus particle display a peptide derived from known neuron-related proteins on the surface of an AAV2 capsid. The packaged viral genome encodes a unique barcode sequence to facilitate capsid identification. 92398 unique oligos encoding 14-amino-acid peptides derived from 135 proteins were synthesized using microarray. Four-fragment Gibson assembly and novel emulsion PCR was then used to generate a plasmid library by inserting oligos into the capsid gene and barcodes between the inverted terminal repeats. This plasmid library was then used to assemble a diverse library of AAV capsids, such that particles were composed of only peptide-modified capsid proteins which package an expression cassette containing RNA expressed barcodes providing post hoc identification of the capsid structure.In parallel, the plasmid library was sequences using Illumina paired-end sequencing to link the RNA expressed barcodes to the de novo capsid structures. The successfully generated AAV library efficiently infected neurons and astrocytes in vitro and displayed a subset of peptides that had efficient retrograde transport ability in neurons in vivo (e.g., transported from striatum to substantia nigra). Functional peptides, which successfully promoted neuronal infectivity or retrograde transport, were identified by Illumina sequencing of RNA expressed barcodes both in vitro and in vivo and efficacy modeled through barcode counting. In conclusion, we developed a high-throughput combinatorial method to generate peptide-modified AAV libraries that are valuable for evaluation of receptor expression of neuronal populations and have the potential to generate novel vectors with unique properties for in vivo gene transfer in the CNS.Figure 1Polar plot showing the number of unique peptides found in custom array and plasmid library followed by the number found in infective AAV particles, at injection site and for transported AAVs.View Large Image | Download PowerPoint Slide

345. A Novel Toolbox for RNA-Editing Based on Massively Parallel Screening Using Barcoded Viral Vectors

Recent technical advances have opened up new avenues in molecular science with great impact on both in vitro and in vivo applications. Next generation sequencing has become available to a greater extent and advanced molecular techniques and methods within RNA/DNA-editing, barcoding and generation of high diversity DNA libraries are constantly evolving, giving rise to novel applications and discoveries.The aim of this study was to establish an unbiased, totally randomized screening assay for RNA-editing that can be universally utilized. By creating high resolution DNA-libraries from genomic sequences where each fragment is uniquely labelled with a DNA-barcode, we were able to map the biological function of each complete sequence. Here, we have utilized this approach to identify the most promising intronic sequences for RNA-editing. The screening assay was performed by deep sequencing of a lentiviral library, where each fragment was linked to a corresponding barcode. mRNA from transduced cells in vitro and in vivo was sequenced to identify and count the expressed barcodes, thus we could identify specific fragments suitable for RNA-editing. View Large Image | Download PowerPoint SlideRNA-editing was achieved via trans-splicing, where two different pre-mRNAs (one endogenous and one virally derived) hybridize through complementary sequences and form one mRNA. In trans-splicing, only the downstream segment of the mRNA is replaced, thus the gene is still under the control of its native promoter and other endogenous regulatory elements remain unaltered. Trans-splicing is inherently specific since the part of the mRNA that is delivered requires endogenous expression to exert an effect. View Large Image | Download PowerPoint SlideBy combining advanced cloning methods, DNA-barcoding and Next generation sequencing, we could screen extensive DNA libraries and select fragments highly efficient in RNA-editing. Our method can be used for correction of mutations, protein tracing and cell type identification.

18. Novel Approach Using Fetal Dopaminergic Grafts In Situ Transduced with AAV-DREADDs Significantly Increases Behavioral Motor Recovery in a Rat Model of Parkinson's Disease

Over the past decades, cell replacement therapy has emerged as a possible therapeutic alternative for the treatment of Parkinsons disease. Although early trials showed great promise, later double blinded trials displayed varied clinical benefits. In order to improve therapeutic potential, we combined dopaminergic cell replacement therapy with designer receptors exclusively activated by designer drugs (DREADDs) which enable non-invasive, orthogonal, chemical modulation of neuronal activity. To date, most research has focused on optogenetics to achieve such control and though highly useful in pre-clinical research, broad clinical applications may be limited.In order to evaluate if activation of transplanted dopaminergic cells is sufficient to potentiate functional recovery, we grafted embryonic dopaminergic neurons from the ventral mesencephalon of TH-Cre expressing knock-in rats into the striatum of 6-OHDA lesioned wild-type rats View Large Image | Download PowerPoint SlideFigure 1 | Schematic showing the location of TH-Cre positive dopaminergic fetal grafts within the striatum as well as the injection site of the AAV-8 viral vector expressing either Dq or Ds between the grafts.Using AAV vectors, we expressed DREADDs capable of either increasing or silencing neuronal activity in Cre expressing neurons. Two different pathways (Gq and Gs coupled) were used to increase neuronal activity, either Gq alone or the combination of Gq/Gs. Systemically administered CNO, which serves as the ligand for the DREADDs significantly increased motor recovery in animals expressing Dq and Ds, while animals expressing receptors inducing neuronal silencing, displayed a significant loss of functional recovery. Interestingly, animals expressing the Gs coupled receptor displayed a larger functional recovery while also displaying motor behavior reminiscent of graft induced dyskinesia and increased rotational behavior. View Large Image | Download PowerPoint SlideFigure 2 | Animals expressing Dq/Ds receptors displayed a significant increase in rotational behavior following s.c. CNO administration, while Dq only and lesion controls displayed no significant increase.Taken together, our findings suggest that modulating activity of neurons used in cell replacement therapy may be able to increase functional recovery in patients with Parkinsons disease and the use of DREADDs may be a potent tool to dissect intracellular regulatory pathways.