A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism.

Adeno-associated virus (AAV) capsid modification enables the generation of recombinant vectors with tailored properties and tropism. Most approaches to date depend on random screening, enrichment, and serendipity. The approach explored here, called BRAVE (barcoded rational AAV vector evolution), enables efficient selection of engineered capsid structures on a large scale using only a single screening round in vivo. The approach stands in contrast to previous methods that require multiple generations of enrichment. With the BRAVE approach, each virus particle displays a peptide, derived from a protein, of known function on the AAV capsid surface, and a unique molecular barcode in the packaged genome. The sequencing of RNA-expressed barcodes from a single-generation in vivo screen allows the mapping of putative binding sequences from hundreds of proteins simultaneously. Using the BRAVE approach and hidden Markov model-based clustering, we present 25 synthetic capsid variants with refined properties, such as retrograde axonal transport in specific subtypes of neurons, as shown for both rodent and human dopaminergic neurons.

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.

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 Parkinson's 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.

Visualizing Arc protein dynamics and localization in the mammalian brain using AAV-mediated in situ gene labeling.

The activity-regulated cytoskeleton-associated (Arc) protein is essential for synaptic plasticity and memory formation. The Arc gene, which contains remnants of a structural GAG retrotransposon sequence, produces a protein that self-assembles into capsid-like structures harboring Arc mRNA. Arc capsids, released from neurons, have been proposed as a novel intercellular mechanism for mRNA transmission. Nevertheless, evidence for intercellular transport of Arc in the mammalian brain is still lacking. To enable the tracking of Arc molecules from individual neurons in vivo, we devised an adeno-associated virus (AAV) mediated approach to tag the N-terminal of the mouse Arc protein with a fluorescent reporter using CRISPR/Cas9 homologous independent targeted integration (HITI). We show that a sequence coding for mCherry can successfully be knocked in at the 5' end of the Arc open reading frame. While nine spCas9 gene editing sites surround the Arc start codon, the accuracy of the editing was highly sequence-dependent, with only a single target resulting in an in-frame reporter integration. When inducing long-term potentiation (LTP) in the hippocampus, we observed an increase of Arc protein highly correlated with an increase in fluorescent intensity and the number of mCherry-positive cells. By proximity ligation assay (PLA), we demonstrated that the mCherry-Arc fusion protein retains the Arc function by interacting with the transmembrane protein stargazin in postsynaptic spines. Finally, we recorded mCherry-Arc interaction with presynaptic protein Bassoon in mCherry-negative surrounding neurons at close proximity to mCherry-positive spines of edited neurons. This is the first study to provide support for inter-neuronal in vivo transfer of Arc in the mammalian brain.

Deconvolution of spatial sequencing provides accurate characterization of hESC-derived DA transplants in vivo.

Cell therapy for Parkinson's disease has experienced substantial growth in the past decades with several ongoing clinical trials. Despite increasing refinement of differentiation protocols and standardization of the transplanted neural precursors, the transcriptomic analysis of cells in the transplant after its full maturation in vivo has not been thoroughly investigated. Here, we present spatial transcriptomics analysis of fully differentiated grafts in their host tissue. Unlike earlier transcriptomics analyses using single-cell technologies, we observe that cells derived from human embryonic stem cells (hESCs) in the grafts adopt mature dopaminergic signatures. We show that the presence of phenotypic dopaminergic genes, which were found to be differentially expressed in the transplants, is concentrated toward the edges of the grafts, in agreement with the immunohistochemical analyses. Deconvolution shows dopamine neurons being the dominating cell type in many features beneath the graft area. These findings further support the preferred environmental niche of TH-positive cells and confirm their dopaminergic phenotype through the presence of multiple dopaminergic markers.

A comparison of machine learning approaches for the quantification of microglial cells in the brain of mice, rats and non-human primates.

Microglial cells are brain-specific macrophages that swiftly react to disruptive events in the brain. Microglial activation leads to specific modifications, including proliferation, morphological changes, migration to the site of insult, and changes in gene expression profiles. A change in inflammatory status has been linked to many neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. For this reason, the investigation and quantification of microglial cells is essential for better understanding their role in disease progression as well as for evaluating the cytocompatibility of novel therapeutic approaches for such conditions. In the following study we implemented a machine learning-based approach for the fast and automatized quantification of microglial cells; this tool was compared with manual quantification (ground truth), and with alternative free-ware such as the threshold-based ImageJ and the machine learning-based Ilastik. We first trained the algorithms on brain tissue obtained from rats and non-human primate immunohistochemically labelled for microglia. Subsequently we validated the accuracy of the trained algorithms in a preclinical rodent model of Parkinson's disease and demonstrated the robustness of the algorithms on tissue obtained from mice, as well as from images provided by three collaborating laboratories. Our results indicate that machine learning algorithms can detect and quantify microglial cells in all the three mammalian species in a precise manner, equipotent to the one observed following manual counting. Using this tool, we were able to detect and quantify small changes between the hemispheres, suggesting the power and reliability of the algorithm. Such a tool will be very useful for investigation of microglial response in disease development, as well as in the investigation of compatible novel therapeutics targeting the brain. As all network weights and labelled training data are made available, together with our step-by-step user guide, we anticipate that many laboratories will implement machine learning-based quantification of microglial cells in their research.

Automated quantification of neuronal swellings in a preclinical rodent model of Parkinson's disease detects region-specific changes in pathology.

BACKGROUND: The development of axonal pathology is a key characteristic of many neurodegenerative disease such as Parkinson's disease and Alzheimer's disease. With advanced disease progression, affected axons do display several signs of pathology such as swelling and fragmentation. In the AAV vector-mediated alpha-synuclein overexpression model of Parkinson's disease, large (> 20 µm2) pathological swellings are prominent characteristics in cortical and subcortical structures. NEW METHOD: This report describes a novel, macro-based workflow to quantify axonal pathology in the form of axonal swellings in the AAV vector-based alpha-synuclein overexpression model. Specifically, the approach is using background correction and thresholding before quantification of structures in 3D throughout a tissue stack. RESULTS: The method was used to quantify TH and aSYN axonal swellings in the prefrontal cortex, striatum, and hippocampus. Regional differences in volume and number of axonal swellings were observed for both in TH and aSYN, with the striatum displaying the greatest signs of pathology. COMPARISON WITH EXISTING METHODS: Existing methods for the quantification of axonal pathology do either rely on proprietary software or are based on manual quantification. The ImageJ workflow described here provides a method to objectively quantify axonal swellings both in volume and number. CONCLUSION: The method described can readily assess axonal pathology in preclinical rodent models of Parkinson's disease and can be easily adapted to other model systems and/or markers.

Sequential or Simultaneous Injection of Preformed Fibrils and AAV Overexpression of Alpha-Synuclein Are Equipotent in Producing Relevant Pathology and Behavioral Deficits.

BACKGROUND: Preclinical rodent models for Parkinson's disease (PD) based on viral human alpha-synuclein (h-αSyn) overexpression recapitulate some of the pathological hallmarks as it presents in humans, such as progressive cell loss and additional synucleinopathy in cortical and subcortical structures. Recent studies have combined viral vector-based overexpression of human wild-type αSyn with the sequential or simultaneous inoculation of preformed fibrils (PFFs) derived from human αSyn. OBJECTIVE: The goal of the study was to investigate whether sequential or combined delivery of the AAV vector and the PFFs are equipotent in inducing stable neurodegeneration and behavioral deficits. METHODS: Here we compare between four experimental paradigms (PFFs only, AAV-h-αSyn only, AAV-h-αSyn with simultaneous PFFs, and AAV-h-αSyn with sequential PFFs) and their respective GFP control groups. RESULTS: We observed reduction of TH expression and loss of neurons in the midbrain in all AAV (h-αSyn or GFP) injected groups, with or without additional PFFs inoculation. The overexpression of either h-αSyn or GFP alone induced motor deficits and dysfunctional dopamine release/reuptake in electrochemical recordings in the ipsilateral striatum. However, we observed a substantial formation of insoluble h-αSyn aggregates and inflammatory response only when h-αSyn and PFFs were combined. Moreover, the presence of h-αSyn induced higher axonal pathology compared to control groups. CONCLUSION: Simultaneous AAV and PFFs injections are equipotent in the presented experimental setup in inducing histopathological and behavioral changes. This model provides new and interesting possibilities for characterizing PD pathology in preclinical models and means to assess future therapeutic interventions.

A novel two-factor monosynaptic TRIO tracing method for assessment of circuit integration of hESC-derived dopamine transplants.

Transplantation in Parkinson's disease using human embryonic stem cell (hESC)-derived dopaminergic (DA) neurons is a promising future treatment option. However, many of the mechanisms that govern their differentiation, maturation, and integration into the host circuitry remain elusive. Here, we engrafted hESCs differentiated toward a ventral midbrain DA phenotype into the midbrain of a preclinical rodent model of Parkinson's disease. We then injected a novel DA-neurotropic retrograde MNM008 adeno-associated virus vector capsid, into specific DA target regions to generate starter cells based on their axonal projections. Using monosynaptic rabies-based tracing, we demonstrated for the first time that grafted hESC-derived DA neurons receive distinctly different afferent inputs depending on their projections. The similarities to the host DA system suggest a previously unknown directed circuit integration. By evaluating the differential host-to-graft connectivity based on projection patterns, this novel approach offers a tool to answer outstanding questions regarding the integration of grafted hESC-derived DA neurons.

Next-Generation Gene Therapy for Parkinson's Disease Using Engineered Viral Vectors.

Recent technological and conceptual advances have resulted in a plethora of exciting novel engineered adeno associated viral (AAV) vector variants. They all have unique characteristics and abilities. This review summarizes the development and their potential in treating Parkinson's disease (PD). Clinical trials in PD have shown over the last decade that AAV is a safe and suitable vector for gene therapy but that it also is a vehicle that can benefit significantly from improvement in specificity and potency. This review provides a concise collection of the state-of-the-art for synthetic capsids and their utility in PD. We also summarize what therapeutical strategies may become feasible with novel engineered vectors, including genome editing and neuronal rejuvenation.

AAV Production Everywhere: A Simple, Fast, and Reliable Protocol for In-house AAV Vector Production Based on Chloroform Extraction.

Recombinant adeno-associated virus (rAAV) is a mammalian virus that has been altered to be used as a gene delivery vehicle. Several changes to the viral genome have made them replication deficient so that this aspect of the viral infection cycle is under full control of the experimenter, while maintaining gene expression machinery. Over the last decades, rAAVs have become the gold standard for studying in vivo gene function and are especially favorable for gene transfer in the central nervous system. AAVs have been proven safe and provide stable gene expression over a long period of time. They are extensively used in preclinical experiments and show great potential for clinical applications. However, the use of AAVs in preclinical settings are often held back due to availability. Waiting lines are long at commercial production facilities, and in-lab production is hindered due to lack of specific laboratory equipment needed. Here we present a novel production method that can be carried out in any molecular biology laboratory using standard laboratory equipment. We provide a simple, fast, and streamlined protocol for production that can result in titers comparable with the more time-consuming iodixanol gradient ultracentrifugation method. The yield using this protocol is high enough for any type of study where AAV is the vector of choice. © 2020 The Authors.

A comparison of AAV-vector production methods for gene therapy and preclinical assessment.

Adeno Associated Virus (AAV)-mediated gene expression in the brain is widely applied in the preclinical setting to investigate the therapeutic potential of specific molecular targets, characterize various cellular functions, and model central nervous system (CNS) diseases. In therapeutic applications in the clinical setting, gene therapy offers several advantages over traditional pharmacological based therapies, including the ability to directly manipulate disease mechanisms, selectively target disease-afflicted regions, and achieve long-term therapeutic protein expression in the absence of repeated administration of pharmacological agents. Next to the gold-standard iodixanol-based AAV vector production, we recently published a protocol for AAV production based on chloroform-precipitation, which allows for fast in-house production of small quantities of AAV vector without the need for specialized equipment. To validate our recent protocol, we present here a direct side-by-side comparison between vectors produced with either method in a series of in vitro and in vivo assays with a focus on transgene expression, cell loss, and neuroinflammatory responses in the brain. We do not find differences in transduction efficiency nor in any other parameter in our in vivo and in vitro panel of assessment. These results suggest that our novel protocol enables most standardly equipped laboratories to produce small batches of high quality and high titer AAV vectors for their experimental needs.

The Financial Implications of a Well-Hidden and Ignored Chronic Lyme Disease Pandemic.

1 million people are predicted to get infected with Lyme disease in the USA in 2018. Given the same incidence rate of Lyme disease in Europe as in the USA, then 2.4 million people will get infected with Lyme disease in Europe in 2018. In the USA by 2050, 55.7 million people (12% of the population) will have been infected with Lyme disease. In Europe by 2050, 134.9 million people (17% of the population) will have been infected with Lyme disease. Most of these infections will, unfortunately, become chronic. The estimated treatment cost for acute and chronic Lyme disease for 2018 for the USA is somewhere between 4.8 billion USD and 9.6 billion USD and for Europe somewhere between 10.1 billion EUR and 20.1 billion EUR. If governments do not finance IV treatment with antibiotics for chronic Lyme disease, then the estimated government cost for chronic Lyme disease for 2018 for the USA is 10.1 billion USD and in Europe 20.1 billion EUR. If governments in the USA and Europe want to minimize future costs and maximize future revenues, then they should pay for IV antibiotic treatment up to a year even if the estimated cure rate is as low as 25%. The cost for governments of having chronic Lyme patients sick in perpetuity is very large.

Destabilizing Domains Enable Long-Term and Inert Regulation of GDNF Expression in the Brain.

Regulation of therapeutic transgene expression can increase the safety of gene therapy interventions, especially when targeting critical organs such as the brain. Although several gene expression systems have been described, none of the current systems has the required safety profile for clinical applications. Our group has previously adapted a system for novel gene regulation based on the destabilizing domain degron technology to successfully regulate glial cell-line derived neurotrophic factor in the brain (GDNF-F-DD). In the present study, we used GDNF-F-DD as a proof-of-principle molecule to fully characterize DD regulation in the brain. Our results indicate that DD could be regulated in a dose-dependent manner. In addition, GDNF-F-DD could also be induced in vivo repeatedly, without loss of activity or efficacy in vivo. Finally, DD regulation was able to be sustained for 24 weeks without loss of expression or any overt toxicity. The present study shows that DD has great potential to regulate gene expression in the brain.

A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing.

Detailed characterization and mapping of oligonucleotide function in vivo is generally a very time consuming effort that only allows for hypothesis driven subsampling of the full sequence to be analysed. Recent advances in deep sequencing together with highly efficient parallel oligonucleotide synthesis and cloning techniques have, however, opened up for entirely new ways to map genetic function in vivo. Here we present a novel, optimized protocol for the generation of universally applicable, barcode labelled, plasmid libraries. The libraries are designed to enable the production of viral vector preparations assessing coding or non-coding RNA function in vivo. When generating high diversity libraries, it is a challenge to achieve efficient cloning, unambiguous barcoding and detailed characterization using low-cost sequencing technologies. With the presented protocol, diversity of above 3 million uniquely barcoded adeno-associated viral (AAV) plasmids can be achieved in a single reaction through a process achievable in any molecular biology laboratory. This approach opens up for a multitude of in vivo assessments from the evaluation of enhancer and promoter regions to the optimization of genome editing. The generated plasmid libraries are also useful for validation of sequencing clustering algorithms and we here validate the newly presented message passing clustering process named Starcode.

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 Parkinson's 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.

Skeletal muscle atrophy in R6/2 mice - altered circulating skeletal muscle markers and gene expression profile changes.

BACKGROUND: In addition to classical neurological symptoms, Huntington's disease (HD) is complicated by peripheral pathology, including progressive skeletal muscle wasting, and common skeletal muscle gene expression changes have been shown in HD mice and human HD. OBJECTIVE: To highlight possible mechanisms underlying muscle wasting in HD, we examined gene expression in pathways governing skeletal muscle contractility, skeletal myogenesis, skeletal muscle wasting, apoptosis and the NFκB signaling pathway in two HD mouse models (the transgenic R6/2 and full-length knock-in Q175). In addition, we assessed circulating markers that increase in response to skeletal muscle injury, skeletal Troponin I (sTnI), fatty acid binding protein 3 (FABP3), and Myosin light chain 3 (Myl3). METHODS: We measured gene expression in muscle tissue as well as in cultured primary myocytes using qPCR. Concentrations of cytokines and muscle proteins were obtained using multiplex ELISA. RESULTS: Circulating markers of muscle injury (sTnI, FABP3, and Myl3) were significantly increased in mouse serum. In skeletal muscle, we observed reduced gene expression of components involved in muscle contractility, with pronounced downregulation of Acta1, Myh2 and Tnni2, among others. Alongside, we found increased expression of caspases (3 and 8) and key elements of the NFκB signaling pathway, p65/RelA, Tradd, and TRAF5. We also found similar gene expression alterations in cultured primary myocytes from R6/2 mice stimulated with TNF-α. CONCLUSIONS: Our results indicate that activation of apoptotic and NFκB pathways occur alongside down-regulation of key compartments of the muscle contractility unit in skeletal muscle of HD mice, and muscle atrophy could possibly be a source of circulating disease progression markers.