large-scale-sars-cov-2-testing-utilizing-saliva-transposition-sample

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. ...

Michigan State University

The discovery of the involvement of alpha-synuclein (α-syn) in Parkinson's disease (PD) pathogenesis has resulted in the development and use of viral vector-mediated α-syn overexpression rodent models. The goal of these series of experiments was to characterize the neurodegeneration and functional deficits resulting from injection of recombinant adeno-associated virus (rAAV) serotype 2/5-expressing human wildtype α-syn in the rat substantia nigra (SN). Rats were unilaterally injected into two sites in the SN with either rAAV2/5-expressing green fluorescent protein (GFP, 1.2 x 10(13)) or varying titers (2.2 x 10(12), 1.0 x 10(13), 5.9 x 10(13), or 1.0 x 10(14)) of rAAV2/5-α-syn. Cohorts of rats were euthanized 4, 8, or 12 weeks following vector injection. The severity of tyrosine hydroxylase immunoreactive (THir) neuron death in the SN pars compacta (SNpc) was dependent on vector titer. An identical magnitude of nigrostriatal degeneration (60-70% SNpc THir neuron degeneration and 40-50% loss of striatal TH expression) was observed four weeks following 1.0 x 10(14) titer rAAV2/5-α-syn injection and 8 weeks following 1.0 x 10(13) titer rAAV2/5-α-syn injection. THir neuron degeneration was relatively uniform throughout the rostral-caudal axis of the SNpc. Despite equivalent nigrostriatal degeneration between the 1.0 x 10(13) and 1.0 x 10(14) rAAV2/5-α-syn groups, functional impairment in the cylinder test and the adjusting steps task was only observed in rats with the longer 8 week duration of α-syn expression. Motor impairment in the cylinder task was highly correlated to striatal TH loss. Further, 8 weeks following 5.9 x 10(13) rAAV2/5-α-syn injection deficits in ultrasonic vocalizations were observed. In conclusion, our rAAV2/5-α-syn overexpression model demonstrates robust nigrostriatal α-syn overexpression, induces significant nigrostriatal degeneration that is both vector and duration dependent and under specific parameters can result in motor impairment that directly relates to the level of striatal TH denervation.

Morphological and behavioral impact of AAV2/5-mediated overexpression of human wildtype alpha-synuclein in the rat nigrostriatal system.

Clinical trials are examining the efficacy of viral vector-mediated gene delivery for treating Parkinson's disease. Although viral vector strategies have been successful in preclinical studies, to date clinical trials have disappointed. This may be because of the fact that preclinical studies fail to account for aging. Aging is the single greatest risk factor for developing Parkinson's disease and age alters cellular processes utilized by viral vectors. We hypothesized that the aged brain would be relatively resistant to transduction when compared with the young adult. We examined recombinant adeno-associated virus 2/5-mediated green fluorescent protein (rAAV2/5 GFP) expression in the young adult and aged rat nigrostriatal system. GFP overexpression was produced in both age groups. However, following rAAV2/5 GFP injection to the substantia nigra aged rats displayed 40%-60% less GFP protein in the striatum, regardless of rat strain or duration of expression. Furthermore, aged rats exhibited 40% fewer cells expressing GFP and 4-fold less GFP messenger RNA. rAAV2/5-mediated gene transfer is compromised in the aged rat midbrain, with deficiencies in early steps of transduction leading to significantly less messenger RNA and protein expression. 

Recombinant adenoassociated virus 2/5-mediated gene transfer is reduced in the aged rat midbrain.

Recombinant adeno-associated virus (AAV) vectors are one of the most widely used gene transfer systems in research and clinical trials. AAV can transduce a wide range of biological tissues, however to date, there has been no investigation on targeted AAV transduction of the enteric nervous system (ENS). Here, we examined the efficiency, tropism, spread, and immunogenicity of AAV transduction in the ENS. Rats received direct injections of various AAV serotypes expressing green fluorescent protein (GFP) into the descending colon. AAV serotypes tested included; AAV 1, 2, 5, 6, 8, or 9 and the AAV2 and AAV8 capsid mutants, AAV2-Y444F, AAV2-tripleY-F, AAV2-tripleY-F+T-V, AAV8-Y733F, and AAV8-doubeY-F+T-V. Transduction, as determined by GFP-positive cells, occurred in neurons and enteric glia within the myenteric and submucosal plexuses of the ENS. AAV6 and AAV9 showed the highest levels of transduction within the ENS. Transduction efficiency scaled with titer and time, was translated to the murine ENS, and produced no vector-related immune response. A single injection of AAV into the colon covered an area of ~47 mm(2). AAV9 primarily transduced neurons, while AAV6 transduced enteric glia and neurons. This is the first report on targeted AAV transduction of neurons and glia in the ENS. 

Targeted gene delivery to the enteric nervous system using AAV: a comparison across serotypes and capsid mutants.

Alpha-Synuclein (α-syn) is by far the most highly vetted pathogenic and therapeutic target in Parkinson's disease. Aggregated α-syn is present in sporadic Parkinson's disease, both in the central nervous system (CNS) and peripheral nervous system (PNS). The enteric division of the PNS is of particular interest because 1) gastric dysfunction is a key clinical manifestation of Parkinson's disease, and 2) Lewy pathology in myenteric and submucosal neurons of the enteric nervous system (ENS) has been referred to as stage zero in the Braak pathological staging of Parkinson's disease. The presence of Lewy pathology in the ENS and the fact that patients often experience enteric dysfunction before the onset of motor symptoms has led to the hypothesis that α-syn pathology starts in the periphery, after which it spreads to the CNS via interconnected neural pathways. Here we sought to directly test this hypothesis in rodents and non-human primates (NHP) using two distinct models of α-syn pathology: the α-syn viral overexpression model and the preformed fibril (PFF) model. Subjects (rat and NHP) received targeted enteric injections of PFFs or adeno-associated virus overexpressing the Parkinson's disease associated A53T α-syn mutant. Rats were evaluated for colonic motility monthly and sacrificed at 1, 6, or 12 months, whereas NHPs were sacrificed 12 months following inoculation, after which the time course and spread of pathology was examined in all animals. Rats exhibited a transient GI phenotype that resolved after four months. Minor α-syn pathology was observed in the brainstem (dorsal motor nucleus of the vagus and locus coeruleus) 1 month after PFF injections; however, no pathology was observed at later time points (nor in saline or monomer treated animals). Similarly, a histopathological analysis of the NHP brains revealed no pathology despite the presence of robust α-syn pathology throughout the ENS which persisted for the entirety of the study (12 months). Our study shows that induction of α-syn pathology in the ENS is sufficient to induce GI dysfunction. Moreover, our data suggest that sustained spread of α-syn pathology from the periphery to the CNS and subsequent propagation is a rare event, and that the presence of enteric α-syn pathology and dysfunction may represent an epiphenomenon.

Induction of alpha-synuclein pathology in the enteric nervous system of the rat and non-human primate results in gastrointestinal dysmotility and transient CNS pathology.

Today any researcher with the desire can easily purchase a viral vector. However, despite the availability of viral vectors themselves, the requisite knowledge that is absolutely essential to conducting a gene therapy experiment remains somewhat obscure and esoteric. To utilize viral vectors to their full potential, a large number of decisions must be made, in some instances prior to even obtaining the vector itself. For example, critical decisions include selection of the proper virus, selection of the proper expression cassette, whether to produce or purchase a viral vector, proper viral handling and storage, the most appropriate delivery method, selecting the proper controls, how to ensure your virus is expressing properly, and many other complex decisions that are essential to performing a successful gene therapy experiment. The need to make so many important decisions can be overwhelming and potentially prohibitive, especially to the novice gene therapist. In order to aid in this challenging process, here we provide an overview of basic gene therapy modalities and a decision tree that can be used to make oneself aware of the options available to the beginning gene therapist. This information can be used as a road map to help navigate the complex and perhaps confusing process of designing a successful gene therapy experiment.

Basic Concepts in Viral Vector-Mediated Gene Therapy.

Adeno-associated virus (AAV) is an increasingly popular tool in the research laboratory, and use of this viral vector clinically is occurring at an accelerated pace. Nevertheless, despite its popularity, AAV is a relatively cumbersome virus to produce; however, significant efforts have been invested to develop, optimize, and simplify methodology that allows the generation of high-quality AAV with significantly increased production yields. Here we describe multiple modalities for production and purification of AAV particles produced in HEK293 cell cultures using an iodixanol density gradient. We include two methods adapted for harvesting virus from the culture media: tangential flow filtration (TFF) and polyethylene glycol precipitation (PEGylation). Moreover, we also describe the protocol for anion exchange chromatography, which can be used after the iodixanol gradient as an additional purification step. Last, we provide various protocols for determining virus titer.

Multimodal Production of Adeno-Associated Virus.

Human and animal studies have shown that exposure to the organochlorine pesticide dieldrin is associated with increased risk of Parkinson's disease (PD). Previous work showed that developmental dieldrin exposure increased neuronal susceptibility to MPTP toxicity in male C57BL/6 mice, possibly via changes in dopamine (DA) packaging and turnover. However, the relevance of the MPTP model to PD pathophysiology has been questioned. We therefore studied dieldrin-induced neurotoxicity in the α-synuclein (α-syn)-preformed fibril (PFF) model, which better reflects the α-syn pathology and toxicity observed in PD pathogenesis. Specifically, we used a "two-hit" model to determine whether developmental dieldrin exposure increases susceptibility to α-syn PFF-induced synucleinopathy. Dams were fed either dieldrin (0.3 mg/kg, every 3-4 days) or vehicle corn oil starting 1 month prior to breeding and continuing through weaning of pups at postnatal day 22. At 12 weeks of age, male and female offspring received intrastriatal α-syn PFF or control saline injections. Consistent with the male-specific increased susceptibility to MPTP, our results demonstrate that developmental dieldrin exposure exacerbates PFF-induced toxicity in male mice only. Specifically, in male offspring, dieldrin exacerbated PFF-induced motor deficits on the challenging beam and increased DA turnover in the striatum 6 months after PFF injection. However, male offspring showed neither exacerbation of phosphorylated α-syn aggregation (pSyn) in the substantia nigra (SN) at 1 or 2 months post-PFF injection, nor exacerbation of PFF-induced TH and NeuN loss in the SN 6 months post-PFF injection. Collectively, these data indicate that developmental dieldrin exposure produces a male-specific exacerbation of synucleinopathy-induced behavioral and biochemical deficits. This sex-specific result is consistent with both previous work in the MPTP model, our previously reported sex-specific effects of this exposure paradigm on the male and female epigenome, and the higher prevalence and more severe course of PD in males. The novel two-hit environmental toxicant/PFF exposure paradigm established in this project can be used to explore the mechanisms by which other PD-related exposures alter neuronal vulnerability to synucleinopathy in sporadic PD.

Developmental exposure to the organochlorine pesticide dieldrin causes male-specific exacerbation of α-synuclein-preformed fibril-induced toxicity and motor deficits.

The gastrointestinal tract may be a site of origin for α-synuclein pathology in idiopathic Parkinson's disease (PD). Disruption of the autophagy-lysosome pathway (ALP) may contribute to α-synuclein aggregation. Here we examined epigenetic alterations in the ALP in the appendix by deep sequencing DNA methylation at 521 ALP genes. We identified aberrant methylation at 928 cytosines affecting 326 ALP genes in the appendix of individuals with PD and widespread hypermethylation that is also seen in the brain of individuals with PD. In mice, we find that DNA methylation changes at ALP genes induced by chronic gut inflammation are greatly exacerbated by α-synuclein pathology. DNA methylation changes at ALP genes induced by synucleinopathy are associated with the ALP abnormalities observed in the appendix of individuals with PD specifically involving lysosomal genes. Our work identifies epigenetic dysregulation of the ALP which may suggest a potential mechanism for accumulation of α-synuclein pathology in idiopathic PD.

Epigenetic inactivation of the autophagy-lysosomal system in appendix in Parkinson's disease.

Positron emission tomography (PET) imaging in early Parkinson's disease (PD) subjects reveals that increased dopamine (DA) turnover and reduced dopamine transporter (DAT) density precede decreases in DA synthesis and storage. The rat α-synuclein preformed fibril (α-syn PFF) model provides a platform to investigate DA dynamics during multiple stages of α-syn inclusion-triggered nigrostriatal degeneration.

Article	Year
Morphological and behavioral impact of AAV2/5-mediated overexpression of human wildtype alpha-synuclein in the rat nigrostriatal system. PloS one\| PubMed ID: 24312298	2013
Recombinant adenoassociated virus 2/5-mediated gene transfer is reduced in the aged rat midbrain. Neurobiology of aging\| PubMed ID: 25457558	2015
Targeted gene delivery to the enteric nervous system using AAV: a comparison across serotypes and capsid mutants. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 25592336	2015
Induction of alpha-synuclein pathology in the enteric nervous system of the rat and non-human primate results in gastrointestinal dysmotility and transient CNS pathology. Neurobiology of disease\| PubMed ID: 29341898	2018
Basic Concepts in Viral Vector-Mediated Gene Therapy. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30706387	2019
Multimodal Production of Adeno-Associated Virus. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30706392	2019
Developmental exposure to the organochlorine pesticide dieldrin causes male-specific exacerbation of α-synuclein-preformed fibril-induced toxicity and motor deficits. Neurobiology of disease\| PubMed ID: 32422283	2020
Epigenetic inactivation of the autophagy-lysosomal system in appendix in Parkinson's disease. Nature communications\| PubMed ID: 34446734	2021
Dopaminergic Positron Emission Tomography Imaging in the Alpha-Synuclein Preformed Fibril Model Reveals Similarities to Early Parkinson's Disease. Movement disorders : official journal of the Movement Disorder Society\| PubMed ID: 35524682	2022

Nathan Kuhn

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