Characterization and biodistribution of under-employed gene therapy vector AAV7.

IMPORTANCE: The use of adeno-associated viruses (AAVs) as gene delivery vectors has vast potential for the treatment of many severe human diseases. Over one hundred naturally existing AAV capsid variants have been described and classified into phylogenetic clades based on their sequences. AAV8, AAV9, AAVrh.10, and other intensively studied capsids have been propelled into pre-clinical and clinical use, and more recently, marketed products; however, less-studied capsids may also have desirable properties (e.g., potency differences, tissue tropism, reduced immunogenicity, etc.) that have yet to be thoroughly described. These data will help build a broader structure-function knowledge base in the field, present capsid engineering opportunities, and enable the use of novel capsids with unique properties.

Brain pharmacology of intrathecal antisense oligonucleotides revealed through multimodal imaging.

Intrathecal (IT) delivery and pharmacology of antisense oligonucleotides (ASOs) for the CNS have been successfully developed to treat spinal muscular atrophy. However, ASO pharmacokinetic (PK) and pharmacodynamic (PD) properties remain poorly understood in the IT compartment. We applied multimodal imaging techniques to elucidate the IT PK and PD of unlabeled, radioactively labeled, or fluorescently labeled ASOs targeting ubiquitously expressed or neuron-specific RNAs. Following lumbar IT bolus injection in rats, all ASOs spread rostrally along the neuraxis, adhered to meninges, and were partially cleared to peripheral lymph nodes and kidneys. Rapid association with the pia and arterial walls preceded passage of ASOs across the glia limitans, along arterial intramural basement membranes, and along white-matter axonal bundles. Several neuronal and glial cell types accumulated ASOs over time, with evidence of probable glial accumulation preceding neuronal uptake. IT doses of anti-GluR1 and anti-Gabra1 ASOs markedly reduced the mRNA and protein levels of their respective neurotransmitter receptor protein targets by 2 weeks and anti-Gabra1 ASOs also reduced binding of the GABAA receptor PET ligand 18F-flumazenil in the brain over 4 weeks. Our multimodal imaging approaches elucidate multiple transport routes underlying the CNS distribution, clearance, and efficacy of IT-dosed ASOs.

Direct visualization of CHIP-mediated degradation of alpha-synuclein in vivo: implications for PD therapeutics.

Parkinson's disease is a neurodegenerative disorder characterized by Lewy bodies, a pathological hallmark comprised mostly of aggregated alpha synuclein. Accumulating evidence demonstrates the association of smaller oligomeric aggregates to disease etiology and many therapeutic approaches are aimed at inhibiting and reducing the aggregation process. Molecular chaperones and co-chaperones play a key role in protein homeostasis and have potential as therapeutics to inhibit alpha synuclein associated toxicity. Here we use a gene therapy approach to evaluate the applicability of the Hsp70 co-chaperone CHIP (C-terminal Hsp70 interacting protein) as a therapeutic candidate and examine its direct effect on alpha synuclein aggregates in vivo. Utilizing a novel viral vector mediated rat model to directly detect alpha synuclein aggregates, we show that CHIP can mediate the degradation of alpha synuclein aggregates in vivo. However, our studies also reveal that CHIP may potentially degrade tyrosine hydroxylase which would compromise the applicability of CHIP as a therapeutic approach for Parkinson's disease.

Chronic treatment with novel small molecule Hsp90 inhibitors rescues striatal dopamine levels but not α-synuclein-induced neuronal cell loss.

Hsp90 inhibitors such as geldanamycin potently induce Hsp70 and reduce cytotoxicity due to α-synuclein expression, although their use has been limited due to toxicity, brain permeability, and drug design. We recently described the effects of a novel class of potent, small molecule Hsp90 inhibitors in cells overexpressing α-synuclein. Screening yielded several candidate compounds that significantly reduced α-synuclein oligomer formation and cytotoxicity associated with Hsp70 induction. In this study we examined whether chronic treatment with candidate Hsp90 inhibitors could protect against α-synuclein toxicity in a rat model of parkinsonism. Rats were injected unilaterally in the substantia nigra with AAV8 expressing human α-synuclein and then treated with drug for approximately 8 weeks by oral gavage. Chronic treatment with SNX-0723 or the more potent, SNX-9114 failed to reduce dopaminergic toxicity in the substantia nigra compared to vehicle. However, SNX-9114 significantly increased striatal dopamine content suggesting a positive neuromodulatory effect on striatal terminals. Treatment was generally well tolerated, but higher dose SNX-0723 (6-10 mg/kg) resulted in systemic toxicity, weight loss, and early death. Although still limited by potential toxicity, Hsp90 inhibitors tested herein demonstrate oral efficacy and possible beneficial effects on dopamine production in a vertebrate model of parkinsonism that warrant further study.

Direct detection of alpha synuclein oligomers in vivo.

BACKGROUND: Rat models of Parkinson's disease are widely used to elucidate the mechanisms underlying disease etiology or to investigate therapeutic approaches. Models were developed using toxins such as MPTP or 6-OHDA to specifically target dopaminergic neurons resulting in acute neuronal loss in the substantia nigra or by using viral vectors to induce the specific and gradual expression of alpha synuclein in the substantia nigra. The detection of alpha- synuclein oligomers, the presumed toxic species, in these models and others has been possible using only indirect biochemical approaches to date. Here we coinjected AAVs encoding alpha-synuclein fused to the N- or C-terminal half of VenusYFP in rat substantia nigra pars compacta and describe for the first time a novel viral vector rodent model with the unique ability to directly detect and track alpha synuclein oligomers ex vivo and in vivo. RESULTS: Viral coinjection resulted in widespread VenusYFP signal within the nigrostriatal pathway, including cell bodies in the substantia nigra and synaptic accumulation in striatal terminals, suggestive of in vivo alpha-synuclein oligomers formation. Transduced rats showed alpha-synuclein induced dopaminergic neuron loss in the substantia nigra, the appearance of dystrophic neurites, and gliosis in the striatum. Moreover, we have applied in vivo imaging techniques in the living mouse to directly image alpha-synuclein oligomers in the cortex. CONCLUSION: We have developed a unique animal model that provides a tool for the Parkinson's disease research community with which to directly detect alpha- synuclein oligomers in vivo and screen therapeutic approaches targeting alpha-synuclein oligomers.

Molecular chaperones and co-chaperones in Parkinson disease.

Parkinson disease, a progressive neurodegenerative disorder, is caused by the pathological accumulation of proteins, including the ubiquitous presynaptic protein α-synuclein. Alterations in the metabolism of α-synuclein have clearly been linked to neurodegeneration, and early steps in the pathological sequence of this protein include the formation of oligomers, fibrils, and small aggregates. Targeting these early steps of oligomerization is one of the main therapeutic approaches in the quest to develop disease-modifying agents. Molecular chaperones, molecules that can mediate the proper folding and refolding of client proteins, are vital to cell function and survival and thus have been explored as potential therapeutic agents. Important to Parkinson disease, chaperones are capable of preventing α-synuclein misfolding, oligomerization, and aggregate formation as shown in vitro and in Parkinson disease animal models. Furthermore, chaperones and associated co-chaperones are closely linked to pathways of protein degradation, like the ubiquitin-proteasome system and autophagy, and are thus able to remove irreversibly misfolded proteins. In this review, we summarize the role of molecular chaperones in Parkinson disease models and discuss the importance of preserving protein homeostasis to prevent neurodegeneration. We also review the growing number of exciting studies that have targeted molecular chaperone function as a novel therapeutic approach.

Filamentous phages reduce alpha-synuclein oligomerization in the membrane fraction of SH-SY5Y cells.

BACKGROUND: alpha-Synuclein (AS) is an abundant neuronal protein predominantly localized in presynaptic terminals in the central nervous system. AS aggregation is a molecular hallmark of several neurodegenerative diseases, including Parkinson's disease, and is thought to play a significant role in the etiology of the disease. Recent experimental evidence indicates that AS exists in two forms, a membrane-bound form and a disordered cytosolic form. Much effort is dedicated to prevent and dissolve AS aggregates, specifically AS oligomers and protofibrils, which are thought to be the more toxic form of aggregates. METHODS: The effect of filamentous phages on AS aggregation in SH-SY5Y cells overexpressing wild-type AS was quantified in ELISA designed to detect and quantify AS oligomers. RESULTS: We found reduced levels of AS oligomers in the membrane fraction in cells treated with filamentous phages compared to nontreated cells. CONCLUSION: The reduction in AS oligomers from the plasma membrane in treated cells may suggest further therapeutic application.

TAR1, a human anti-p53 single-chain antibody, restores tumor suppressor function to mutant p53 variants.

The tumor suppressor gene p53 is mutated in more than half of human tumors. One important characteristic of p53 mutants is their accumulation in the nucleus of cancer cells. Thus, reactivation of mutant p53 proteins may trigger massive apoptosis in tumor cells. Pharmacologic methods are currently under development to induce mutant p53 proteins to resume their wild-type function. We have identified a human single-chain Fv fragment, designated as transcriptional transactivation and apoptosis restoring (TAR1), which specifically and with high affinity binds to mutant p53 and restores its wild-type active conformation. Binding of TAR1 to mutant p53 induced transcriptional transactivation of p53 target genes and down-regulation of mutant p53 transcriptional target genes. TAR1 treatment induced apoptosis in a variety of cell lines endogenously expressing p53 carrying different point mutations DNA contact or structural p53 mutants. Moreover, in an animal model of mice carrying human xenografts, TAR1 induced tumor regression with no apparent deleterious side effects. Thus, it may be considered as a potential candidate for anticancer treatment, targeting tumors with mutant p53.

Modulation effect of filamentous phage on alpha-synuclein aggregation.

Conversion of soluble peptides and proteins into amyloid fibrils and/or intermediate oligomers is believed to be the central event in the pathogenesis of most human neurodegenerative diseases, including Parkinson's disease (PD). Here we describe the modulating effect of filamentous phages on aggregation of alpha-synuclein (AS) in vitro and in a PD cellular model. Filamentous phages, well understood at both structural and genetic levels, have a nanotubular appearance, showing conformational similarities to amyloid fibrils. Since filamentous phages can infect only bacteria and have no tropism to mammalian cells, we utilized the f88 system to present a peptide containing a cyclic RGD (arg-gly-asp), which enabled phage internalization into the cells. Detection of intracellular AS oligomers, in differentiated SH-SY5Y cells, stably transfected with wild type AS gene, was performed using Western blot and ELISA measurements. Data presented here show reduced levels of AS soluble aggregates in phage treated cells compared to non-treated cells, suggesting new therapeutics for PD.