Reduced D2 /D3 Receptor Binding and Glucose Metabolism in a Macaque Model of Huntington's Disease.

BACKGROUND: Dopamine system dysfunction and altered glucose metabolism are implicated in Huntington's disease (HD), a neurological disease caused by mutant huntingtin (mHTT) expression. OBJECTIVE: The aim was to characterize alterations in cerebral dopamine D2 /D3 receptor density and glucose utilization in a newly developed AAV-mediated NHP model of HD that expresses mHTT throughout numerous brain regions. METHODS: Positron emission tomography (PET) imaging was performed using [18 F]fallypride to quantify D2 /D3 receptor density and 2-[18 F]fluoro-2-deoxy-d-glucose ([18 F]FDG) to measure cerebral glucose utilization in these HD macaques. RESULTS: Compared to controls, HD macaques showed significantly reduced dopamine D2 /D3 receptor densities in basal ganglia (P < 0.05). In addition, HD macaques displayed significant glucose hypometabolism throughout the cortico-basal ganglia network (P < 0.05). CONCLUSIONS: [18 F]Fallypride and [18 F]FDG are PET imaging biomarkers of mHTT-mediated disease progression that can be used as noninvasive outcome measures in future therapeutic studies with this AAV-mediated HD macaque model. © 2022 International Parkinson and Movement Disorder Society.

Enhanced CNS transduction from AAV.PHP.eB infusion into the cisterna magna of older adult rats compared to AAV9.

The development of high efficiency, central nervous system (CNS) targeting AAV-based gene therapies is necessary to address challenges in both pre-clinical and clinical investigations. The engineered capsids, AAV.PHP.B and AAV.PHP.eB, show vastly improved blood-brain barrier penetration compared to their parent serotype, AAV9, but with variable effect depending on animal system, strain, and delivery route. As most characterizations of AAV.PHP variants have been performed in mice, it is currently unknown whether AAV.PHP variants improve CNS targeting when delivered intrathecally in rats. We evaluated the comparative transduction efficiencies of equititer doses (6 × 1011vg) of AAV.PHP.eB-CAG-GFP and AAV9-CAG-GFP when delivered into the cisterna magna of 6-9-month old rats. Using both quantitative and qualitative assessments, we observed consistently superior biodistribution of GFP+ cells and fibers in animals treated with AAV.PHP.eB compared to those treated with AAV9. Enhanced GFP signal was uniformly observed throughout rostrocaudal brain regions in AAV.PHP.eB-treated animals with matching GFP protein expression detected in the forebrain, midbrain, and cerebellum. Collectively, these data illustrate the benefit of intracisternal infusions of AAV.PHP.eB as an optimal system to distribute CNS gene therapies in preclinical investigations of rats, and may have important translational implications for the clinical CNS targeting.

The macaque brain ONPRC18 template with combined gray and white matter labelmap for multimodal neuroimaging studies of Nonhuman Primates.

Macaques are the most common nonhuman primate (NHP) species used in neuroscience research. With the advancement of many neuroimaging techniques, new studies are beginning to apply multiple types of in vivo magnetic resonance imaging (MRI), such as structural imaging (sMRI) with T1 and T2 weighted contrasts alongside diffusion weighed (DW) imaging. In studies involving rhesus macaques, this approach can be used to better understand micro-structural changes that occur during development, in various disease states or with normative aging. However, many of the available rhesus brain atlases have been designed for only one imaging modality, making it difficult to consistently define the same brain regions across multiple imaging modalities in the same subject. To address this, we created a brain atlas from 18 adult rhesus macaques that includes co-registered templates constructed from images frequently used to characterize macroscopic brain structure (T2/SPACE and T1/MP-RAGE), and a diffusion tensor imaging (DTI) template. The DTI template was up-sampled from 1 mm isotropic resolution to resolution match to the T1 and T2-weighted images (0.5 mm isotropic), and the parameter maps were derived for FA, AD, RD and MD.The labelmap volumes delineate 57 gray matter regions of interest (ROIs; 36 cortical regions and 21 subcortical structures), as well as 74 white matter tracts. Importantly, the labelmap overlays both the structural and diffusion templates, enabling the same regions to be consistently identified across imaging modalities. A specialized condensed version of the labelmap ROIs are also included to further extend the usefulness of this tool for imaging data with lower spatial resolution, such as functional MRI (fMRI) or positron emission tomography (PET).

AAV-PHP.B Administration Results in a Differential Pattern of CNS Biodistribution in Non-human Primates Compared with Mice.

The ability of recombinant adeno-associated virus (AAV) to deliver transgenes to the CNS has allowed for several advancements in the field of gene therapy to treat brain disorders. Although most AAVs do not readily cross the blood-brain barrier and transduce the CNS following peripheral administration, AAV-PHP.B has recently been shown to transduce brains of mice with higher efficiency compared with its parent serotype, AAV9, following injection into the retro-orbital sinus. Here, we extended this foundational work by comparing AAV-PHP.B transduction efficiency in wild-type C57BL/6J mice using four clinically applicable delivery strategies including two intravascular (intra-jugular vein and intra-carotid artery) and two intra-cerebral spinal fluid (CSF) routes (intra-cisterna magna and intra-lateral ventricle). We scaled up these comparisons in a larger-animal model and evaluated transduction efficiency of AAV-PHP.B in the rhesus macaque. We found widespread and largely equal CNS transduction in mice following all four injection strategies, whereas we observed a differential pattern of transduction in macaques with broad cortical and spinal cord transduction seen after intrathecal administration and only very low transduction following intravascular administration. Taken together, these results suggest that AAV-PHP.B may be a useful gene therapy vector for neurological disorders, particularly those stemming from broad cortical or spinal cord neuropathology.

Normalizing glucocorticoid levels attenuates metabolic and neuropathological symptoms in the R6/2 mouse model of huntington's disease.

Huntington's disease (HD) is a fatal genetic neurological disorder caused by a mutation in the human Huntingtin (HTT) gene. This mutation confers a toxic gain of function of the encoded mutant huntingtin (mHTT) protein, leading to widespread neuropathology including the formation of mHTT-positive inclusion bodies, gene dysregulation, reduced levels of adult dentate gyrus neurogenesis and neuron loss throughout many regions of the brain. Additionally, because HTT is ubiquitously expressed, several peripheral tissues are also affected. HD patients suffer from progressive motor, cognitive, psychiatric, and metabolic symptoms, including weight loss and skeletal muscle wasting. HD patients also show neuroendocrine changes including a robust, significant elevation in circulating levels of the glucocorticoid, cortisol. Previously, we confirmed that the R6/2 mouse model of HD exhibits elevated corticosterone (the rodent homolog of cortisol) levels and demonstrated that experimentally elevated corticosterone exacerbates R6/2 HD symptomology, resulting in severe and rapid weight loss and a shorter latency to death. Given that efficacious therapeutics are lacking for HD, here we investigated whether normalizing glucocorticoid levels could serve as a viable therapeutic approach for this disease. We tested the hypothesis that normalizing glucocorticoids to wild-type levels would ameliorate HD symptomology. Wild-type (WT) and transgenic R6/2 mice were allocated to three treatment groups: 1) adrenalectomy with normalized, WT-level corticosterone replacement (10 µg/ml), 2) adrenalectomy with high HD-level corticosterone replacement (35 µg/ml), or 3) sham surgery with no corticosterone replacement. Normalizing corticosterone to WT levels led to an improvement in metabolic rate in male R6/2 mice, as indicated by indirect calorimetry, including a reduction in oxygen consumption and normalization of respiratory exchange ratio values (p < .05 for both). Normalizing corticosterone also ameliorated brain atrophy in female R6/2 mice and skeletal muscle wasting in both male and female R6/2 mice (p < .05 for all). Female R6/2 mice given WT-level corticosterone replacement also showed a reduction in HD neuropathological markers, including a reduction in mHTT inclusion burden in the striatum, cortex, and hippocampus (p < .05 for all). This data illustrates that ameliorating glucocorticoid dysregulation leads to a significant improvement in HD symptomology in the R6/2 mouse model and suggests that cortisol-reducing therapeutics may be of value in improving HD patient quality of life.

Discovery of a CLN7 model of Batten disease in non-human primates.

We have identified a natural Japanese macaque model of the childhood neurodegenerative disorder neuronal ceroid lipofuscinosis, commonly known as Batten Disease, caused by a homozygous frameshift mutation in the CLN7 gene (CLN7-/-). Affected macaques display progressive neurological deficits including visual impairment, tremor, incoordination, ataxia and impaired balance. Imaging, functional and pathological studies revealed that CLN7-/- macaques have reduced retinal thickness and retinal function early in disease, followed by profound cerebral and cerebellar atrophy that progresses over a five to six-year disease course. Histological analyses showed an accumulation of cerebral, cerebellar and cardiac storage material as well as degeneration of neurons, white matter fragmentation and reactive gliosis throughout the brain of affected animals. This novel CLN7-/- macaque model recapitulates key behavioral and neuropathological features of human Batten Disease and provides novel insights into the pathophysiology linked to CLN7 mutations. These animals will be invaluable for evaluating promising therapeutic strategies for this devastating disease.

Gene suppression strategies for dominantly inherited neurodegenerative diseases: lessons from Huntington's disease and spinocerebellar ataxia.

RNA-targeting approaches are emerging as viable therapeutics that offer an alternative method to modulate traditionally 'undrugable' targets. In the case of dominantly inherited neurodegenerative diseases, gene suppression strategies can target the underlying cause of these intractable disorders. Polyglutamine diseases are caused by CAG expansions in discrete genes, making them ideal candidates for gene suppression therapies. Here, we discuss the current state of gene suppression approaches for Huntington's disease and the spinocerebellar ataxias, including the use of antisense oligonucleotides, short-interfering RNAs, as well as viral vector-mediated delivery of short hairpin RNAs and artificial microRNAs. We focus on lessons learned from preclinical studies investigating gene suppression therapies for these disorders, particularly in rodent models of disease and in non-human primates. In animal models, recent advances in gene suppression technologies have not only prevented disease progression in a number of cases, but have also reversed existing disease, providing evidence that reducing the expression of disease-causing genes may be of benefit in symptomatic patients. Both allele- and non-allele-specific approaches to gene suppression have made great strides over the past decade, showing efficacy and safety in both small and large animal models. Advances in delivery techniques allow for broad and durable suppression of target genes, have been validated in non-human primates and in some cases, are currently being evaluated in human patients. Finally, we discuss the challenges of developing and delivering gene suppression constructs into the CNS and recent advances of potential therapeutics into the clinic.

Single nucleotide seed modification restores in vivo tolerability of a toxic artificial miRNA sequence in the mouse brain.

Huntington's disease is a fatal neurodegenerative disease caused by polyglutamine-expansion in huntingtin (HTT). Recent work showed that gene silencing approaches, including RNA interference (RNAi), improve disease readouts in mice. To advance RNAi to the clinic, we designed miHDS1, with robust knockdown of human HTT and minimized silencing of unintended transcripts. In Rhesus macaque, AAV delivery of miHDS1 to the putamen reduced HTT expression with no adverse effects on neurological status including fine and gross motor skills, no immune activation and no induction of neuropathology out to 6 weeks post injection. Others showed safety of a different HTT-targeting RNAi in monkeys for 6 months. Application of miHDS1 to Huntington's patients requires further safety testing in normal rodents, despite the fact that it was optimized for humans. To satisfy this regulatory requirement, we evaluated normal mice after AAV.miHDS1 injection. In contrast to monkeys, neurological deficits occurred acutely in mice brain and was attributed to off-target silencing through interactions of miHDS1 with the 3'UTR of other transcripts. While we resolved miHDS1 toxicity in mouse brain and maintained miHDS1-silencing efficacy, these studies highlight that optimizing nucleic acid-based medicines for safety in humans presents challenges for safety testing in rodents or other distantly related species.

Intrajugular vein delivery of AAV9-RNAi prevents neuropathological changes and weight loss in Huntington's disease mice.

Huntington's disease (HD) is a fatal neurological disorder caused by a CAG repeat expansion in the HTT gene, which encodes a mutant huntingtin protein (mHTT). The mutation confers a toxic gain of function on huntingtin, leading to widespread neurodegeneration and inclusion formation in many brain regions. Although the hallmark symptom of HD is hyperkinesia stemming from striatal degeneration, several other brain regions are affected which cause psychiatric, cognitive, and metabolic symptoms. Additionally, mHTT expression in peripheral tissue is associated with skeletal muscle atrophy, cardiac failure, weight loss, and diabetes. We, and others, have demonstrated a prevention of motor symptoms in HD mice following direct striatal injection of adeno-associated viral vector (AAV) serotype 1 encoding an RNA interference (RNAi) construct targeting mutant HTT mRNA (mHTT). Here, we expand these efforts and demonstrate that an intrajugular vein injection of AAV serotype 9 (AAV9) expressing a mutant HTT-specific RNAi construct significantly reduced mHTT expression in multiple brain regions and peripheral tissues affected in HD. Correspondingly, this approach prevented atrophy and inclusion formation in key brain regions as well as the severe weight loss germane to HD transgenic mice. These results demonstrate that systemic delivery of AAV9-RNAi may provide more widespread clinical benefit for patients suffering from HD.

Mutant huntingtin's interaction with mitochondrial protein Drp1 impairs mitochondrial biogenesis and causes defective axonal transport and synaptic degeneration in Huntington's disease.

The purpose of this study was to investigate the link between mutant huntingtin (Htt) and neuronal damage in relation to mitochondria in Huntington's disease (HD). In an earlier study, we determined the relationship between mutant Htt and mitochondrial dynamics/synaptic viability in HD patients. We found mitochondrial loss, abnormal mitochondrial dynamics and mutant Htt association with mitochondria in HD patients. In the current study, we sought to expand on our previous findings and further elucidate the relationship between mutant Htt and mitochondrial and synaptic deficiencies. We hypothesized that mutant Htt, in association with mitochondria, alters mitochondrial dynamics, leading to mitochondrial fragmentation and defective axonal transport of mitochondria in HD neurons. In this study, using postmortem HD brains and primary neurons from transgenic BACHD mice, we identified mutant Htt interaction with the mitochondrial protein Drp1 and factors that cause abnormal mitochondrial dynamics, including GTPase Drp1 enzymatic activity. Further, using primary neurons from BACHD mice, for the first time, we studied axonal transport of mitochondria and synaptic degeneration. We also investigated the effect of mutant Htt aggregates and oligomers in synaptic and mitochondrial deficiencies in postmortem HD brains and primary neurons from BACHD mice. We found that mutant Htt interacts with Drp1, elevates GTPase Drp1 enzymatic activity, increases abnormal mitochondrial dynamics and results in defective anterograde mitochondrial movement and synaptic deficiencies. These observations support our hypothesis and provide data that can be utilized to develop therapeutic targets that are capable of inhibiting mutant Htt interaction with Drp1, decreasing mitochondrial fragmentation, enhancing axonal transport of mitochondria and protecting synapses from toxic insults caused by mutant Htt.

Systemic delivery of mutant huntingtin lowering antisense oligonucleotides to the brain using apolipoprotein A-I nanodisks for Huntington disease.

Efficient delivery of therapeutics to the central nervous system (CNS) remains a major challenge for the treatment of neurological diseases. Huntington disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion mutation in the HTT gene which codes for a toxic mutant huntingtin (mHTT) protein. Pharmacological reduction of mHTT in the CNS using antisense oligonucleotides (ASO) ameliorates HD-like phenotypes in rodent models of HD, with such therapies being investigated in clinical trials for HD. In this study, we report the optimization of apolipoprotein A-I nanodisks (apoA-I NDs) as vehicles for delivery of a HTT-targeted ASO (HTT ASO) to the brain and peripheral organs for HD. We demonstrate that apoA-I wild type (WT) and the apoA-I K133C mutant incubated with a synthetic lipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, can self-assemble into monodisperse discoidal particles with diameters <20 nm that transmigrate across an in vitro blood-brain barrier model of HD. We demonstrate that apoA-I NDs are well tolerated in vivo, and that apoA-I K133C NDs show enhanced distribution to the CNS and peripheral organs compared to apoA-I WT NDs following systemic administration. ApoA-I K133C conjugated with HTT ASO forms NDs (HTT ASO NDs) that induce significant mHTT lowering in the liver, skeletal muscle and heart as well as in the brain when delivered intravenously in the BACHD mouse model of HD. Furthermore, HTT ASO NDs increase the magnitude of mHTT lowering in the striatum and cortex compared to HTT ASO alone following intracerebroventricular administration. These findings demonstrate the potential utility of apoA-I NDs as biocompatible vehicles for enhancing delivery of mutant HTT lowering ASOs to the CNS and peripheral organs for HD.

Transvascular delivery of small interfering RNA to the central nervous system.

A major impediment in the treatment of neurological diseases is the presence of the blood-brain barrier, which precludes the entry of therapeutic molecules from blood to brain. Here we show that a short peptide derived from rabies virus glycoprotein (RVG) enables the transvascular delivery of small interfering RNA (siRNA) to the brain. This 29-amino-acid peptide specifically binds to the acetylcholine receptor expressed by neuronal cells. To enable siRNA binding, a chimaeric peptide was synthesized by adding nonamer arginine residues at the carboxy terminus of RVG. This RVG-9R peptide was able to bind and transduce siRNA to neuronal cells in vitro, resulting in efficient gene silencing. After intravenous injection into mice, RVG-9R delivered siRNA to the neuronal cells, resulting in specific gene silencing within the brain. Furthermore, intravenous treatment with RVG-9R-bound antiviral siRNA afforded robust protection against fatal viral encephalitis in mice. Repeated administration of RVG-9R-bound siRNA did not induce inflammatory cytokines or anti-peptide antibodies. Thus, RVG-9R provides a safe and noninvasive approach for the delivery of siRNA and potentially other therapeutic molecules across the blood-brain barrier.

Alterations of fractional anisotropy throughout cortico-basal ganglia gray matter in a macaque model of Huntington's Disease.

We recently generated a nonhuman primate (NHP) model of the neurodegenerative disorder Huntington's disease (HD) using adeno-associated viral vectors to express a fragment of mutant HTT protein (mHTT) throughout the cortico-basal ganglia circuit. Previous work by our group established that mHTT-treated NHPs exhibit progressive motor and cognitive phenotypes which are accompanied by mild volumetric reductions of cortical-basal ganglia structures and reduced fractional anisotropy (FA) in the white matter fiber pathways interconnecting these regions, mirroring findings observed in early-stage HD patients. Given the mild structural atrophy observed in cortical and sub-cortical gray matter regions characterized in this model using tensor-based morphometry, the current study sought to query potential microstructural alterations in the same gray matter regions using diffusion tensor imaging (DTI), to define early biomarkers of neurodegenerative processes in this model. Here, we report that mHTT-treated NHPs exhibit significant microstructural changes in several cortical and subcortical brain regions that comprise the cortico-basal ganglia circuit; with increased FA in the putamen and globus pallidus and decreased FA in the caudate nucleus and several cortical regions. DTI measures also correlated with motor and cognitive deficits such that animals with increased basal ganglia FA, and decreased cortical FA, had more severe motor and cognitive impairment. These data highlight the functional implications of microstructural changes in the cortico-basal ganglia circuit in early-stage HD.

In Vivo Cerebral Imaging of Mutant Huntingtin Aggregates Using 11C-CHDI-180R PET in a Nonhuman Primate Model of Huntington Disease.

Huntington disease (HD) is a neurodegenerative disorder caused by an expanded polyglutamine (CAG) trinucleotide expansion in the huntingtin (HTT) gene that encodes the mutant huntingtin protein (mHTT). Visualization and quantification of cerebral mHTT will provide a proxy for target engagement and a means to evaluate therapeutic interventions aimed at lowering mHTT in the brain. Here, we validated the novel radioligand 11C-labeled 6-(5-((5-methoxypyridin-2-yl)methoxy)benzo[d]oxazol-2-yl)-2-methylpyridazin-3(2H)-one (11C-CHDI-180R) using PET imaging to quantify cerebral mHTT aggregates in a macaque model of HD. Methods: Rhesus macaques received MRI-guided intrastriatal delivery of a mixture of AAV2 and AAV2.retro viral vectors expressing an HTT fragment bearing 85 CAG repeats (85Q, n = 5), a control HTT fragment bearing 10 CAG repeats (10Q, n = 4), or vector diluent only (phosphate-buffered saline, n = 5). Thirty months after surgery, 90-min dynamic PET/CT imaging was used to investigate 11C-CHDI-180R brain kinetics, along with serial blood sampling to measure input function and stability of the radioligand. The total volume of distribution was calculated using a 2-tissue-compartment model as well as Logan graphical analysis for regional quantification. Immunostaining for mHTT was performed to corroborate the in vivo findings. Results: 11C-CHDI-180R displayed good metabolic stability (51.4% ± 4.0% parent in plasma at 60 min after injection). Regional time-activity curves displayed rapid uptake and reversible binding, which were described by a 2-tissue-compartment model. Logan graphical analysis was associated with the 2-tissue-compartment model (r 2 = 0.96, P < 0.0001) and used to generate parametric volume of distribution maps. Compared with controls, animals administered the 85Q fragment exhibited significantly increased 11C-CHDI-180R binding in several cortical and subcortical brain regions (group effect, P < 0.0001). No difference in 11C-CHDI-180R binding was observed between buffer and 10Q animals. The presence of mHTT aggregates in the 85Q animals was confirmed histologically. Conclusion: We validated 11C-CHDI-180R as a radioligand to visualize and quantify mHTT aggregated species in a HD macaque model. These findings corroborate our previous work in rodent HD models and show that 11C-CHDI-180R is a promising tool to assess the mHTT aggregate load and the efficacy of therapeutic strategies.

GDNF gene therapy for alcohol use disorder in male non-human primates.

Alcohol use disorder (AUD) exacts enormous personal, social and economic costs globally. Return to alcohol use in treatment-seeking patients with AUD is common, engendered by a cycle of repeated abstinence-relapse episodes even with use of currently available pharmacotherapies. Repeated ethanol use induces dopaminergic signaling neuroadaptations in ventral tegmental area (VTA) neurons of the mesolimbic reward pathway, and sustained dysfunction of reward circuitry is associated with return to drinking behavior. We tested this hypothesis by infusing adeno-associated virus serotype 2 vector encoding human glial-derived neurotrophic factor (AAV2-hGDNF), a growth factor that enhances dopaminergic neuron function, into the VTA of four male rhesus monkeys, with another four receiving vehicle, following induction of chronic alcohol drinking. GDNF expression ablated the return to alcohol drinking behavior over a 12-month period of repeated abstinence-alcohol reintroduction challenges. This behavioral change was accompanied by neurophysiological modulations to dopamine signaling in the nucleus accumbens that countered the hypodopaminergic signaling state associated with chronic alcohol use, indicative of a therapeutic modulation of limbic circuits countering the effects of alcohol. These preclinical findings suggest gene therapy targeting relapse prevention may be a potential therapeutic strategy for AUD.

Preclinical safety of RNAi-mediated HTT suppression in the rhesus macaque as a potential therapy for Huntington's disease.

To date, a therapy for Huntington's disease (HD), a genetic, neurodegenerative disorder, remains elusive. HD is characterized by cell loss in the basal ganglia, with particular damage to the putamen, an area of the brain responsible for initiating and refining motor movements. Consequently, patients exhibit a hyperkinetic movement disorder. RNA interference (RNAi) offers therapeutic potential for this disorder by reducing the expression of HTT, the disease-causing gene. We have previously demonstrated that partial suppression of both wild-type and mutant HTT in the striatum prevents behavioral and neuropathological abnormalities in rodent models of HD. However, given the role of HTT in various cellular processes, it remains unknown whether a partial suppression of both alleles will be safe in mammals whose neurophysiology, basal ganglia anatomy, and behavioral repertoire more closely resembles that of a human. Here, we investigate whether a partial reduction of HTT in the normal non-human primate putamen is safe. We demonstrate that a 45% reduction of rhesus HTT expression in the mid- and caudal putamen does not induce motor deficits, neuronal degeneration, astrogliosis, or an immune response. Together, these data suggest that partial suppression of wild-type HTT expression is well tolerated in the primate putamen and further supports RNAi as a therapy for HD.

Cerebrospinal fluid biomarkers for assessing Huntington disease onset and severity.

The identification of molecular biomarkers in CSF from individuals affected by Huntington disease may help improve predictions of disease onset, better define disease progression and could facilitate the evaluation of potential therapies. The primary objective of our study was to investigate novel CSF protein candidates and replicate previously reported protein biomarker changes in CSF from Huntington disease mutation carriers and healthy controls. Our secondary objective was to compare the discriminatory potential of individual protein analytes and combinations of CSF protein markers for stratifying individuals based on the severity of Huntington disease. We conducted a hypothesis-driven analysis of 26 pre-specified protein analytes in CSF from 16 manifest Huntington disease subjects, eight premanifest Huntington disease mutation carriers and eight healthy control individuals using parallel-reaction monitoring mass spectrometry. In addition to reproducing reported changes in previously investigated CSF biomarkers (NEFL, PDYN, and PENK), we also identified novel exploratory CSF proteins (C1QB, CNR1, GNAL, IDO1, IGF2, and PPP1R1B) whose levels were altered in Huntington disease mutation carriers and/or across stages of disease. Moreover, we report strong associations of select CSF proteins with clinical measures of disease severity in manifest Huntington disease subjects (C1QB, CNR1, NEFL, PDYN, PPP1R1B, and TTR) and with years to predicted disease onset in premanifest Huntington disease mutation carriers (ALB, C4B, CTSD, IGHG1, and TTR). Using receiver operating characteristic curve analysis, we identified PENK as being the most discriminant CSF protein for stratifying Huntington disease mutation carriers from controls. We also identified exploratory multi-marker CSF protein panels that improved discrimination of premanifest Huntington disease mutation carriers from controls (PENK, ALB and NEFL), early/mid-stage Huntington disease from premanifest mutation carriers (PPP1R1B, TTR, CHI3L1, and CTSD), and late-stage from early/mid-stage Huntington disease (CNR1, PPP1R1B, BDNF, APOE, and IGHG1) compared with individual CSF proteins. In this study, we demonstrate that combinations of CSF proteins can outperform individual markers for stratifying individuals based on Huntington disease mutation status and disease severity. Moreover, we define exploratory multi-marker CSF protein panels that, if validated, may be used to improve the accuracy of disease-onset predictions, complement existing clinical and imaging biomarkers for monitoring the severity of Huntington disease, and potentially for assessing therapeutic response in clinical trials. Additional studies with CSF collected from larger cohorts of Huntington disease mutation carriers are needed to replicate these exploratory findings.

A novel rhesus macaque model of Huntington's disease recapitulates key neuropathological changes along with motor and cognitive decline.

We created a new nonhuman primate model of the genetic neurodegenerative disorder Huntington's disease (HD) by injecting a mixture of recombinant adeno-associated viral vectors, serotypes AAV2 and AAV2.retro, each expressing a fragment of human mutant HTT (mHTT) into the caudate and putamen of adult rhesus macaques. This modeling strategy results in expression of mutant huntingtin protein (mHTT) and aggregate formation in the injected brain regions, as well as dozens of other cortical and subcortical brain regions affected in human HD patients. We queried the disruption of cortico-basal ganglia circuitry for 30 months post-surgery using a variety of behavioral and imaging readouts. Compared to controls, mHTT-treated macaques developed working memory decline and progressive motor impairment. Multimodal imaging revealed circuit-wide white and gray matter degenerative processes in several key brain regions affected in HD. Taken together, we have developed a novel macaque model of HD that may be used to develop disease biomarkers and screen promising therapeutics.

Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi.

Huntington's disease (HD) is a fatal, dominant neurodegenerative disease caused by a polyglutamine repeat expansion in exon 1 of the HD gene, which encodes the huntingtin protein. We and others have shown that RNAi is a candidate therapy for HD because expression of inhibitory RNAs targeting mutant human HD transgenes improved neuropathology and behavioral deficits in HD mouse models. Here, we developed shRNAs targeting conserved sequences in human HD and mouse HD homolog (HDh) mRNAs to initiate preclinical testing in a knockin mouse model of HD. We screened 35 shRNAs in vitro and subsequently narrowed our focus to three candidates for in vivo testing. Unexpectedly, two active shRNAs induced significant neurotoxicity in mouse striatum, although HDh mRNA expression was reduced to similar levels by all three. Additionally, a control shRNA containing mismatches also induced toxicity, although it did not reduce HDh mRNA expression. Interestingly, the toxic shRNAs generated higher antisense RNA levels, compared with the nontoxic shRNA. These results demonstrate that the robust levels of antisense RNAs emerging from shRNA expression systems can be problematic in the mouse brain. Importantly, when sequences that were toxic in the context of shRNAs were placed into artificial microRNA (miRNA) expression systems, molecular and neuropathological readouts of neurotoxicity were significantly attenuated without compromising mouse HDh silencing efficacy. Thus, miRNA-based approaches may provide more appropriate biological tools for expressing inhibitory RNAs in the brain, the implications of which are crucial to the development of RNAi for both basic biological and therapeutic applications.

Nonallele-specific silencing of mutant and wild-type huntingtin demonstrates therapeutic efficacy in Huntington's disease mice.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by mutant huntingtin (htt) protein, and there are currently no effective treatments. Recently, we and others demonstrated that silencing mutant htt via RNA interference (RNAi) provides therapeutic benefit in HD mice. We have since found that silencing wild-type htt in adult mouse striatum is tolerated for at least 4 months. However, given the role of htt in various cellular processes, it remains unknown whether nonallele-specific silencing of both wild-type and mutant htt is a viable therapeutic strategy for HD. Here, we tested whether cosilencing wild-type and mutant htt provides therapeutic benefit and is tolerable in HD mice. After treatment, HD mice showed significant reductions in wild-type and mutant htt, and demonstrated improved motor coordination and survival. We performed transcriptional profiling to evaluate the effects of reducing wild-type htt in adult mouse striatum. We identified gene expression changes that are concordant with previously described roles for htt in various cellular processes. Also, several abnormally expressed transcripts associated with early-stage HD were differentially expressed in our studies, but intriguingly, those involved in neuronal function changed in opposing directions. Together, these encouraging and surprising findings support further testing of nonallele-specific RNAi therapeutics for HD.