Polyglutamine-Specific Gold Nanoparticle Complex Alleviates Mutant Huntingtin-Induced Toxicity.

Huntington's disease (HD) belongs to protein misfolding disorders associated with polyglutamine (polyQ)-rich mutant huntingtin (mHtt) protein inclusions. Currently, it is indicated that the aggregation of polyQ-rich mHtt participates in neuronal toxicity and dysfunction. Here, we designed and synthesized a polyglutamine-specific gold nanoparticle (AuNP) complex, which specifically targeted mHtt and alleviated its toxicity. The polyglutamine-specific AuNPs were prepared by decorating the surface of AuNPs with an amphiphilic peptide (JLD1) consisting of both polyglutamine-binding sequences and negatively charged sequences. By applying the polyQ aggregation model system, we demonstrated that AuNPs-JLD1 dissociated the fibrillary aggregates from the polyQ peptide and reduced its ß-sheet content in a concentration-dependent manner. By further integrating polyethyleneimine (PEI) onto AuNPs-JLD1, we generated a complex (AuNPs-JLD1-PEI). We showed that this complex could penetrate cells, bind to cytosolic mHtt proteins, dissociate mHtt inclusions, reduce mHtt oligomers, and ameliorate mHtt-induced toxicity. AuNPs-JLD1-PEI was also able to be transported to the brain and improved the functional deterioration in the HD Drosophila larva model. Our results revealed the feasibility of combining AuNPs, JLD1s, and cell-penetrating polymers against mHtt protein aggregation and oligomerization, which hinted on the early therapeutic strategies against HD.

Inhibition of huntingtin aggregation by its N-terminal 17-residue peptide and its analogs.

The N-terminal 17-residue stretch of huntingtin (httN17) folds into an amphipathic α-helix. The httN17-harboring polyQ peptides form oligomers that are mediated via the assembly of the httN17 α-helices. The oligomerization results in higher local concentration of the polyglutamine (polyQ) region, thereby facilitating amyloid formation. The httN17 co-assembles with the httN17-harbouring polyQ peptides, thereby reducing the local polyQ concentration, and consequently inhibiting aggregation. This study presents the aggregation inhibition of the exon I region of pathogenic huntingtin by httN17 and its analogs. The C-terminal amidation of httN17 is found to be essential for activity. The httN17 peptides with free amino terminus and the acetylated amino terminus possess comparable activity. The httN17 analog, wherein the Leu7 and Ala10 are substituted with 2-aminoisobutyric acid residues, exhibits significantly higher activity than the native httN17.

Specific non-genetic IAP-based protein erasers (SNIPERs) as a potential therapeutic strategy.

As a newly emerged technology, PROTAC (proteolysis targeting chimera) is a promising therapeutic strategy for varieties of diseases. Unlike small molecule inhibitors, PROTACs catalytically induce target proteins degradation, including currently "undruggable" target proteins. In addition, PROTACs can be a potentially successful strategy to overcome drug resistance. IAPs can inhibit apoptosis by inhibiting caspase, and also exhibits the activity of E3 ubiquitin ligase. Specific and nongenetic IAP-based protein erasers (SNIPERs) are hybrid molecules that designed based on IAPs, and used to degrade the target proteins closely associated with diseases. Their structures consist of three parts, including target protein ligand, E3 ligase ligand and the linker between them. SNIPERs (PROTACs) degrade diseases-associated proteins through human inherent ubiquitin-proteasome system. So far, many SNIPERs have been developed to treat diseases that difficult to handle by traditional methods, such as radiotherapy, chemotherapy and small molecule inhibitors, and showed promising prospects in application. In this paper, the recent advances of SNIPERs were summarized, and the chances and challenges associated with this area were also highlighted.

Discovery of the First Druggable GPR52 Antagonist to Treat Huntington's Disease.

GPR52 is an orphan G protein-coupled receptor (GPCR) highly expressed in the brain, especially in the striatum, and represents an emerging therapeutic target for Huntington's disease (HD), an incurable monogenic neurodegenerative disorder caused by the mutation of the huntingtin (mHTT) gene. This Viewpoint discusses the discovery, published in this journal, that a highly potent and specific GPR52 antagonist was identified through high-throughput screening and structure-activity relationship study, which diminishes not only mHTT protein levels, but also ameliorates HD-like phenotypes in the animal disease models. This strategy offers intriguing promise as a surprising approach for HD therapy, where nucleic acid medicine approaches such as small interference RNAs have been the main focus and encounter obstacles such as delivery efficiency.

Therapeutic Update on Huntington's Disease: Symptomatic Treatments and Emerging Disease-Modifying Therapies.

Huntington's disease (HD) is a monogenic neurodegenerative disorder that presents with progressive motor, behavior, and cognitive symptoms leading to early disability and mortality. HD is caused by an expanded CAG repeats in exon 1 of the huntingtin (HTT) gene. The corresponding genetic test allows a clinical, definite diagnosis in life and the identification of a fully penetrant mutation carrier in a premanifest stage. In addition to the development of symptomatic treatments that attempt to address unmet care needs such as apathy, irritability, and cognition, novel therapies that target pathways specific to HD biology are being developed with the intent of slowing disease progression. Among these approaches, HTT protein lowering therapies hold great promise. There are currently active programs using antisense oligonucleotides (ASOs), RNA interference, small-molecule splicing modulators, and zinc-finger protein transcription factor. Except for ASOs and RNA interference approaches, the remaining therapeutic strategies are at a preclinical stage of development. While the current therapeutic landscape in HD may bring an unparalleled change in the lives of people with HD and their families with the first-ever disease-modifying therapy, the evaluation of these therapies requires novel tools that enable a more efficient and expedited discovery and evaluative process. Examples are biomarkers targeting the HTT protein to measure target engagement or disease progression and rating scales more sensitive to the earliest clinical changes. These tools will be instrumental in the next phase of disease-modifying clinical trials in HD likely to target the phenoconversion period of the disease, including the prodromal HD stage.

Huntingtin-Lowering Therapies for Huntington Disease: A Review of the Evidence of Potential Benefits and Risks.

Huntington disease (HD) is caused by a cytosine-adenine-guanine trinucleotide repeat expansion in the huntingtin gene, HTT, that results in expression of variant (mutant) huntingtin protein (HTT). Therapeutic strategies that reduce HTT levels are currently being pursued to slow or stop disease progression in people with HD. These approaches are supported by robust preclinical data indicating that reducing variant huntingtin protein is associated with decreased HD pathology. However, the risk-benefit profile of reducing either variant HTT or both variant and wild-type HTT is currently an open question that is being addressed in ongoing clinical trials. This review aims to examine the current data available regarding altered HTT in humans, normal animals, and animal models of HD. Studies indexed in PubMed were searched using the MeSH term Huntington disease or the text words huntington or huntingtin from August 31, 1999, to August 31, 2019, with no language restrictions. Additional studies were included from the reference lists of relevant studies and the authors' personal files. Articles describing at least 1 aspect of HTT reduction were included, prioritizing those published within the last 10 years. In vivo studies were also prioritized, with a focus on studies that examined the consequences of wild-type HTT reduction in adults. In a recently completed phase 1/2a study of RG6042 in 46 adults with early manifest HD, antisense oligonucleotide-mediated partial reduction of HTT was reported to be generally safe and well tolerated over the course of 4-monthly RG6042 doses. In case studies of people with rare genetic variations in huntingtin alleles, the loss of 1 wild-type allele was not associated with HD. People with homozygous cytosine-adenine-guanine expansions developed normally until the onset of HD, although they may have experienced a more aggressive disease course. In mouse models of HD, partial reduction of HTT was beneficial, with improvements in motor, cognitive, and behavioral phenotypes. The partial reduction of wild-type HTT in normal adult rodents and nonhuman primates was generally safe and well tolerated. The body of evidence reviewed in this article indicates a positive risk-benefit profile for the partial reduction of either variant HTT alone or both variant and wild-type HTT. These strategies target the underlying cause of HD and are currently being tested in several investigational clinical trials.

Potent and sustained huntingtin lowering via AAV5 encoding miRNA preserves striatal volume and cognitive function in a humanized mouse model of Huntington disease.

Huntington disease (HD) is a fatal neurodegenerative disease caused by a pathogenic expansion of a CAG repeat in the huntingtin (HTT) gene. There are no disease-modifying therapies for HD. Artificial microRNAs targeting HTT transcripts for degradation have shown preclinical promise and will soon enter human clinical trials. Here, we examine the tolerability and efficacy of non-selective HTT lowering with an AAV5 encoded miRNA targeting human HTT (AAV5-miHTT) in the humanized Hu128/21 mouse model of HD. We show that intrastriatal administration of AAV5-miHTT results in potent and sustained HTT suppression for at least 7 months post-injection. Importantly, non-selective suppression of huntingtin was generally tolerated, however high dose AAV5-miHTT did induce astrogliosis. We observed an improvement of select behavioural and modest neuropathological HD-like phenotypes in Hu128/21 mice, suggesting a potential therapeutic benefit of miRNA-mediated non-selective HTT lowering. Finally, we also observed that potent reduction of wild type HTT (wtHTT) in Hu21 control mice was tolerated up to 7 months post-injection but may induce impairment of motor coordination and striatal atrophy. Taken together, our data suggests that in the context of HD, the therapeutic benefits of mHTT reduction may outweigh the potentially detrimental effects of wtHTT loss following non-selective HTT lowering.

A Comprehensive Haplotype-Targeting Strategy for Allele-Specific HTT Suppression in Huntington Disease.

Huntington disease (HD) is a fatal neurodegenerative disorder caused by a gain-of-function mutation in HTT. Suppression of mutant HTT has emerged as a leading therapeutic strategy for HD, with allele-selective approaches targeting HTT SNPs now in clinical trials. Haplotypes associated with the HD mutation (A1, A2, A3a) represent panels of allele-specific gene silencing targets for efficient treatment of individuals with HD of Northern European and indigenous South American ancestry. Here we extend comprehensive haplotype analysis of the HD mutation to key populations of Southern European, South Asian, Middle Eastern, and admixed African ancestry. In each of these populations, the HD mutation occurs predominantly on the A2 HTT haplotype. Analysis of HD haplotypes across all affected population groups enables rational selection of candidate target SNPs for development of allele-selective gene silencing therapeutics worldwide. Targeting SNPs on the A1 and A2 haplotypes in parallel is essential to achieve treatment of the most HD-affected subjects in populations where HD is most prevalent. Current allele-specific approaches will leave a majority of individuals with HD untreated in populations where the HD mutation occurs most frequently on the A2 haplotype. We further demonstrate preclinical development of potent and selective ASOs targeting SNPs on the A2 HTT haplotype, representing an allele-specific treatment strategy for these individuals. On the basis of comprehensive haplotype analysis, we show the maximum proportion of HD-affected subjects that may be treated with three or four allele targets in different populations worldwide, informing current allele-specific HTT silencing strategies.

Allele-selective lowering of mutant HTT protein by HTT-LC3 linker compounds.

Accumulation of mutant proteins is a major cause of many diseases (collectively called proteopathies), and lowering the level of these proteins can be useful for treatment of these diseases. We hypothesized that compounds that interact with both the autophagosome protein microtubule-associated protein 1A/1B light chain 3 (LC3)1 and the disease-causing protein may target the latter for autophagic clearance. Mutant huntingtin protein (mHTT) contains an expanded polyglutamine (polyQ) tract and causes Huntington's disease, an incurable neurodegenerative disorder2. Here, using small-molecule-microarray-based screening, we identified four compounds that interact with both LC3 and mHTT, but not with the wild-type HTT protein. Some of these compounds targeted mHTT to autophagosomes, reduced mHTT levels in an allele-selective manner, and rescued disease-relevant phenotypes in cells and in vivo in fly and mouse models of Huntington's disease. We further show that these compounds interact with the expanded polyQ stretch and could lower the level of mutant ataxin-3 (ATXN3), another disease-causing protein with an expanded polyQ tract3. This study presents candidate compounds for lowering mHTT and potentially other disease-causing proteins with polyQ expansions, demonstrating the concept of lowering levels of disease-causing proteins using autophagosome-tethering compounds.

Discovery of Arginine Ethyl Ester as Polyglutamine Aggregation Inhibitor: Conformational Transitioning of Huntingtin N-Terminus Augments Aggregation Suppression.

Huntington's disease (HD) is a genetic disorder caused by a CAG expansion mutation in the huntingtin gene leading to polyglutamine (polyQ) expansion in the N-terminal part of huntingtin (Httex1). Expanded polyQ, through a complex aggregation pathway, forms aggregates in neurons and presents a potential therapeutic target. Here we show Httex1 aggregation suppression by arginine and arginine ethyl ester (AEE) in vitro, as well as in yeast and mammalian cell models of HD, bearing expanded polyQ. These molecules also rescue locomotion dysfunction in HD Drosophila model. Both molecules alter the hydrogen bonding network of polyQ to enhance its aqueous solubility and delay aggregation. AEE shows direct binding with the NT17 part of Httex1 to induce structural changes to impart an enhanced inhibitory effect. This study provides a platform for the development of better arginine based therapeutic molecules against polyQ-rich Httex1 aggregation.

Antibody-based therapies for Huntington's disease: current status and future directions.

The types of treatments and interventions being developed for chronic neurodegenerative disorders have expanded considerably in recent years. In addition to the variety of targets being pursued, strategies have moved from symptom management to more directed disease-modifying approaches. Among them are antibody-based therapies, which are not only being evaluated for a range of tauopathies and synucleinopathies, but are also emerging as a potential application for monogenic disorders of the central nervous system (CNS), including Huntington's disease (HD). Despite the excitement around the early trial data of anti-sense oligonucleotides (ASO) treatment for such disorders, antibody therapies may hold the key to tackling another aspect of the disease that could be critical to its pathogenesis. While gene-based methodologies are designed to lower, predominantly within cellular elements, mutant huntingtin protein (mHtt) - the genetic product of HD - the pathological protein is abundant in free forms and in several compartments including the cerebrospinal fluid, the plasma and the extracellular matrix. With accumulating evidence for the spreading and seeding capacities of mHtt, it may indeed be essential to target the protein both intracellularly and extracellularly. Therefore, free forms of mHtt not only represents an ideal target for antibodies, but one that needs to be addressed if meaningful and maximal clinical benefits are to be expected. This review explores the potential use of antibody-based therapies to treat HD, including the rationale for this approach as well as the pre-clinical data supporting it. The potential challenges that will need to be considered if such route is to be pursued clinically are also discussed.

Lowering Mutant Huntingtin Using Tricyclo-DNA Antisense Oligonucleotides As a Therapeutic Approach for Huntington's Disease.

Huntington's disease is a neurodegenerative disorder caused by a CAG repeat expansion in the first exon of huntingtin gene (HTT) encoding for a toxic polyglutamine protein. This disease is characterized by motor, psychiatric, and cognitive impairments. Currently, there is no disease modifying treatment. However, reducing the expression of the huntingtin protein (HTT) using antisense oligonucleotides (ASOs) has been shown as a promising therapeutic strategy. In this study, we explore the therapeutic potential of ASO made of tricyclo-DNA (tcDNA), a conformationally constrained DNA analog, to silence HTT. We used a gapmer ASO, containing central DNA nucleotides flanked by tcDNA modifications on 5' and 3' ends, allowing the recruitment of RNAse H and subsequent degradation of the messenger RNA. After transfection of tcDNA-ASO in patient-derived fibroblast cell lines, we show a strong decrease of HTT mRNA and protein levels. As a control, 2'O-methyl-RNA targeting the same region of HTT was also tested and did not induce a significant effect. tcDNA-ASO were also evaluated in vivo in the YAC128 mice, containing the full-length human HTT gene with 128 CAG repeat expansion. Single intracerebroventricular (ICV) injections of tcDNA induce a significant decrease of HTT messenger and protein levels in the cortex, hippocampus, striatum, and cerebellum of treated mice. tcDNA-ASO were found well distributed in the central nervous system (CNS) and show long lasting effect with protein levels still low, 12 weeks after a single ICV injection. This proof of concept study suggests the therapeutic potential of gapmer tcDNA ASO to downregulate huntingtin in vitro and in vivo.

Biocompatible Inhibitor Based on Chitosan and Amphiphilic Peptide against Mutant Huntingtin Toxicity.

Huntington's disease (HD) is classified as a protein-misfolding disease correlated with the mutant Huntingtin (mHtt) protein with abnormally expanded polyglutamine (polyQ) domains. Because no effective drugs have yet been reported, attempts to develop better therapy to delay the age of onset are in urgent demand. In this study, an amphiphilic peptide consisting of negatively charged hexaglutamic acid and a stretch of decaglutamine (E6 Q10 ) was chemically synthesized as an inhibitor against polyQ and mHtt toxicity. It is found that E6 Q10 selfassembles into spherical vesicles, as shown by means of TEM, cryoelectron microscopy, and dynamic light scattering. Assembled E6 Q10 prevented the polyQ-rich peptide (KKWQ20 AKK) from forming amyloid fibrils. To enable the cell-penetration ability of E6 Q10 , the E6 Q10 ⋅chitosan complex was generated. It is demonstrated that the complex penetrates cells, interferes with the mHtt oligomerization and aggregation process, and prevents mHtt cytotoxicity. By combining positively charged chitosan and amphiphilic peptides with a negatively charge moiety, a new strategy is provided to develop biocompatible and biodegradable inhibitors against mHtt toxicity.

Astrocyte transduction is required for rescue of behavioral phenotypes in the YAC128 mouse model with AAV-RNAi mediated HTT lowering therapeutics.

Huntington's disease (HD) is a fatal autosomal dominant neurodegenerative disease caused by a CAG expansion, which translates into an elongated polyglutamine (polyQ) repeat near the amino-terminus of the huntingtin protein (HTT). This results in production of a toxic mutant huntingtin protein (mHTT) that leads to neuronal dysfunction and death. Currently, no disease-modifying treatments are available; however, numerous therapeutic strategies aimed at lowering HTT levels in the brain are under development. To date, studies have not closely examined the contribution of mHTT in neurons vs astrocytes to disease pathophysiology. To better understand the role of astrocytes in HD pathophysiology and the need for cell type specific targeting of HTT lowering therapeutic strategies, AAV capsids were employed that selectively transduce neurons, or both neurons and astrocytes. These vectors carrying miRNA sequences directed against HTT were injected into the YAC128 mouse model of HD to selectively lower HTT expression in neurons alone versus neurons and astrocytes. The results suggested that HTT lowering in neurons alone was not sufficient to rescue the motor phenotype in YAC128 mice. Furthermore, HTT lowering in both cell types was required to achieve maximal functional benefit. The study suggested that astrocyte dysfunction may play a critical role in HD pathogenesis, and thus astrocytes represent an important therapeutic target.

Targeting Huntingtin Expression in Patients with Huntington's Disease.

BACKGROUND: Huntington's disease is an autosomal-dominant neurodegenerative disease caused by CAG trinucleotide repeat expansion in HTT, resulting in a mutant huntingtin protein. IONIS-HTTRx (hereafter, HTTRx) is an antisense oligonucleotide designed to inhibit HTT messenger RNA and thereby reduce concentrations of mutant huntingtin. METHODS: We conducted a randomized, double-blind, multiple-ascending-dose, phase 1-2a trial involving adults with early Huntington's disease. Patients were randomly assigned in a 3:1 ratio to receive HTTRx or placebo as a bolus intrathecal administration every 4 weeks for four doses. Dose selection was guided by a preclinical model in mice and nonhuman primates that related dose level to reduction in the concentration of huntingtin. The primary end point was safety. The secondary end point was HTTRx pharmacokinetics in cerebrospinal fluid (CSF). Prespecified exploratory end points included the concentration of mutant huntingtin in CSF. RESULTS: Of the 46 patients who were enrolled in the trial, 34 were randomly assigned to receive HTTRx (at ascending dose levels of 10 to 120 mg) and 12 were randomly assigned to receive placebo. Each patient received all four doses and completed the trial. Adverse events, all of grade 1 or 2, were reported in 98% of the patients. No serious adverse events were seen in HTTRx-treated patients. There were no clinically relevant adverse changes in laboratory variables. Predose (trough) concentrations of HTTRx in CSF showed dose dependence up to doses of 60 mg. HTTRx treatment resulted in a dose-dependent reduction in the concentration of mutant huntingtin in CSF (mean percentage change from baseline, 10% in the placebo group and -20%, -25%, -28%, -42%, and -38% in the HTTRx 10-mg, 30-mg, 60-mg, 90-mg, and 120-mg dose groups, respectively). CONCLUSIONS: Intrathecal administration of HTTRx to patients with early Huntington's disease was not accompanied by serious adverse events. We observed dose-dependent reductions in concentrations of mutant huntingtin. (Funded by Ionis Pharmaceuticals and F. Hoffmann-La Roche; ClinicalTrials.gov number, NCT02519036.).

Nucleic Acid Therapeutics in Huntington's Disease.

BACKGROUND: Protein misfolding is a critical factor in the progression of a large number of neurodegenerative diseases. The incorrectly folded protein is prone to aggregation, leading to aberrant interaction with other cellular proteins, elevated oxidative stress, impaired cellular machinery, finally resulting in cell death. Due to its monogenic origin, Huntington's disease (HD) is a poster child of protein misfolding neurodegenerative disorders. The presence of neuronal inclusions of mutant huntingtin N-terminal fragments, mainly in the cortex and striatum, is a neuropathological hallmark of HD. Inhibition of protein misfolding and aggregation has been attempted using a variety of conventional protein stabilizers. METHODS: This review describes how, in recent times, nucleic acid therapeutics has emerged as a selective tool to downregulate the aberrant transcript and reduce expression of mutant huntingtin, thereby alleviating protein aggregation. Different strategies of use of nucleic acids, including antisense oligonucleotides, short inhibitory RNA sequences and aptamers have been discussed. The following patent databases were consulted: European Patent Office (EPO), the United States Patent and Trademark Office (USPTO), Patent scope Search International and National Patent Collections (WIPO) and Google Patents. RESULTS: Tools such as RNA interference (RNAi) and antisense oligonucleotides (ASOs) are potential therapeutic agents which target the post-transcriptional step, accelerating mRNA degradation and inhibiting the production of the mutant protein. These nucleic acid sequences not only target the elongated CAG triplet repeat translating to an expanded polyglutamine tract in the mutant protein, but have also been used to target single nucleotide polymorphisms associated with the mutant allele. The therapeutic sequences have been investigated in a number of cells and animal models of HD. One antisense sequence, with desirable safety properties, has recently shown downregulation of huntingtin protein in a limited clinical trial. RNA aptamers have also shown promising results in inhibiting protein aggregation in a yeast model of HD. Novel drug delivery techniques have been employed to overcome the blood brain barrier for the use of these therapeutic sequences. CONCLUSION: The selectivity and specificity imparted by nucleic acids, along with novel delivery techniques, make them hopeful candidates for the development of a curative strategy for HD.

Huntingtin associates with the actin cytoskeleton and α-actinin isoforms to influence stimulus dependent morphology changes.

One response of cells to growth factor stimulus involves changes in morphology driven by the actin cytoskeleton and actin associated proteins which regulate functions such as cell adhesion, motility and in neurons, synaptic plasticity. Previous studies suggest that Huntingtin may be involved in regulating morphology however, there has been limited evidence linking endogenous Huntingtin localization or function with cytoplasmic actin in cells. We found that depletion of Huntingtin in human fibroblasts reduced adhesion and altered morphology and these phenotypes were made worse with growth factor stimulation, whereas the presence of the Huntington's Disease mutation inhibited growth factor induced changes in morphology and increased numbers of vinculin-positive focal adhesions. Huntingtin immunoreactivity localized to actin stress fibers, vinculin-positive adhesion contacts and membrane ruffles in fibroblasts. Interactome data from others has shown that Huntingtin can associate with α-actinin isoforms which bind actin filaments. Mapping studies using a cDNA encoding α-actinin-2 showed that it interacts within Huntingtin aa 399-969. Double-label immunofluorescence showed Huntingtin and α-actinin-1 co-localized to stress fibers, membrane ruffles and lamellar protrusions in fibroblasts. Proximity ligation assays confirmed a close molecular interaction between Huntingtin and α-actinin-1 in human fibroblasts and neurons. Huntingtin silencing with siRNA in fibroblasts blocked the recruitment of α-actinin-1 to membrane foci. These studies support the idea that Huntingtin is involved in regulating adhesion and actin dependent functions including those involving α-actinin.

Introducing an expanded CAG tract into the huntingtin gene causes a wide spectrum of ultrastructural defects in cultured human cells.

Modeling of neurodegenerative diseases in vitro holds great promise for biomedical research. Human cell lines harboring a mutations in disease-causing genes are thought to recapitulate early stages of the development an inherited disease. Modern genome-editing tools allow researchers to create isogenic cell clones with an identical genetic background providing an adequate "healthy" control for biomedical and pharmacological experiments. Here, we generated isogenic mutant cell clones with 150 CAG repeats in the first exon of the huntingtin (HTT) gene using the CRISPR/Cas9 system and performed ultrastructural and morphometric analyses of the internal organization of the mutant cells. Electron microscopy showed that deletion of three CAG triplets or an HTT gene knockout had no significant influence on the cell structure. The insertion of 150 CAG repeats led to substantial changes in quantitative and morphological parameters of mitochondria and increased the association of mitochondria with the smooth and rough endoplasmic reticulum while causing accumulation of small autolysosomes in the cytoplasm. Our data indicate for the first time that expansion of the CAG repeat tract in HTT introduced via the CRISPR/Cas9 technology into a human cell line initiates numerous ultrastructural defects that are typical for Huntington's disease.

Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier.

Effective transvascular delivery of therapeutic oligonucleotides to the brain presents a major hurdle to the development of gene silencing technologies for treatment of genetically defined neurological disorders. Distribution to the brain after systemic administrations is hampered by the low permeability of the blood-brain barrier (BBB) and the rapid clearance kinetics of these drugs from the blood. Here we show that transient osmotic disruption of the BBB enables transvascular delivery of hydrophobically modified small interfering RNA (hsiRNA) to the rat brain. Intracarotid administration of 25% mannitol and hsiRNA conjugated to phosphocholine-docosahexanoic acid (PC-DHA) resulted in broad ipsilateral distribution of PC-DHA-hsiRNAs in the brain. PC-DHA conjugation enables hsiRNA retention in the parenchyma proximal to the brain vasculature and enabled active internalization by neurons and astrocytes. Moreover, transvascular delivery of PC-DHA-hsiRNAs effected Htt mRNA silencing in the striatum (55%), hippocampus (51%), somatosensory cortex (52%), motor cortex (37%), and thalamus (33%) 1 week after administration. Aside from mild gliosis induced by osmotic disruption of the BBB, transvascular delivery of PC-DHA-hsiRNAs was not associated with neurotoxicity. Together, these findings provide proof-of-concept that temporary disruption of the BBB is an effective strategy for the delivery of therapeutic oligonucleotides to the brain.