small molecules

2026-06-04 | Neuronal VPS13A depletion links diacylglycerol PKC signaling and synaptic spines.

Intermembrane lipid transfer protein VPS13A (VPS13A) is a large protein whose cellular functions are still under investigation. VPS13A gene mutations leading to the absence of protein expression cause Chorea-acanthocytosis (ChAc), an ultra-rare inherited neurodegenerative movement disorder. Although the molecular mechanisms linking VPS13A loss to neuronal dysfunction remain unclear, its role as a bulk lipid transfer protein suggests that impaired lipid distribution may represent a primary pathogenic mechanism leading to neurodegeneration in ChAc. In this study, we investigated the effect of neuronal silencing of VPS13A in a murine model to phenocopy the human disease. Lipidomics analysis revealed an increase in the concentration of several diacylglycerol species induced by VPS13A knockdown. We then explored the downstream molecular pathways related to the altered diacylglycerol levels and found that VPS13A knockdown induces a decrease in protein kinase C (PKC)βII concentration but an increase in PKCα/βII phosphorylation in cultured neurons. Finally, pharmacological inhibition of PKCβII reverted aberrant neuronal morphology and loss of spine density induced by VPS13A KD. These results underscore the importance of VPS13A in regulating neuronal lipid distribution, showing that its absence perturbs the diacylglycerol/PKC signaling pathway, with measurable effects on neuronal structure and synaptic density. Overall, our results underscore a previously underappreciated role for VPS13A in the structural organization of neurons through lipid-mediated signaling mechanisms, providing insight into the cellular dysfunction underlying ChAc.

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2026-04-01 | Chorea-acanthocytosis masquerading as a progressive seizure disorder with apparent early immunotherapy responsiveness.

Chorea-acanthocytosis (ChAc) is a rare genetic disorder characterised by a hyperkinetic movement disorder, dystonia, cognitive and neuropsychiatric deficits and seizures. We report the case of a 30-year-old patient who presented with a decade of episodic neurological dysfunction and seizures. The condition was initially suspected to be an immunotherapy-responsive seronegative autoimmune encephalitis but progressed to oromandibular dystonia, raising suspicion of a neurodegenerative condition. Neuroimaging showed bilateral caudate atrophy and acanthocytes were seen on blood film microscopy in association with raised creatine kinase. Genetic testing revealed our patient to be compound heterozygous for two pathogenic variants in the VPS13A gene and confirmed the diagnosis of ChAc. This case highlights the importance of considering ChAc in the differential diagnosis of a progressive treatment-refractory seizure disorder in the context of oromandibular dystonia.

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2025-04-25 | Premature skeletal muscle aging in VPS13A deficiency relates to impaired autophagy.

VPS13A disease (chorea-acanthocytosis), is an ultra-rare autosomal recessive neurodegenerative disorder caused by mutations of the VPS13A gene encoding Vps13A. Increased serum levels of the muscle isoform of creatine kinase associated with often asymptomatic muscle pathology are among the poorly understood early clinical manifestations of VPS13A disease. Here, we carried out an integrated analysis of skeletal muscle from Vps13a-/- mice and from VPS13A disease patient muscle biopsies. The absence of Vps13A impaired autophagy, resulting in pathologic metabolic remodeling characterized by cellular energy depletion, increased protein/lipid oxidation and a hyperactivated unfolded protein response. This was associated with defects in myofibril stability and the myofibrillar regulatory proteome, with accumulation of the myocyte senescence marker, NCAM1. In Vps13a-/- mice, the impairment of autophagy was further supported by the lacking effect of starvation alone or in combination with colchicine on autophagy markers. As a proof of concept, we showed that rapamycin treatment rescued the accumulation of terminal phase autophagy markers LAMP1 and p62 as well as NCAM1, supporting a connection between impaired autophagy and accelerated aging in the absence of VPS13A. The premature senescence was also corroborated by local activation of pro-inflammatory NF-kB-related pathways in both Vps13a-/- mice and patients with VPS13A disease. Our data link for the first time impaired autophagy and inflammaging with muscle dysfunction in the absence of VPS13A. The biological relevance of our mouse findings, supported by human muscle biopsy data, shed new light on the role of VPS13A in muscle homeostasis.

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2025-03-09 | VPS13A disease: Bridging motor dysfunction and psychiatric symptoms – A case report

Neuroacanthocytosis encompasses a group of disorders with basal ganglia pathology leading to characteristic movement disorders and a high prevalence of psychiatric manifestations. VPS13A disease (VPS13A-D), formerly known as Chorea-acanthocytosis (ChAc), is a rare autosomal recessive disorder caused by pathogenic variants in the vacuolar protein sorting 13 homolog A (VPS13A) gene that leads to chorein loss and affects the basal ganglia, especially the caudate nucleus. The available literature reveals a gap in the exploration of the coexistence of neuropsychiatric symptoms, with only a few clinical reports described in psychiatric literature. To improve the understanding of this particular genetic disease, this article reports a clinical case of VPS13A-D and discusses its comorbid neuropsychiatric manifestations by analyzing the presentation, laboratory and imaging findings, and by briefly addressing the distinct neurobiology and neuropathology of this disorder concerning psychiatric manifestations, according to the relevant literature. We present a 37-year-old male diagnosed with VPS13A-D with an involuntary movement presentation and neuropsychiatric symptoms, including orobuccal self-mutilation, anxiety and obsessive-compulsive traits. The patient received treatment with tetrabenazine (for better control of chorea) and fluvoxamine (to address psychiatric symptoms), showing symptomatic improvement during outpatient follow-up. The discussion delves into the neurobiological framework of VPS13A-D, emphasizing the role of the basal ganglia in both motor and neuropsychiatric manifestations. Psychopathological aspects such as dysexecutive syndrome and obsessive‒compulsive symptoms are explored, highlighting the impact of frontal-subcortical circuits in these presentations. This case underscores the complexity of neuropsychiatric symptoms associated with VPS13A-D and highlights the challenges in distinguishing between movement anomalies and psychopathology, accentuating the need for a comprehensive approach through multidisciplinary collaboration for improved patient care and outcomes.

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2024-11-13 | Chorein deficiency promotes ferroptosis.

Ferroptosis is a type of programmed cell death owed to an intracellular accumulation of iron resulting in the generation reactive oxygen species, which in turn can cause peroxidation of plasma membrane lipids and ultimately result in cell death. We investigated the potential involvement of VPS13A deficiency in ferroptosis. The VPS13A gene encodes for chorein, and its deficiency is a molecular cause of chorea-acanthocytosis (ChAc), a Huntington-like disease with neurodegeneration in the striatum. In our previous study, we found male infertility characterized by increased malondialdehyde staining of the spermatozoa in the testes of the ChAc model mice. Thus, in this study we performed metabolome analysis of sperm extracted from the epididymis of the ChAc model mice, which revealed decreased cystine levels, suggesting an association between chorein deficiency and ferroptosis. We then investigated the role of chorein in ferroptosis using VPS13A knockdown (VPS13A-KD) HEK293 cells. We found that VPS13A-KD cells displayed a significantly diminished resistance to tert-Butyl hydroperoxide (tBHP)-induced lipid peroxidation and cell death compared to control cells, which could be rescued by treatment with ferrostatin-1. Moreover, VPS13A-KD cells showed Fe(II) accumulation, suggesting an impaired capacity for divalent iron removal. In the cytosolic fraction of VPS13A-KD cells, the protein level of glutathione peroxidase 4 (GPX4) was significantly reduced, suggesting that dysfunction of chorein impairs GPX4 transport, thereby facilitating ferroptosis. These results suggest that ferroptosis may contribute to neurodegeneration in ChAc caused by loss of chorein function.

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cell therapies

2026-01-10 | Subthalamic Deep Brain Stimulation for Chorea-Acanthocytosis: A Single-Center Case Series.

Chorea-acanthocytosis (ChAc) is a rare autosomal recessive neurodegenerative disorder characterized by progressive movement disorders. Deep brain stimulation (DBS) targeting the globus pallidus internus has shown efficacy in managing ChAc. However, evidence regarding subthalamic nucleus (STN) DBS remains limited, with only two cases previously reported from our center. We analyzed seven consecutive patients with ChAc who underwent STN DBS at Ruijin Hospital (2010-2024). Motor symptoms were assessed using the Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS) and the Unified Huntington's Disease Rating Scale (UHDRS). Quality of life was measured with the 36-Item Short Form Survey (SF-36). Examinations occurred at baseline, early follow-up (EFU,

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2024-08-09 | A case of chorea-acanthocytosis with significant improvement of symptoms at one year with deep brain stimulation: case report and literature review.

Chorea-acanthocytosis (ChAc) is a rare, neurodegenerative disorder caused by mutations in the VPS13A gene. In this article, we report on a 32-year-old man diagnosed with ChAc, with involuntary movements of the mouth and trunk, drooling of the mouth, slurred speech, and abnormal vocalizations as the main clinical manifestations. Three weeks after implantation of globus pallidus internal (GPi)-deep brain stimulation (DBS), the patient's symptoms improved significantly. For example, articulation is clear, involuntary trunk movements and salivation have largely disappeared, and abnormal vocalizations have been significantly reduced. After 1 year of follow-up, the improvement in involuntary movement symptoms is essentially the same as before. As far as we know, we are the first to report the relief of involuntary vocalizations in a patient with GPi-DBS treatment, and that salivation and involuntary trunk movements have almost disappeared, and all other symptoms are significantly relieved, which is rare in previous cases. All of the above proves that the treatment of our case with DBS was very successful and that longer term follow-up is critical. We also hope that our case will provide new references and therapeutic ideas for the future treatment of patients with ChAc.

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2023-01-17 | Tongue‐biting ataxia that appeared to be a psychiatric disorder: a case of neuroacanthocytosis

A 32-year-old hearing-impaired woman with epilepsy presented with loss of consciousness, seizures, and tongue biting. She was hemodynamically stable and had communication difficulties because of her hearing loss. She required endotracheal intubation to prevent airway obstruction by blood. Head computed tomography showed no atrophy of the bilateral caudate nucleus and lateral ventricular enlargement. On day 1 after hospitalization, she was extubated and involuntary perioral movements were observed. She had a seizure on day 8. Endotracheal intubation and mechanical ventilation were performed again to prevent involuntary perioral movements and tongue biting (Fig. 1A). She wrote that she did not need her tongue, which suggested a psychiatric disorder. She had visited many hospitals over several years and was prescribed anticonvulsants; the hospitals failed to diagnose a physical illness. However, the presence of tongue and lip deformities because of self-harm, involuntary perioral movements, truncal ataxia, a history of epilepsy, and the similar symptoms exhibited by only her brother, among relatives, implied acanthocytosis.1 Acanthocytes were identified on the peripheral blood smear (Fig. 1B). We identified and excluded symptomatic chorea, drug-induced chorea, metabolic disease, and other neurodegenerative diseases based on age, family history, medications used, blood tests, and imaging findings. She was referred to a university hospital for a definitive diagnosis of neuroacanthocytosis and deep brain stimulation surgery for her involuntary movements.2 After surgery, her involuntary movements improved, the gait stabilized, and the tongue biting disappeared. Although psychiatric disorders may present with self-harm and suspected epilepsy, involuntary movements should prompt consideration of a hereditary disorder. Approval of the Research Protocol: N/A. Registry and the Registration No. of the Study/Trial: N/A. Informed Consent: Written informed consent was obtained from the patient. Animal Studies: N/A. Conflict of Interest: None declared.

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2022-06-03 | Deep brain stimulation for chorea-acanthocytosis: a systematic review.

Deep brain stimulation (DBS) is a reversible treatment for chorea-acanthocytosis (ChAc). Its safety and efficacy remain elusive due to the low prevalence of ChAc. We aimed to investigate the safety and efficacy of DBS for ChAc by systematically reviewing literature through PubMed and EMBASE. Inclusion criteria were reports on the efficacy or safety of DBS for ChAc and English language articles, and exclusion criteria were other movement disorders, non-human subjects, and studies without original data. Most studies were published as case reports, and we therefore pooled these cases in one cohort. Twenty studies with 34 patients were included. The mean age of symptom onset was 29.3 years (range, 17-48). The median follow-up was 12 months (range, 2-84). Twenty-nine patients underwent GPi-DBS, two received STN-DBS, and one underwent Vop-DBS. Electrodes were implanted into the ventralis oralis complex of the thalamus and the pallidal in two patients. Symptoms seemed to be easier relieved in chorea (88.5%) and dystonia (76.9%) but dysarthria of most patients (85.7%) was no response after DBS. The Unified Huntington's Disease Rating Scale-Motor Score was used to assess the efficacy of DBS in 25 patients; the mean score decreased from 43.2 to 22.3 and the median improvement rate was 46.7%. Of 24 patients with data on adverse events, complications occurred in 9 patients (37.5%; mostly transient and mild events). DBS is a promising treatment for ChAc with satisfactory efficacy and safety based on the review. Pallidal and thalamic DBS have been applied in ChAc; GPi-DBS seems to be more widely used.

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2019-08-19 | Progress in the Diagnosis and Management of Chorea-acanthocytosis.

Chorea-acanthocytosis (ChAc) is the most common subtype of neuroacanthocytosis syndrome, characterized by the presence of acanthocytes and neurological disorders. It is thought to be caused by VPS13A mutations. Characteristic movement disorders in ChAc is choreiform movements affecting both trunk and extremities and prominent orolingual dyskinesia is pathognomonic. Acanthocytosis in peripheral blood smear, elevated serum creatine kinase and atrophy of heads of caudate nuclei and dilation of the anterior horn of the lateral ventricles in magnetic resonance imaging could assist the diagnosis of ChAc. Botulinum toxin injection is a possible treatment for the typical orofacial dystonia. Deep brain stimulation is a novel surgical treatment modality. Most cases chose globus pallidus internus as target. Patients with dystonia as a major manifestation will benefit more from high-frequency stimulation and those with major findings of chorea and dysarthria are suitable for low-frequency stimulation. More evidence of long-term outcomes is warranted.

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proteins

2024-09-09 | Treatment of a lip defect in a patient with chorea-acanthocytosis using a combination of surgical and adjuvant onabotulinumtoxinA therapy: a case report.

Chorea-acanthocytosis (ChAc) is an extremely rare neurodegenerative disorder characterized by movement disorders and acanthocytosis. Orofacial dyskinesia is a distinct symptom of this disorder that can lead to lip injuries and feeding difficulties. This paper reports the first case of a patient with ChAc presenting with a lip defect, who was managed with surgical and adjuvant onabotulinumtoxinA (BTX-A) therapy. A 43-year-old woman diagnosed with ChAc was referred to our clinic because of a 5× 5 mm lip defect resulting from orofacial dyskinesia. Wedge resection of the scar tissue was carried out, followed by reconstruction by suturing. Postoperatively, BTX-A injections were administered to ameliorate dyskinesia. Thirty units of BTX-A were injected into each masseter muscle, and 40 units were injected into the orbicularis oris muscle. At 1, 2, and 4 weeks after the injections, assessments were performed using the Abnormal Involuntary Movement Scale, and the patient's impression of change was assessed using the Global Rating of Change Scale. Subsequent adjuvant BTX-A treatment yielded subjective and objective improvements in orofacial dyskinesia. In conclusion, lip reconstruction and adjuvant BTX-A injections were effective in treating lip defects associated with orofacial dyskinesia in patients with ChAc, which highlights the need for a multimodal treatment approach.

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2023-09-16 | VPS13A knockdown impairs corticostriatal synaptic plasticity and locomotor behavior in a new mouse model of chorea-acanthocytosis.

Chorea-acanthocytosis (ChAc) is an inherited neurodegenerative movement disorder caused by VPS13A gene mutations leading to the absence of protein expression. The striatum is the most affected brain region in ChAc patients. However, the study of the VPS13A function in the brain has been poorly addressed. Here we generated a VPS13A knockdown (KD) model and aimed to elucidate the contribution of VPS13A to synaptic plasticity and neuronal communication in the corticostriatal circuit. First, we infected primary cortical neurons with miR30-shRNA against VPS13A and analyzed its effects on neuronal plasticity. VPS13A-KD neurons showed a higher degree of branching than controls, accompanied by decreased BDNF and PSD-95 levels, indicative of synaptic alterations. We then injected AAV-KD bilaterally in the frontal cortex and two different regions of the striatum of mice and analyzed the effects of VPS13A-KD on animal behavior and synaptic plasticity. VPS13A-KD mice showed modification of the locomotor behavior pattern, with increased exploratory behavior and hyperlocomotion. Corticostriatal dysfunction in VPS13A-KD mice was evidenced by impaired striatal long-term depression (LTD) after stimulation of cortical afferents, which was partially recovered by BDNF administration. VPS13A-KD did not lead to neuronal loss in the cortex or the striatum but induced a decrease in the neuronal release of CX3CL1 and triggered a microglial reaction, especially in the striatum. Notably, CX3CL1 administration partially restored the impaired corticostriatal LTD in VPS13A-KD mice. Our results unveil the involvement of VPS13A in neuronal connectivity modifying BDNF and CX3CL1 release. Moreover, the involvement of VPS13A in synaptic plasticity and motor behavior provides key information to further understand not only ChAc pathophysiology but also other neurological disorders.

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2022-06-23 | VPS13A and VPS13C influence lipid droplet abundance

ABSTRACT Lipid transfer proteins mediate the exchange of lipids between closely apposed membranes at organelle contact sites and play key roles in lipid metabolism, membrane homeostasis, and cellular signaling. A recently discovered novel family of lipid transfer proteins, which includes the VPS13 proteins (VPS13A-D), adopt a rod-like bridge conformation with an extended hydrophobic groove that enables the bulk transfer of membrane lipids for membrane growth. Loss of function mutations in VPS13A and VPS13C cause chorea acanthocytosis and Parkinson’s disease, respectively. VPS13A and VPS13C localize to multiple organelle contact sites, including endoplasmic reticulum (ER) – lipid droplet (LD) contact sites, but the functional roles of these proteins in LD regulation remains mostly unexplored. Here, we employ CRISPR-Cas9 genome editing to generate VPS13A and VPS13C knockout cell lines in U-2 OS cells via deletion of exon 2 and introduction of an early frameshift. Analysis of LD content in these cell lines revealed that loss of either VPS13A or VPS13C results in reduced LD abundance under oleate-stimulated conditions. These data implicate VPS13A and VPS13C in LD regulation and raise the intriguing possibility that VPS13A and VPS13C-mediated lipid transfer facilitates LD biogenesis.

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2017-10-17 | Management of oromandibular dystonia on a chorea acanthocytosis: a brief review of the literature and a clinical case.

Chorea acanthocytosis is an extremely rare neurodegenerative condition characterized by neuropsychiatric disturbances, movement disorders, neuropathy, seizures, and acanthocytosis. In this case report, the authors will present the management of the oromandibular movement disorders associated with this disease. This case report describes the focal management of the severe orofacial manifestations associated with this condition. The therapeutic approach adopted to reduce the severe oromandibular movements, dysphagia, and the numerous oral ulcers was selective electromyography (EMG)-guided botulinum toxin application to the inferior head of the lateral pterygoid muscles and masseters. This would be applied to control severe and sudden oromandibular dystonia. Through this procedure, the authors were able to reduce these severe oral manifestations, which had a major impact on the patient's quality of life, and temporarily improve vital functions, such as mastication, deglutition, and speech articulation. Electromyography-guided botulinum toxin application may be a useful tool in the multimodal management of this condition.

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2017-01-20 | Drosophila Vps13 Is Required for Protein Homeostasis in the Brain

Chorea-Acanthocytosis is a rare, neurodegenerative disorder characterized by progressive loss of locomotor and cognitive function. It is caused by loss of function mutations in the Vacuolar Protein Sorting 13A (VPS13A) gene, which is conserved from yeast to human. The consequences of VPS13A dysfunction in the nervous system are still largely unspecified. In order to study the consequences of VPS13A protein dysfunction in the ageing central nervous system we characterized a Drosophila melanogaster Vps13 mutant line. The Drosophila Vps13 gene encoded a protein of similar size as human VPS13A. Our data suggest that Vps13 is a peripheral membrane protein located to endosomal membranes and enriched in the fly head. Vps13 mutant flies showed a shortened life span and age associated neurodegeneration. Vps13 mutant flies were sensitive to proteotoxic stress and accumulated ubiquitylated proteins. Levels of Ref(2)P, the Drosophila orthologue of p62, were increased and protein aggregates accumulated in the central nervous system. Overexpression of the human Vps13A protein in the mutant flies partly rescued apparent phenotypes. This suggests a functional conservation of human VPS13A and Drosophila Vps13. Our results demonstrate that Vps13 is essential to maintain protein homeostasis in the larval and adult Drosophila brain. Drosophila Vps13 mutants are suitable to investigate the function of Vps13 in the brain, to identify genetic enhancers and suppressors and to screen for potential therapeutic targets for Chorea-Acanthocytosis.

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antibodies

2025-12-11 | A Novel VPS13A Deletion in VPS13A Disease (Chorea-Acanthocytosis): A Case Report with Brief Literature Summary.

VPS13A disease is a rare, autosomal-recessive, neurodegenerative disorder characterized by involuntary movements, orofacial dystonia, seizures, psychiatric symptoms, and the presence of spiky, deformed red blood cells (acanthocytes). The disease is caused by mutations in the VPS13A gene, which encodes the VPS13A protein (previously known as chorein). This protein is a member of the family of bridge-like lipid transport proteins, involved in bulk lipid transfer between membranes and intracellular vesicle trafficking. We describe the case of a 37-year-old woman with gait instability, semi-flexed legs, and involuntary distal muscle movements. Genetic testing was performed using next-generation sequencing (NGS), followed by molecular analysis. Fibroblasts from the patient, her mother, and a healthy control were analyzed by immunofluorescence and Western blotting. NGS identified a novel homozygous 2.8 kb deletion encompassing exons 69-70 (69-70del) of the VPS13A gene (NM_033305.3). The same variant was detected in the patient's mother in a heterozygous state and her brother in a homozygous state. Although other deletions in the gene have been described, a comprehensive search of population variant databases and the existing literature did not reveal previous reports of this deletion. Fibroblasts from the patient, her mother and a healthy control were characterized. Functional assays showed a complete absence of the VPS13A protein in the patient's fibroblasts. This study expands the mutational spectrum of VPS13A-linked VPS13A disease and underlines the importance of comprehensive genetic analysis in atypical cases.

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2025-07-11 | Various Gait Patterns in Chorea‐Acanthocytosis

Ethical Compliance Statement: We confirm that we have read the Journal's position on issues involved in ethical publication and affirm that this work is consistent with those guidelines. Patients gave informed consent before inclusion in this report. The authors confirm that the approval of an institutional review board was not required for this work. Funding Sources and Conflicts of Interest: The authors report no sources of funding and no conflicts of interest. Financial Disclosures for Previous 12 Months: The authors declare that there are no disclosures to report. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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2025-06-30 | Editorial: Progress in neuroacanthocytosis syndromes and related diseases including other bulk lipid transfer disorders

The rare conditions XK disease (McLeod syndrome) and VPS13A disease (chorea-acanthocytosis), 25 have historically been termed "neuroacanthocytosis", due to the association of neurodegeneration, 26 particularly of the basal ganglia, with spiky deformed red blood cells (acanthocytes). Even more 27 obsolete is the 1960s' designation "Levine-Critchley syndrome": genetic analyses of the reported 28 families have determined that Levine´s patients were affected by mutations of XK and those of 29Critchley by VPS13A variants. Two multi-author books summarize early developments (Danek,30 2004; The articles collected here represent a number of recent advances in this rapidly-evolving field. The 94 ultimate goal is to develop and validate therapies for these devasting neurological and multi-organ 95 disorders, and additionally to add to understanding of cellular processes which may benefit other 96 fields. 97

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2025-04-07 | Occam’s Razor or Hickam’s Dictum: GAD65 Temporal Lobe Epilepsy with Subsequent Movement Disorder and Coincidental Mutations in the VPS13A Gene (P2-8.012)

We present a case with coexisting GAD65 CNS autoimmunity and coincidental variants of uncertain significance (VUS) in the VPS13A gene which is associated with autosomal recessive chorea acanthocytosis.

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2016-04-26 | ChAcNLS, a Novel Modification to Antibody-Conjugates Permitting Target Cell-Specific Endosomal Escape, Localization to the Nucleus, and Enhanced Total Intracellular Accumulation

The design of antibody-conjugates (ACs) for delivering molecules for targeted applications in humans has sufficiently progressed to demonstrate clinical efficacy in certain malignancies and reduced systemic toxicity that occurs with standard nontargeted therapies. One area that can advance clinical success for ACs will be to increase their intracellular accumulation. However, entrapment and degradation in the endosomal-lysosomal pathway, on which ACs are reliant for the depositing of their molecular payload inside target cells, leads to reduced intracellular accumulation. Innovative approaches that can manipulate this pathway may provide a strategy for increasing accumulation. We hypothesized that escape from entrapment inside the endosomal-lysosomal pathway and redirected trafficking to the nucleus could be an effective approach to increase intracellular AC accumulation in target cells. Cholic acid (ChAc) was coupled to the peptide CGYGPKKKRKVGG containing the nuclear localization sequence (NLS) from SV-40 large T-antigen, which is termed ChAcNLS. ChAcNLS was conjugated to the mAb 7G3 (7G3-ChAcNLS), which has nanomolar affinity for the cell-surface leukemic antigen interleukin-3 receptor-α (IL-3Rα). Our aim was to determine whether 7G3-ChAcNLS increased intracellular accumulation while retaining nanomolar affinity and IL-3Rα-positive cell selectivity. Competition ELISA and cell treatment assays were performed. Cell fractionation, confocal microscopy, flow cytometry, and Western blot techniques were used to determine the level of antibody accumulation inside cells and in corresponding nuclei. In addition, the radioisotope copper-64 (64Cu) was also utilized as a surrogate molecular cargo to evaluate nuclear and intracellular accumulation by radioactivity counting. 7G3-ChAcNLS effectively escaped endosome entrapment and degradation resulting in a unique intracellular distribution pattern. mAb modification with ChAcNLS maintained 7G3 nM affinity and produced high selectivity for IL-3Rα-positive cells. In contrast, 7G3 ACs with the ability to either escape endosome entrapment or traffic to the nucleus was not superior to 7G3-ChAcNLS for increasing intracellular accumulation. Transportation of 64Cu when complexed to 7G3-ChAcNLS also resulted in increased nuclear and intracellular radioactivity accumulation. Thus, ChAcNLS is a novel mAb functionalizing technology that demonstrates its ability to increase AC intracellular accumulation in target cells through escaping endosome entrapment coupled to nuclear trafficking.

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