Testing Of Potential Therapeutics Research Articles

Abstract 4257: A novel 3D co-culture platform for patient-specific immune-oncology treatment

Abstract Lung cancer is the leading cause of global cancer-related mortality resulting in ~1.8 million deaths. annually. Over the past decade, the contribution of tumor microenvironment (TME) in lung cancer progression has been highly appreciated and provided significant improvements of survival outcomes for patients. That includes the complexity of the tumor immune microenvironment (TIME) playing a critical role in cancer progression. However, drug resistance usually arises and there is an urgent need for novel immune cell-based therapies. 3D tumor cell structures that recapitulate the architecture and functionality of the original tumor have revolutionized the understanding of disease mechanisms and testing potential therapeutics. However, there are certain drawbacks that need to be overcome. For example, these models mainly consist of only the epithelial compartment and lack signals from the microenvironment. Recent advances with engineering co-culture systems with immune cells at Champions Oncology help to emulate cell-cell and immune cell infiltration. We have successfully established a biobank of TumorGraft3D (CTG3Ds) derived from non-small cell lung cancer (NSCLC) patient-derived xenografts as well as their autologous and allogeneic immune cells (TILs, PBMCs) to establish a proprietary co-culture platform to investigate disease mechanisms and test potential therapeutics. In brief, surgical resections from patients were used to generate patient-derived xenografts organoids, and matched immune cells were expanded using standard rapid expansion protocol. The combination of a comprehensive staining system and high-content imaging as an endpoint allowed for 3D confocal analysis and deep evaluation of cancer-immune cellular interaction at the single cell level. Our pipeline of image analysis not only differentiates cancer-immune cell spatial distribution and cellular death but also allows for the evaluation of tumor infiltration and drug cytotoxicity. The characterization of the platform showed proliferation of the cancer cells and functional immune profiling by flow cytometry. The molecular profile of the organoids composing the platform showed a tight overlap with the original tumor highlighting the clinical relevance of the model. In addition, this novel co-culture platform has been tested with several NSCLC models to test the efficacy of various therapeutics such as immune-checkpoint inhibitors (ICIs) in reproducible, cost-effective, and patient-specific manner. In summary, Champion’s novel co-culture platform allows screening of novel immune targeting agents to determine impacts tumor cytotoxicity. Furthermore, our lung cancer tumorGraft3D platform serves as a powerful tool to facilitate the practice of precision medicine by investigating a variety of therapeutic modalities, alone or in combination, to identify patient-specific treatment strategies. Citation Format: Taylor Light, Samaneh Kamali, Mara Gilardi, Abhay Andar, Karin Abarca-Heidemann, Maria Mancini. A novel 3D co-culture platform for patient-specific immune-oncology treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4257.

Altered ubiquitin signaling induces Alzheimer’s disease-like hallmarks in a three-dimensional human neural cell culture model

Alzheimer’s disease (AD) is characterized by toxic protein accumulation in the brain. Ubiquitination is essential for protein clearance in cells, making altered ubiquitin signaling crucial in AD development. A defective variant, ubiquitin B + 1 (UBB+1), created by a non-hereditary RNA frameshift mutation, is found in all AD patient brains post-mortem. We now detect UBB+1 in human brains during early AD stages. Our study employs a 3D neural culture platform derived from human neural progenitors, demonstrating that UBB+1 alone induces extracellular amyloid-β (Aβ) deposits and insoluble hyperphosphorylated tau aggregates. UBB+1 competes with ubiquitin for binding to the deubiquitinating enzyme UCHL1, leading to elevated levels of amyloid precursor protein (APP), secreted Aβ peptides, and Aβ build-up. Crucially, silencing UBB+1 expression impedes the emergence of AD hallmarks in this model system. Our findings highlight the significance of ubiquitin signalling as a variable contributing to AD pathology and present a nonclinical platform for testing potential therapeutics.

Heroin- and Fentanyl-Induced Respiratory Depression in a Rat Plethysmography Model: Potency, Tolerance, and Sex Differences.

The opioid overdose death toll in the United States is an ongoing public health crisis. We characterized the magnitude and duration of respiratory depression, the leading cause of death in opioid overdose cases, induced by heroin or fentanyl and the development of tolerance in male and female rats. We used whole-body plethysmography to first establish dose-response curves by recording breathing for 60 minutes post-intravenous opioid injection. We then tested the development of respiratory tolerance to acute heroin or fentanyl over several weeks and to chronic fentanyl with acute fentanyl or heroin challenge. Heroin and fentanyl each provoked dose-dependent respiratory depression. Heroin caused prolonged (45-60 minute) respiratory depression in female and male rats, characterized by decreased frequency, tidal volume, and minute ventilation and increased inspiratory time and apneic pause. Fentanyl produced similar changes with a shorter duration (10-15 minutes). High-dose heroin or fentanyl produced robust respiratory depression that was slightly more severe in females and, when given intermittently (acute doses 2 to 3 weeks apart), did not lead to tolerance. In contrast, chronic fentanyl delivered with an osmotic minipump resulted in tolerance to acute fentanyl and heroin, characterized by a shorter duration of respiratory depression. This effect persisted during withdrawal in males only. Our model and experimental design will allow for investigation of the neurobiology of opioid-induced respiratory depression and for testing potential therapeutics to reverse respiratory depression or stimulate breathing. SIGNIFICANCE STATEMENT: Fentanyl was more potent and had shorter duration in producing respiratory depression than heroin in both sexes, whereas female rats were more sensitive than males to heroin-induced respiratory depression. Tolerance/cross-tolerance develops in chronic fentanyl administration but is minimized with long interadministration intervals.

Porcine Model of Spinal Cord Injury: A Systematic Review.

Spinal cord injury (SCI) is a devastating disease with limited effective treatment options. Animal paradigms are vital for understanding the pathogenesis of SCI and testing potential therapeutics. The porcine model of SCI is increasingly favored because of its greater similarity to humans. However, its adoption is limited by the complexities of care and range of testing parameters. Researchers need to consider swine selection, injury method, post-operative care, rehabilitation, behavioral outcomes, and histology metrics. Therefore, we systematically reviewed full-text English-language articles to evaluate study characteristics used in developing a porcine model and summarize the interventions that have been tested using this paradigm. A total of 63 studies were included, with 33 examining SCI pathogenesis and 30 testing interventions. Studies had an average sample size of 15 pigs with an average weight of 26 kg, and most used female swine with injury to the thoracic cord. Injury was most commonly induced by weight drop with compression. The porcine model is amenable to testing various interventions, including mean arterial pressure augmentation (n = 7), electrical stimulation (n = 6), stem cell therapy (n = 5), hypothermia (n = 2), biomaterials (n = 2), gene therapy (n = 2), steroids (n = 1), and nanoparticles (n = 1). It is also notable for its clinical translatability and is emerging as a valuable pre-clinical study tool. This systematic review can serve as a guideline for researchers implementing and testing the porcine SCI model.

Use of Translational, Genetically Modified Porcine Models to Ultimately Improve Intestinal Disease Treatment.

For both human and veterinary patients, non-infectious intestinal disease is a major cause of morbidity and mortality. To improve treatment of intestinal disease, large animal models are increasingly recognized as critical tools to translate the basic science discoveries made in rodent models into clinical application. Large animal intestinal models, particularly porcine, more closely resemble human anatomy, physiology, and disease pathogenesis; these features make them critical to the pre-clinical study of intestinal disease treatments. Previously, large animal model use has been somewhat precluded by the lack of genetically altered large animals to mechanistically investigate non-infectious intestinal diseases such as colorectal cancer, cystic fibrosis, and ischemia-reperfusion injury. However, recent advances and increased availability of gene editing technologies has led to both novel use of large animal models in clinically relevant intestinal disease research and improved testing of potential therapeutics for these diseases.

Human Mesenchymal Stem Cell-Derived Miniature Joint System for Disease Modeling and Drug Testing.

Diseases of the knee joint such as osteoarthritis (OA) affect all joint elements. An in vitro human cell‐derived microphysiological system capable of simulating intraarticular tissue crosstalk is desirable for studying etiologies/pathogenesis of joint diseases and testing potential therapeutics. Herein, a human mesenchymal stem cell‐derived miniature joint system (miniJoint) is generated, in which engineered osteochondral complex, synovial‐like fibrous tissue, and adipose tissue are integrated into a microfluidics‐enabled bioreactor. This novel design facilitates different tissues communicating while still maintaining their respective phenotypes. The miniJoint exhibits physiologically relevant changes when exposed to interleukin‐1β mediated inflammation, which are similar to observations in joint diseases in humans. The potential of the miniJoint in predicting in vivo efficacy of drug treatment is confirmed by testing the “therapeutic effect” of the nonsteroidal anti‐inflammatory drug, naproxen, as well as four other potential disease‐modifying OA drugs. The data demonstrate that the miniJoint recapitulates complex tissue interactions, thus providing a robust organ chip model for the study of joint pathology and the development of novel therapeutic interventions.

Recapitulating lipid accumulation and related metabolic dysregulation in human liver-derived organoids.

Fatty liver disease has grown into a major global health burden, attributed to multi-factors including sedentary lifestyle, obesogenic diet and prevalence of metabolic disorders. The lack of robust experimental models is hampering the research and therapeutic development for fatty liver disease. This study aims to develop an organoid-based 3D culture model to recapitulate key features of fatty liver disease focusing on intracellular lipid accumulation and metabolic dysregulation. We used human liver-derived intrahepatic cholangiocyte organoids and hepatocyte differentiated organoids. These organoids were exposed to lactate, pyruvate, and octanoic acid (LPO) for inducing lipid accumulation and mitochondrial impairment. Lipid accumulation resulted in alternations of gene transcription with major effects on metabolic pathways, including triglyceride and glucose level increase, which is consistent with metabolic changes in fatty liver disease patients. Interestingly, lipid accumulation affected mitochondria as shown by morphological transitions, alternations in expression of mitochondrial encoded genes, and reduction of ATP production. Meanwhile, we found treatment with obeticholic acid and metformin can alleviate fat accumulation in organoids. This study demonstrated that LPO exposure can induce lipid accumulation and associated metabolic dysregulation in human liver-derived organoids. This provides an innovative model for studying fatty liver disease and testing potential therapeutics. KEY MESSAGES: Lactate, pyruvate, and octanoic acid induce lipid accumulation in liver organoids. Organoids of human compared to mouse origin are more efficient in lipid accumulation. Lipid accumulation dysregulates metabolic pathway and impairs mitochondrial function. Demonstrating a proof-of-concept for testing medications in organoids.

In vivo Evaluation of Mucociliary Clearance in Mice.

Respiratory motile cilia, specialized organelles of the cell, line the apical surface of epithelial cells lining the respiratory tract. By beating in a metachronal, synchronal fashion, these multiple, motile, actin-based organelles generate a cephalad fluid flow clearing the respiratory tract of inhaled pollutants and pathogens. With increasing environmental pollution, novel viral pathogens and emerging multi-drug resistant bacteria, cilia generated mucociliary clearance (MCC) is essential for maintaining lung health. MCC is also depressed in multiple congenital disorders like primary ciliary dyskinesia, cystic fibrosis as well as acquired disorders like chronic obstructive pulmonary disease. All these disorders have established, in some case multiple, mouse models. In this publication, we detail a method using a small amount of radioactivity and dual-modality SPECT/CT imaging to accurately and reproducibly measure MCC in mice in vivo. The method allows for recovery of mice after imaging, making serial measurements possible, and testing potential therapeutics longitudinally over time. The data in wild-type mice demonstrates the reproducibility of the MCC measurement as long as adequate attention to detail is paid, and the protocol strictly adhered to.

Pristane-induced mammary carcinomas.

Animal models for studying human breast cancer carcinogenesis and testing drug candidates on human breast cancer have extensively been proposed. Especially, chemically induced breast tumor models have been used because they can mimic the progression of clinical cancer from the beginning and can be generated with a facile procedure. Pristane is a hydrocarbon oil that is used as a chemical carcinogen to induce tumorigenesis in mice as well as arthritis and lupus nephritis in rats. In only a few studies, pristane-induced breast cancer models have been reported. This chapter is designated to describe pristane-induced mammary carcinoma models. Here, we provide a protocol for generating pristane-induced breast tumors in mice models for analyzing and for testing potential therapeutics on them. The given protocol can be applied to other animal models with some changes.

Livestock Challenge Models of Rift Valley Fever for Agricultural Vaccine Testing.

Since the discovery of Rift Valley Fever virus (RVFV) in Kenya in 1930, the virus has become widespread throughout most of Africa and is characterized by sporadic outbreaks. A mosquito-borne pathogen, RVFV is poised to move beyond the African continent and the Middle East and emerge in Europe and Asia. There is a risk that RVFV could also appear in the Americas, similar to the West Nile virus. In light of this potential threat, multiple studies have been undertaken to establish international surveillance programs and diagnostic tools, develop models of transmission dynamics and risk factors for infection, and to develop a variety of vaccines as countermeasures. Furthermore, considerable efforts to establish reliable challenge models of Rift Valley fever virus have been made and platforms for testing potential vaccines and therapeutics in target species have been established. This review emphasizes the progress and insights from a North American perspective to establish challenge models in target livestock such as cattle, sheep, and goats in comparisons to other researchers' reports. A brief summary of the potential role of wildlife, such as buffalo and white-tailed deer as reservoir species will also be discussed.

Pathogenesis of SARS-CoV-2 in Transgenic Mice Expressing Human Angiotensin-Converting Enzyme 2

COVID-19 has spread worldwide since 2019 and is now a severe threat to public health. We previously identified the causative agent as a novel SARS-related coronavirus (SARS-CoV-2) that uses human angiotensin-converting enzyme 2 (hACE2) as the entry receptor. Here, we successfully developed a SARS-CoV-2 hACE2 transgenic mouse (HFH4-hACE2 in C3B6 mice) infection model. The infected mice generated typical interstitial pneumonia and pathology that were similar to those of COVID-19 patients. Viral quantification revealed the lungs as the major site of infection, although viral RNA could also be found in the eye, heart, and brain in some mice. Virus identical to SARS-CoV-2 in full-genome sequences was isolated from the infected lung and brain tissues. Last, we showed that pre-exposure to SARS-CoV-2 could protect mice from severe pneumonia. Our results show that the hACE2 mouse would be a valuable tool for testing potential vaccines and therapeutics.

Mouse models of thyroid cancer: Bridging pathogenesis and novel therapeutics.

Due to a global increase in the incidence of thyroid cancer, numerous novel mouse models were established to reveal thyroid cancer pathogenesis and test promising therapeutic strategies, necessitating a comprehensive review of translational medicine that covers (i) the role of mouse models in the research of thyroid cancer pathogenesis, and (ii) preclinical testing of potential anti-thyroid cancer therapeutics. The present review article aims to: (i) describe the current approaches for mouse modeling of thyroid cancer, (ii) provide insight into the biology and genetics of thyroid cancers, and (iii) offer guidance on the use of mouse models for testing potential therapeutics in preclinical settings. Based on research with mouse models of thyroid cancer pathogenesis involving the RTK, RAS/RAF/MEK/ERK, PI3K/AKT/mTOR, SRC, and JAK-STAT signaling pathways, inhibitors of VEGFR, MEK, mTOR, SRC, and STAT3 have been developed as anti-thyroid cancer drugs for "bench-to-bedside" translation. In the future, mouse models of thyroid cancer will be designed to be ''humanized" and "patient-like," offering opportunities to: (i) investigate the pathogenesis of thyroid cancer through target screening based on the CRISPR/Cas system, (ii) test drugs based on new mouse models, and (iii) explore the underlying mechanisms based on multi-omics.

Biomolecular Analysis of Beta Dose-Dependent Cutaneous Radiation Injury in a Porcine Model.

While cutaneous radiation injury (CRI) is generally referenced as a consequence of a nuclear attack, it can also be caused by less dangerous events such as the use of dirty bombs, industrial radiological accidents, or accidental overexposure of beta (β) particle or gamma (γ) radiation sources in medical procedures. Although the gross clinical consequences of these injuries have been well documented, relatively little is known about the molecular changes underlying the progression of pathology. Here we describe a porcine model of cutaneous radiation injury after skin was exposed to strontium-90 b particle at doses of 16-42 Gy and characterize the anatomical and molecular changes over 70 days. The results show that irradiated sites displayed dosedependent increases in erythema and moist desquamation that peaked between days 35 and 42. Dose-dependent histopathological changes were observed, with higher doses exhibiting increased inflammation and epidermal hyperplasia beyond day 35. Furthermore, immunohistochemistry showed that exposure to 37 Gy β-particle radiation decreased epidermal cell proliferation and desmosomal junction proteins at day 70, suggesting compromised epidermal integrity. Metabolomic analysis of biopsies revealed dose- and time-dependent changes as high as 252-fold in several metabolites not previously linked to CRI. These alterations were seen in pathways reflecting protein degradation, oxidative stress, eicosanoid production, collagen matrix remodeling, mitochondrial stress, cell membrane composition and vascular disruption. Taken together, these data show that exposure to high doses of β particle damaged the molecular processes underlying skin integrity to a greater extent and for a longer period of time than has been shown previously. These findings further understanding of radiation-induced skin injury and serve as a foundation for the development and testing of potential therapeutics to treat CRI.

Quantitative comparison of three rat models of Achilles tendon injury: A multidisciplinary approach

The Achilles tendon, while the strongest and largest tendon in the body, is frequently injured. Inconclusive evidence exists regarding treatment strategies for both complete tears and partial tears. Well-characterized animal models of tendon injury are important for understanding physiological processes of tendon repair and testing potential therapeutics. Utilizing three distinct models of rat Achilles tendon injury, the objective of this study was to define and compare the effects and relative impact on tendon properties and ankle function of both tear severity (complete tear versus partial tear, both with post-operative immobilization) and immobilization after partial tear (partial tear with versus without immobilization). We hypothesized that a complete tear would cause inferior post-injury properties compared to a partial tear, and that immediate loading after partial tear would improve post-injury properties compared to immobilization. All models were reproducible and had distinct effects on measured parameters. Injury severity drastically influenced tendon healing, with complete tear causing decreased ankle mobility and tendon mechanics compared to partial tears. One week of plantarflexion immobilization had a strong effect on animals receiving a partial tear. Tendons with partial tears and immobilization failed early during fatigue cycling three weeks post-injury. Partial tear without immobilization had no effect on ankle range of motion through dorsiflexion at any time point compared to the pre-surgery value, while partial tear with immobilization demonstrated diminished function at all post-injury time points. All three models of Achilles injury could be useful for tendon healing investigations, chosen based on the prospective applications of a potential therapeutic.

High-Content Analysis of Mitochondrial Function in iPSC-Derived Neurons.

Mitochondrial dysfunction is linked to many neurological diseases; therefore, the ability to measure mitochondrial function is of great use for researching disease and testing potential therapeutics. Here we describe a high-content assay to simultaneously measure mitochondrial membrane potential, morphology and cell viability in iPSC-derived neurons. Neurons are seeded into plates suitable for fluorescent microscopy, stained with the mitochondrial membrane potential-dependent dye TMRM, cytoplasmic dye Calcein AM, and nuclear stain Hoechst 33342. Images are acquired in live cells and analyzed using automated image analysis software.

Current Models of Fatty Liver Disease; New Insights, Therapeutic Targets and Interventions.

Non-alcoholic fatty liver disease (NAFLD) encompasses a spectrum of disorders ranging from simple steatosis to steatosis with inflammation and fibrosis. NAFLD is currently the most prevalent chronic liver disease worldwide, with a global prevalence of 25%, and is soon projected to be the leading cause for liver transplantation in the US. Alarmingly, few effective pharmacotherapeutic approaches are currently available to block or attenuate development and progression of NAFLD. Preclinical models are critical for unraveling the complex and multi-factorial etiology of NAFLD and for testing potential therapeutics. Here we review preclinical models that have been instrumental in highlighting molecular and cellular mechanisms underlying the pathogenesis of NAFLD and in facilitating early proof-of-concept investigations into novel intervention strategies.

FUS(1-359) transgenic mice as a model of ALS: pathophysiological and molecular aspects of the proteinopathy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder that leads to the eventual death of motor neurons. Described cases of familial ALS have emphasized the significance of protein misfolding and aggregation of two functionally related proteins, FUS (fused in sarcoma) and TDP-43, implicated in RNA metabolism. Herein, we performed a comprehensive analysis of the in vivo model of FUS-mediated proteinopathy (ΔFUS(1-359) mice). First, we used the Noldus CatWalk system and confocal microscopy to determine the time of onset of the first clinical symptoms and the appearance of FUS-positive inclusions in the cytoplasm of neuronal cells. Second, we applied RNA-seq to evaluate changes in the gene expression profile encompassing the pre-symptomatic and the symptomatic stages of disease progression in motor neurons and the surrounding microglia of the spinal cord. The resulting data show that FUS-mediated proteinopathy is virtually asymptomatic in terms of both the clinical symptoms and the molecular aspects of neurodegeneration until it reaches the terminal stage of disease progression (120days from birth). After this time, the pathological process develops very rapidly, resulting in the formation of massive FUS-positive inclusions accompanied by a transcriptional "burst" in the spinal cord cells. Specifically, it manifests in activation of a pro-inflammatory phenotype of microglial cells and malfunction of acetylcholine synapse transmission in motor neurons. Overall, we assume that the highly reproducible course of the pathological process, as well as the described accompanying features, makes ΔFUS(1-359) mice a convenient model for testing potential therapeutics against proteinopathy-induced decay of motor neurons.

Retina-specific loss of Ikbkap/Elp1 causes mitochondrial dysfunction that leads to selective retinal ganglion cell degeneration in a mouse model of familial dysautonomia.

ABSTRACTFamilial dysautonomia (FD) is an autosomal recessive disorder marked by developmental and progressive neuropathies. It is caused by an intronic point-mutation in the IKBKAP/ELP1 gene, which encodes the inhibitor of κB kinase complex-associated protein (IKAP, also called ELP1), a component of the elongator complex. Owing to variation in tissue-specific splicing, the mutation primarily affects the nervous system. One of the most debilitating hallmarks of FD that affects patients' quality of life is progressive blindness. To determine the pathophysiological mechanisms that are triggered by the absence of IKAP in the retina, we generated retina-specific Ikbkap conditional knockout (CKO) mice using Pax6-Cre, which abolished Ikbkap expression in all cell types of the retina. Although sensory and autonomic neuropathies in FD are known to be developmental in origin, the loss of IKAP in the retina did not affect its development, demonstrating that IKAP is not required for retinal development. The loss of IKAP caused progressive degeneration of retinal ganglion cells (RGCs) by 1 month of age. Mitochondrial membrane integrity was breached in RGCs, and later in other retinal neurons. In Ikbkap CKO retinas, mitochondria were depolarized, and complex I function and ATP were significantly reduced. Although mitochondrial impairment was detected in all Ikbkap-deficient retinal neurons, RGCs were the only cell type to degenerate; the survival of other retinal neurons was unaffected. This retina-specific FD model is a useful in vivo model for testing potential therapeutics for mitigating blindness in FD. Moreover, our data indicate that RGCs and mitochondria are promising targets.

Development of a Large Animal Long-Term Intervertebral Disc Organ Culture Model That Includes the Bony Vertebrae for Ex Vivo Studies.

Intervertebral disc (IVD) degeneration is a common cause of low back pain. Testing potential therapeutics in the regeneration of the disc requires the use of model systems. Although several animal models have been developed to investigate IVD degeneration, they are technically challenging to prepare, expensive, present with limitations when performing biomechanical studies on the disc, and are impractical in large-scale screening of novel anabolic and scaffolding agents. An IVD organ culture system offers an inexpensive alternative. In the current paradigm, the bony endplates are removed to allow for nutrient diffusion and maintenance of disc cell viability. Although this is an excellent system for testing biologics, it results in concave cartilage endplates and, as such, requires special platens for loading purposes in a bioreactor as flat ones can overload the annular disc region leading to improper loading. Furthermore, the absence of bone makes it unsuitable for applying complex cyclic loading, a topic of interest in the study of chronic progressive degeneration, as multiaxial loading is more representative of daily forces encountered by the IVD. We have developed and validated a novel long-term IVD organ culture model that retains vertebral bone and is easy to prepare. Our model is ideal for testing potential drugs and alternate-based therapies, in addition to investigating the long-term effects of loading paradigms on disc degeneration and repair.

The HTT CAG-Expansion Mutation Determines Age at Death but Not Disease Duration in Huntington Disease

Huntington disease (HD) is caused by an expanded HTT CAG repeat that leads in a length-dependent, completely dominant manner to onset of a characteristic movement disorder. HD also displays early mortality, so we tested whether the expanded CAG repeat exerts a dominant influence on age at death and on the duration of clinical disease. We found that, as with clinical onset, HD age at death is determined by expanded CAG-repeat length and has no contribution from the normal CAG allele. Surprisingly, disease duration is independent of the mutation's length. It is also unaffected by a strong genetic modifier of HD motor onset. These findings suggest two parsimonious alternatives. (1) HD pathogenesis is driven by mutant huntingtin, but before or near motor onset, sufficient CAG-driven damage occurs to permit CAG-independent processes and then lead to eventual death. In this scenario, some pathological changes and their clinical correlates could still worsen in a CAG-driven manner after disease onset, but these CAG-related progressive changes do not themselves determine duration. Alternatively, (2) HD pathogenesis is driven by mutant huntingtin acting in a CAG-dependent manner with different time courses in multiple cell types, and the cellular targets that lead to motor onset and death are different and independent. In this scenario, processes driven by HTT CAG length lead directly to death but not via the striatal pathology associated with motor manifestations. Each scenario has important ramifications for the design and testing of potential therapeutics, especially those aimed at preventing or delaying characteristic motor manifestations.