Shibi Likhite

Astrocytes from familial and sporadic ALS patients are toxic to motor neurons

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease, with astrocytes implicated as contributing substantially to motor neuron death in familial (F)ALS. However, the proposed role of astrocytes in the pathology of ALS derives in part from rodent models of FALS based upon dominant mutations within the superoxide dismutase 1 (SOD1) gene, which account for <2% of all ALS cases. Their role in sporadic (S)ALS, which affects >90% of ALS patients, remains to be established. Using astrocytes generated from postmortem tissue from both FALS and SALS patients, we show that astrocytes derived from both patient groups are similarly toxic to motor neurons. We also demonstrate that SOD1 is a viable target for SALS, as its knockdown significantly attenuates astrocyte-mediated toxicity toward motor neurons. Our data highlight astrocytes as a non–cell autonomous component in SALS and provide an in vitro model system to investigate common disease mechanisms and evaluate potential therapies for SALS and FALS.

Direct conversion of patient fibroblasts demonstrates non-cell autonomous toxicity of astrocytes to motor neurons in familial and sporadic ALS

Significance Direct conversion is a recently established method to generate neuronal progenitor cells (NPCs) from skin fibroblasts in a fast and efficient manner. In this study, we show that this method can be used to model neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Because the origin of ALS is mainly sporadic with unknown cause, methods to model the disease are urgently needed. The produced NPCs are differentiated into astrocytes, which are involved in motor neuron death in ALS. Strikingly, skin-derived astrocytes show similar toxicity toward motor neurons as astrocytes from autopsies of patients. This tool now allows studying ALS while the patient is still alive and can help in testing potential therapeutics for individual patients. Amyotrophic lateral sclerosis (ALS) causes motor neuron degeneration, paralysis, and death. Accurate disease modeling, identifying disease mechanisms, and developing therapeutics is urgently needed. We previously reported motor neuron toxicity through postmortem ALS spinal cord-derived astrocytes. However, these cells can only be harvested after death, and their expansion is limited. We now report a rapid, highly reproducible method to convert adult human fibroblasts from living ALS patients to induced neuronal progenitor cells and subsequent differentiation into astrocytes (i-astrocytes). Non-cell autonomous toxicity to motor neurons is found following coculture of i-astrocytes from familial ALS patients with mutation in superoxide dismutase or hexanucleotide expansion in C9orf72 (ORF 72 on chromosome 9) the two most frequent causes of ALS. Remarkably, i-astrocytes from sporadic ALS patients are as toxic as those with causative mutations, suggesting a common mechanism. Easy production and expansion of i-astrocytes now enables rapid disease modeling and high-throughput drug screening to alleviate astrocyte-derived toxicity.

Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy

Background Spinal muscular atrophy type 1 (SMA1) is a progressive, monogenic motor neuron disease with an onset during infancy that results in failure to achieve motor milestones and in death or the need for mechanical ventilation by 2 years of age. We studied functional replacement of the mutated gene encoding survival motor neuron 1 (SMN1) in this disease. Methods Fifteen patients with SMA1 received a single dose of intravenous adeno‐associated virus serotype 9 carrying SMN complementary DNA encoding the missing SMN protein. Three of the patients received a low dose (6.7×1013 vg per kilogram of body weight), and 12 received a high dose (2.0×1014 vg per kilogram). The primary outcome was safety. The secondary outcome was the time until death or the need for permanent ventilatory assistance. In exploratory analyses, we compared scores on the CHOP INTEND (Childrens Hospital of Philadelphia Infant Test of Neuromuscular Disorders) scale of motor function (ranging from 0 to 64, with higher scores indicating better function) in the two cohorts and motor milestones in the high‐dose cohort with scores in studies of the natural history of the disease (historical cohorts). Results As of the data cutoff on August 7, 2017, all 15 patients were alive and event‐free at 20 months of age, as compared with a rate of survival of 8% in a historical cohort. A rapid increase from baseline in the score on the CHOP INTEND scale followed gene delivery, with an increase of 9.8 points at 1 month and 15.4 points at 3 months, as compared with a decline in this score in a historical cohort. Of the 12 patients who had received the high dose, 11 sat unassisted, 9 rolled over, 11 fed orally and could speak, and 2 walked independently. Elevated serum aminotransferase levels occurred in 4 patients and were attenuated by prednisolone. Conclusions In patients with SMA1, a single intravenous infusion of adenoviral vector containing DNA coding for SMN resulted in longer survival, superior achievement of motor milestones, and better motor function than in historical cohorts. Further studies are necessary to confirm the safety and efficacy of this gene therapy. (Funded by AveXis and others; ClinicalTrials.gov number, NCT02122952.)

Therapeutic AAV9-mediated Suppression of Mutant SOD1 Slows Disease Progression and Extends Survival in Models of Inherited ALS

Mutations in superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (ALS) resulting in progressive motor neuron death through one or more acquired toxicities. Involvement of wild-type SOD1 has been linked to sporadic ALS, as misfolded SOD1 has been reported in affected tissues of sporadic patients and toxicity of astrocytes derived from sporadic ALS patients to motor neurons has been reported to be reduced by lowering the synthesis of SOD1. We now report slowed disease onset and progression in two mouse models following therapeutic delivery using a single peripheral injection of an adeno-associated virus serotype 9 (AAV9) encoding an shRNA to reduce the synthesis of ALS-causing human SOD1 mutants. Delivery to young mice that develop aggressive, fatal paralysis extended survival by delaying both disease onset and slowing progression. In a later-onset model, AAV9 delivery after onset markedly slowed disease progression and significantly extended survival. Moreover, AAV9 delivered intrathecally to nonhuman primates is demonstrated to yield robust SOD1 suppression in motor neurons and glia throughout the spinal cord and therefore, setting the stage for AAV9-mediated therapy in human clinical trials.

Improving single injection CSF delivery of AAV9-mediated gene therapy for SMA: a dose-response study in mice and nonhuman primates.

Spinal muscular atrophy (SMA) is the most frequent lethal genetic neurodegenerative disorder in infants. The disease is caused by low abundance of the survival of motor neuron (SMN) protein leading to motor neuron degeneration and progressive paralysis. We previously demonstrated that a single intravenous injection (IV) of self-complementary adeno-associated virus-9 carrying the human SMN cDNA (scAAV9-SMN) resulted in widespread transgene expression in spinal cord motor neurons in SMA mice as well as nonhuman primates and complete rescue of the disease phenotype in mice. Here, we evaluated the dosing and efficacy of scAAV9-SMN delivered directly to the cerebral spinal fluid (CSF) via single injection. We found widespread transgene expression throughout the spinal cord in mice and nonhuman primates when using a 10 times lower dose compared to the IV application. Interestingly, in nonhuman primates, lower doses than in mice can be used for similar motor neuron targeting efficiency. Moreover, the transduction efficacy is further improved when subjects are kept in the Trendelenburg position to facilitate spreading of the vector. We present a detailed analysis of transduction levels throughout the brain, brainstem, and spinal cord of nonhuman primates, providing new guidance for translation toward therapy for a wide range of neurodegenerative disorders.

Major histocompatibility complex class I molecules protect motor neurons from astrocyte-induced toxicity in amyotrophic lateral sclerosis.

Astrocytes isolated from individuals with amyotrophic lateral sclerosis (ALS) are toxic to motor neurons (MNs) and play a non–cell autonomous role in disease pathogenesis. The mechanisms underlying the susceptibility of MNs to cell death remain unclear. Here we report that astrocytes derived from either mice bearing mutations in genes associated with ALS or human subjects with ALS reduce the expression of major histocompatibility complex class I (MHCI) molecules on MNs; reduced MHCI expression makes these MNs susceptible to astrocyte-induced cell death. Increasing MHCI expression on MNs increases survival and motor performance in a mouse model of ALS and protects MNs against astrocyte toxicity. Overexpression of a single MHCI molecule, HLA-F, protects human MNs from ALS astrocyte–mediated toxicity, whereas knockdown of its receptor, the killer cell immunoglobulin-like receptor KIR3DL2, on human astrocytes results in enhanced MN death. Thus, our data indicate that, in ALS, loss of MHCI expression on MNs renders them more vulnerable to astrocyte-mediated toxicity.Astrocytes isolated from individuals with amyotrophic lateral sclerosis (ALS) are toxic towards motor neurons (MNs) and play a non-cell autonomous role in disease pathogenesis. The mechanisms underlying the susceptibility of motor neurons to cell death remains unclear. Here, we report that astrocytes derived from mice bearing ALS mutations and from individuals with ALS reduce expression of major histocompatibility complex class I (MHCI) on MNs. Reduced MHCI expression makes these MNs susceptible to astrocyte-induced cell death. Increasing MHCI expression on MNs increases survival and motor performance in a mouse model of ALS and protects MN against astrocyte toxicity. A single MHCI molecule, HLA-F, protects MNs from ALS astrocyte-mediated toxicity, while knockdown of its receptor, the killer cell immunoglobulin-like receptor KIR3DL2, an inhibitory receptor that recognizes MHCI, on astrocytes results in enhanced MN death. These data indicate that in ALS upon loss of MHCI expression MNs become vulnerable to astrocyte-mediated toxicity.

Oligodendrocytes contribute to motor neuron death in ALS via SOD1-dependent mechanism

Significance Oligodendrocytes have been implicated in disease pathology in amyotrophic lateral sclerosis (ALS) using transgenic mouse models. To date there is no human coculture system available to investigate oligodendrocyte involvement in motor neuron (MN) death in ALS. Our data highlight that oligodendrocytes derived from patients with familial and sporadic ALS from induced pluripotent stem cells and induced neural progenitor cells play an active role in MN death. Oligodendrocyte toxicity is mediated through soluble factors and cell-to-cell contact, thus identifying multiple mechanisms of action and therapeutic opportunities. Their pathogenic phenotype can be reversed by achieving superoxide dismutase 1 knockdown in early oligodendrocyte progenitors in both familial and sporadic cases, but not chromosome 9 ORF 72 samples. This study provides important insights for patient subgrouping and timelines for therapeutic approaches. Oligodendrocytes have recently been implicated in the pathophysiology of amyotrophic lateral sclerosis (ALS). Here we show that, in vitro, mutant superoxide dismutase 1 (SOD1) mouse oligodendrocytes induce WT motor neuron (MN) hyperexcitability and death. Moreover, we efficiently derived human oligodendrocytes from a large number of controls and patients with sporadic and familial ALS, using two different reprogramming methods. All ALS oligodendrocyte lines induced MN death through conditioned medium (CM) and in coculture. CM-mediated MN death was associated with decreased lactate production and release, whereas toxicity in coculture was lactate-independent, demonstrating that MN survival is mediated not only by soluble factors. Remarkably, human SOD1 shRNA treatment resulted in MN rescue in both mouse and human cultures when knockdown was achieved in progenitor cells, whereas it was ineffective in differentiated oligodendrocytes. In fact, early SOD1 knockdown rescued lactate impairment and cell toxicity in all lines tested, with the exclusion of samples carrying chromosome 9 ORF 72 (C9orf72) repeat expansions. These did not respond to SOD1 knockdown nor did they show lactate release impairment. Our data indicate that SOD1 is directly or indirectly involved in ALS oligodendrocyte pathology and suggest that in this cell type, some damage might be irreversible. In addition, we demonstrate that patients with C9ORF72 represent an independent patient group that might not respond to the same treatment.

Translating SOD1 Gene Silencing toward the Clinic: A Highly Efficacious, Off-Target-free, and Biomarker-Supported Strategy for fALS

Of familial amyotrophic lateral sclerosis (fALS) cases, 20% are caused by mutations in the gene encoding human cytosolic Cu/Zn superoxide dismutase (hSOD1). Efficient translation of the therapeutic potential of RNAi for the treatment of SOD1-ALS patients requires the development of vectors that are free of significant off-target effects and with reliable biomarkers to discern sufficient target engagement and correct dosing. Using adeno-associated virus serotype 9 to deliver RNAi against hSOD1 in the SOD1G93A mouse model, we found that intrathecal injection of the therapeutic vector via the cisterna magna delayed onset of disease, decreased motor neuron death at end stage by up to 88%, and prolonged the median survival of SOD1G93A mice by up to 42%. To our knowledge, this is the first report to demonstrate no significant off-target effects linked to hSOD1 silencing, providing further confidence in the specificity of this approach. We also report the measurement of cerebrospinal fluid (CSF) hSOD1 protein levels as a biomarker of effective dosing and efficacy of hSOD1 knockdown. Together, these data provide further confidence in the safety of the clinical therapeutic vector. The CSF biomarker will be a useful measure of biological activity for translation into human clinical trials.

Abstract 5504: Development of screening system for Brain Tumor Stem Cell-targeting chemotherapeutic agents

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Malignant glioma remains one of the most devastating diseases despite recent advances in therapies. Standard of care for malignant gliomas consists of maximal surgical resection, followed by external radiation with concurrent chemotherapy with temozolomide. Unfortunately, tumor recurrence is essentially inevitable and most of the therapies have only limited effects on the survival of patients experiencing tumor recurrence or progression. Emerging evidence has indicated that Brain Tumor Stem Cells (BTSC) can be the specific target of therapy and that elimination of BTSC will most likely result in the growth arrest of the entire tumor mass and the prevention of recurrence. The current therapies, however, preferentially target non-stem tumor cells. Thus, identification of BTSC-specific drugs is highly recommended in order to eradicate the entire tumor cells. To discover the prospective compounds from a large number of candidates efficiently, we have developed here the screening system for BTSC-targeting chemotherapeutic agents. Candidate compounds are screened in a stepwise manner based on their inhibitory potential against BTSC by using several in vitro and in vivo preclinical assays. Being different from the conventional compound discovery procedure, we combine multiple assays aiming at detection of the drugs directly targeting BTSC. Thus, this system functions as a rational agent-prioritization process essential for future progress in developing effective novel cancer treatments. Experimental Approach: We use several human BTSC cultures derived from surgical specimens. These samples have been confirmed to be clonogenic and multipotent in culture, and tumorigenic by xenotransplantation in immunodeficient mice brains. To assess the effect of compounds, the initial step is to use clonogenic assay, cell survival and migration assays with slice cultures of mouse brains, and xenotransplantation of pretreated BTSC. Potential compounds are then processed to the in vivo efficiency and efficacy validation assays using the following animal models, 1. BTSC-derived mouse brain tumor model: patient-derived BTSC transplanted in the mouse brain, 2. Genetically-engineered spontaneous mouse brain tumor model, and 3. Subcutaneous xenograft model of surgical specimens of brain tumor tissues: tumor tissue from patient directly transplanted subcutaneously into immunodeficient mice. Effect of the treatment on tumor progression is monitored by several imaging modalities including microMRI, SPECT/CT, and PET/CT. We also carry out pharmacokinetics profiling to determine the serum levels of the tested compounds and the maximum tolerated dose evaluation to determine their toxicities in comparison to their antitumor activities. In this presentation, we describe the strengths and limitations of each of our assays and also our data with the leading compounds from the previous screening. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5504.