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  1. Fulltext AAV9 transduction mediated by systemic delivery of vector via retro-orbital injection in newborn, neonatal and juvenile mice
    Prabhakar S, Lule S, da Hora CC, Breakefield XO, Cheah PS
    Exp Anim, 2021 Nov 10;70(4):450-458.
    PMID: 34039790 DOI: 10.1538/expanim.20-0186
    Adeno-associated virus (AAV)-based gene therapy is gaining popularity owing to its excellent safety profile and effective therapeutic outcomes in a number of diseases. Intravenous (IV) injection of AAV into the tail vein, facial vein and retro-orbital (RO) venous sinus have all been useful strategies to infuse the viral vector systemically. However, tail vein injection is technically challenging in juvenile mice, and injection at young ages (≤ postnatal day-(P)21) is essentially impossible. The temporal or facial vein is localized anterior to the ear bud and is markedly visible in the first couple of days postnatally. However, this method is age-dependent and requires a dissecting microscope. Retro-orbital injection (ROI), on the other hand, is suitable for all murine ages, including newborn and older mice, and is relatively less stressful to animals compared to tail vein injection. Although many reports have shown ROI as an effective route of AAV delivery, herein we aim to highlight and summarize the methods and benefits of ROI. To capture the full spectrum of transduction efficiency mediated by ROI, we transduced the editing-dependent reporter mice (Ai9 Cre reporter mice) with the AAV9 vector, which targets a wide range of peripheral tissues with exceptional brain tropism. We also provide a comprehensive description of the ROI technique to facilitate viral vector administration without complications.
  2. Fulltext Long-Term Therapeutic Efficacy of Intravenous AAV-Mediated Hamartin Replacement in Mouse Model of Tuberous Sclerosis Type 1
    Prabhakar S, Cheah PS, Zhang X, Zinter M, Gianatasio M, Hudry E, et al.
    Mol Ther Methods Clin Dev, 2019 Dec 13;15:18-26.
    PMID: 31534984 DOI: 10.1016/j.omtm.2019.08.003
    Tuberous sclerosis complex (TSC) is a tumor suppressor syndrome caused by mutations in TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins act as a complex that inhibits mammalian target of rapamycin (mTOR)-mediated cell growth and proliferation. Loss of either protein leads to overgrowth in many organs, including subependymal nodules, subependymal giant cell astrocytomas, and cortical tubers in the human brain. Neurological manifestations in TSC include intellectual disability, autism, hydrocephalus, and epilepsy. In a stochastic mouse model of TSC1 brain lesions, complete loss of Tsc1 is achieved in homozygous Tsc1-floxed mice in a subpopulation of neural cells in the brain by intracerebroventricular (i.c.v.) injection at birth of an adeno-associated virus (AAV) vector encoding Cre recombinase. This results in median survival of 38 days and brain pathology, including subependymal lesions and enlargement of neuronal cells. Remarkably, when these mice were injected intravenously on day 21 with an AAV9 vector encoding hamartin, most survived at least up to 429 days in apparently healthy condition with marked reduction in brain pathology. Thus, a single intravenous administration of an AAV vector encoding hamartin restored protein function in enough cells in the brain to extend lifespan in this TSC1 mouse model.
  3. Fulltext Glioma-Derived miRNA-Containing Extracellular Vesicles Induce Angiogenesis by Reprogramming Brain Endothelial Cells
    Lucero R, Zappulli V, Sammarco A, Murillo OD, Cheah PS, Srinivasan S, et al.
    Cell Rep, 2020 02 18;30(7):2065-2074.e4.
    PMID: 32075753 DOI: 10.1016/j.celrep.2020.01.073
    Glioblastoma (GBM) is characterized by aberrant vascularization and a complex tumor microenvironment. The failure of anti-angiogenic therapies suggests pathways of GBM neovascularization, possibly attributable to glioblastoma stem cells (GSCs) and their interplay with the tumor microenvironment. It has been established that GSC-derived extracellular vesicles (GSC-EVs) and their cargoes are proangiogenic in vitro. To further elucidate EV-mediated mechanisms of neovascularization in vitro, we perform RNA-seq and DNA methylation profiling of human brain endothelial cells exposed to GSC-EVs. To correlate these results to tumors in vivo, we perform histoepigenetic analysis of GBM molecular profiles in the TCGA collection. Remarkably, GSC-EVs and normal vascular growth factors stimulate highly distinct gene regulatory responses that converge on angiogenesis. The response to GSC-EVs shows a footprint of post-transcriptional gene silencing by EV-derived miRNAs. Our results provide insights into targetable angiogenesis pathways in GBM and miRNA candidates for liquid biopsy biomarkers.
  4. Fulltext CRISPR-Cas knockout of miR21 reduces glioma growth
    Nieland L, van Solinge TS, Cheah PS, Morsett LM, El Khoury J, Rissman JI, et al.
    Mol Ther Oncolytics, 2022 Jun 16;25:121-136.
    PMID: 35572197 DOI: 10.1016/j.omto.2022.04.001
    Non-coding RNAs, including microRNAs (miRNAs), support the progression of glioma. miR-21 is a small, non-coding transcript involved in regulating gene expression in multiple cellular pathways, including the regulation of proliferation. High expression of miR-21 has been shown to be a major driver of glioma growth. Manipulating the expression of miRNAs is a novel strategy in the development of therapeutics in cancer. In this study we aimed to target miR-21. Using CRISPR genome-editing technology, we disrupted the miR-21 coding sequences in glioma cells. Depletion of this miRNA resulted in the upregulation of many downstream miR-21 target mRNAs involved in proliferation. Phenotypically, CRISPR-edited glioma cells showed reduced migration, invasion, and proliferation in vitro. In immunocompetent mouse models, miR-21 knockout tumors showed reduced growth resulting in an increased overall survival. In summary, we show that by knocking out a key miRNA in glioma, these cells have decreased proliferation capacity both in vitro and in vivo. Overall, we identified miR-21 as a potential target for CRISPR-based therapeutics in glioma.
  5. Fulltext Glioblastoma-Associated Microglia Reprogramming Is Mediated by Functional Transfer of Extracellular miR-21
    Abels ER, Maas SLN, Nieland L, Wei Z, Cheah PS, Tai E, et al.
    Cell Rep, 2019 09 17;28(12):3105-3119.e7.
    PMID: 31533034 DOI: 10.1016/j.celrep.2019.08.036
    Gliomas are primary, diffusely infiltrating brain tumors. Microglia are innate immune cells in the CNS and make up a substantial portion of the tumor mass. Glioma cells shape their microenvironment, communicating with and reprogramming surrounding cells, resulting in enhanced angiogenesis, immune suppression, and remodeling of the extracellular matrix. Glioma cells communicate with microglia, in part by releasing extracellular vesicles (EVs). Mouse glioma cells stably expressing a palmitoylated GFP to label EVs were implanted intracranially into syngeneic miR-21-null mice. Here, we demonstrate functional delivery of miR-21, regulating specific downstream mRNA targets in microglia after uptake of tumor-derived EVs. These findings attest to EV-dependent microRNA delivery as studied in an in vivo-based model and provide insight into the reprograming of microglial cells by tumor cells to create a favorable microenvironment for cancer progression.
  6. Fulltext Gene therapy for tuberous sclerosis complex type 2 in a mouse model by delivery of AAV9 encoding a condensed form of tuberin
    Cheah PS, Prabhakar S, Yellen D, Beauchamp RL, Zhang X, Kasamatsu S, et al.
    Sci Adv, 2021 Jan;7(2).
    PMID: 33523984 DOI: 10.1126/sciadv.abb1703
    Tuberous sclerosis complex (TSC) results from loss of a tumor suppressor gene - TSC1 or TSC2, encoding hamartin and tuberin, respectively. These proteins formed a complex to inhibit mTORC1-mediated cell growth and proliferation. Loss of either protein leads to overgrowth lesions in many vital organs. Gene therapy was evaluated in a mouse model of TSC2 using an adeno-associated virus (AAV) vector carrying the complementary for a "condensed" form of human tuberin (cTuberin). Functionality of cTuberin was verified in culture. A mouse model of TSC2 was generated by AAV-Cre recombinase disruption of Tsc2-floxed alleles at birth, leading to a shortened lifespan (mean 58 days) and brain pathology consistent with TSC. When these mice were injected intravenously on day 21 with AAV9-cTuberin, the mean survival was extended to 462 days with reduction in brain pathology. This demonstrates the potential of treating life-threatening TSC2 lesions with a single intravenous injection of AAV9-cTuberin.
  7. Fulltext Gene replacement therapy in a schwannoma mouse model of neurofibromatosis type 2
    Prabhakar S, Beauchamp RL, Cheah PS, Yoshinaga A, Haidar EA, Lule S, et al.
    Mol Ther Methods Clin Dev, 2022 Sep 08;26:169-180.
    PMID: 35846573 DOI: 10.1016/j.omtm.2022.06.012
    Loss of function of the neurofibromatosis type 2 (NF2) tumor suppressor gene leads to the formation of schwannomas, meningiomas, and ependymomas, comprising ∼50% of all sporadic cases of primary nervous system tumors. NF2 syndrome is an autosomal dominant condition, with bi-allelic inactivation of germline and somatic alleles resulting in loss of function of the encoded protein merlin and activation of mammalian target of rapamycin (mTOR) pathway signaling in NF2-deficient cells. Here we describe a gene replacement approach through direct intratumoral injection of an adeno-associated virus vector expressing merlin in a novel human schwannoma model in nude mice. In culture, the introduction of an AAV1 vector encoding merlin into CRISPR-modified human NF2-null arachnoidal cells (ACs) or Schwann cells (SCs) was associated with decreased size and mTORC1 pathway activation consistent with restored merlin activity. In vivo, a single injection of AAV1-merlin directly into human NF2-null SC-derived tumors growing in the sciatic nerve of nude mice led to regression of tumors over a 10-week period, associated with a decrease in dividing cells and an increase in apoptosis, in comparison with vehicle. These studies establish that merlin re-expression via gene replacement in NF2-null schwannomas is sufficient to cause tumor regression, thereby potentially providing an effective treatment for NF2.
  8. Fulltext Methods for Systematic Identification of Membrane Proteins for Specific Capture of Cancer-Derived Extracellular Vesicles
    Zaborowski MP, Lee K, Na YJ, Sammarco A, Zhang X, Iwanicki M, et al.
    Cell Rep, 2019 Apr 02;27(1):255-268.e6.
    PMID: 30943406 DOI: 10.1016/j.celrep.2019.03.003
    Analysis of cancer-derived extracellular vesicles (EVs) in biofluids potentially provides a source of disease biomarkers. At present there is no procedure to systematically identify which antigens should be targeted to differentiate cancer-derived from normal host cell-derived EVs. Here, we propose a computational framework that integrates information about membrane proteins in tumors and normal tissues from databases: UniProt, The Cancer Genome Atlas, the Genotype-Tissue Expression Project, and the Human Protein Atlas. We developed two methods to assess capture of EVs from specific cell types. (1) We used palmitoylated fluorescent protein (palmtdTomato) to label tumor-derived EVs. Beads displaying antibodies of interest were incubated with conditioned medium from palmtdTomato-expressing cells. Bound EVs were quantified using flow cytometry. (2) We also showed that membrane-bound Gaussia luciferase allows the detection of cancer-derived EVs in blood of tumor-bearing animals. Our analytical and validation platform should be applicable to identify antigens on EVs from any tumor type.
  9. Fulltext Membrane-bound Gaussia luciferase as a tool to track shedding of membrane proteins from the surface of extracellular vesicles
    Zaborowski MP, Cheah PS, Zhang X, Bushko I, Lee K, Sammarco A, et al.
    Sci Rep, 2019 Nov 22;9(1):17387.
    PMID: 31758005 DOI: 10.1038/s41598-019-53554-y
    Extracellular vesicles (EVs) released by cells play a role in intercellular communication. Reporter and targeting proteins can be modified and exposed on the surface of EVs to investigate their half-life and biodistribution. A characterization of membrane-bound Gaussia luciferase (mbGluc) revealed that its signal was detected also in a form smaller than common EVs (<70 nm). We demonstrated that mbGluc initially exposed on the surface of EVs, likely undergoes proteolytic cleavage and processed fragments of the protein are released into the extracellular space in active form. Based on this observation, we developed a new assay to quantitatively track shedding of membrane proteins from the surface of EVs. We used this assay to show that ectodomain shedding in EVs is continuous and is mediated by specific proteases, e.g. metalloproteinases. Here, we present a novel tool to study membrane protein cleavage and release using both in vitro and in vivo models.
  10. Fulltext Mutant Allele-Specific CRISPR Disruption in DYT1 Dystonia Fibroblasts Restores Cell Function
    Cruz L, György B, Cheah PS, Kleinstiver BP, Eimer WA, Garcia SP, et al.
    Mol Ther Nucleic Acids, 2020 Sep 04;21:1-12.
    PMID: 32502938 DOI: 10.1016/j.omtn.2020.05.009
    Most individuals affected with DYT1 dystonia have a heterozygous 3-bp deletion in the TOR1A gene (c.907_909delGAG). The mutation appears to act through a dominant-negative mechanism compromising normal torsinA function, and it is proposed that reducing mutant torsinA may normalize torsinA activity. In this study, we used an engineered Cas9 variant from Streptococcus pyogenes (SpCas9-VRQR) to target the mutation in the TOR1A gene in order to disrupt mutant torsinA in DYT1 patient fibroblasts. Selective targeting of the DYT1 allele was highly efficient with most common non-homologous end joining (NHEJ) edits, leading to a predicted premature stop codon with loss of the torsinA C terminus (delta 302-332 aa). Structural analysis predicted a functionally inactive status of this truncated torsinA due to the loss of residues associated with ATPase activity and binding to LULL1. Immunoblotting showed a reduction of the torsinA protein level in Cas9-edited DYT1 fibroblasts, and a functional assay using HSV infection indicated a phenotypic recovery toward that observed in control fibroblasts. These findings suggest that the selective disruption of the mutant TOR1A allele using CRISPR-Cas9 inactivates mutant torsinA, allowing the remaining wild-type torsinA to exert normal function.
  11. Fulltext Small RNA Sequencing across Diverse Biofluids Identifies Optimal Methods for exRNA Isolation
    Srinivasan S, Yeri A, Cheah PS, Chung A, Danielson K, De Hoff P, et al.
    Cell, 2019 04 04;177(2):446-462.e16.
    PMID: 30951671 DOI: 10.1016/j.cell.2019.03.024
    Poor reproducibility within and across studies arising from lack of knowledge regarding the performance of extracellular RNA (exRNA) isolation methods has hindered progress in the exRNA field. A systematic comparison of 10 exRNA isolation methods across 5 biofluids revealed marked differences in the complexity and reproducibility of the resulting small RNA-seq profiles. The relative efficiency with which each method accessed different exRNA carrier subclasses was determined by estimating the proportions of extracellular vesicle (EV)-, ribonucleoprotein (RNP)-, and high-density lipoprotein (HDL)-specific miRNA signatures in each profile. An interactive web-based application (miRDaR) was developed to help investigators select the optimal exRNA isolation method for their studies. miRDar provides comparative statistics for all expressed miRNAs or a selected subset of miRNAs in the desired biofluid for each exRNA isolation method and returns a ranked list of exRNA isolation methods prioritized by complexity, expression level, and reproducibility. These results will improve reproducibility and stimulate further progress in exRNA biomarker development.
  12. Fulltext TAF1 Transcripts and Neurofilament Light Chain as Biomarkers for X-linked Dystonia-Parkinsonism
    Al Ali J, Vaine CA, Shah S, Campion L, Hakoum A, Supnet ML, et al.
    Mov Disord, 2021 01;36(1):206-215.
    PMID: 32975318 DOI: 10.1002/mds.28305
    BACKGROUND: X-linked dystonia-parkinsonism is a rare neurological disease endemic to the Philippines. Dystonic symptoms appear in males at the mean age of 40 years and progress to parkinsonism with degenerative pathology in the striatum. A retrotransposon inserted in intron 32 of the TAF1 gene leads to alternative splicing in the region and a reduction of the full-length mRNA transcript.

    OBJECTIVES: The objective of this study was to discover cell-based and biofluid-based biomarkers for X-linked dystonia-parkinsonism.

    METHODS: RNA from patient-derived neural progenitor cells and their secreted extracellular vesicles were used to screen for dysregulation of TAF1 expression. Droplet-digital polymerase chain reaction was used to quantify the expression of TAF1 mRNA fragments 5' and 3' to the retrotransposon insertion and the disease-specific splice variant TAF1-32i in whole-blood RNA. Plasma levels of neurofilament light chain were measured using single-molecule array.

    RESULTS: In neural progenitor cells and their extracellular vesicles, we confirmed that the TAF1-3'/5' ratio was lower in patient samples, whereas TAF1-32i expression is higher relative to controls. In whole-blood RNA, both TAF1-3'/5' ratio and TAF1-32i expression can differentiate patient (n = 44) from control samples (n = 18) with high accuracy. Neurofilament light chain plasma levels were significantly elevated in patients (n = 43) compared with both carriers (n = 16) and controls (n = 21), with area under the curve of 0.79.

    CONCLUSIONS: TAF1 dysregulation in blood serves as a disease-specific biomarker that could be used as a readout for monitoring therapies targeting TAF1 splicing. Neurofilament light chain could be used in monitoring neurodegeneration and disease progression in patients. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

  13. Fulltext Discovery and Verification of Extracellular miRNA Biomarkers for Non-invasive Prediction of Pre-eclampsia in Asymptomatic Women
    Srinivasan S, Treacy R, Herrero T, Olsen R, Leonardo TR, Zhang X, et al.
    Cell Rep Med, 2020 05 19;1(2).
    PMID: 32864636 DOI: 10.1016/j.xcrm.2020.100013
    Development of effective prevention and treatment strategies for pre-eclampsia is limited by the lack of accurate methods for identification of at-risk pregnancies. We performed small RNA sequencing (RNA-seq) of maternal serum extracellular RNAs (exRNAs) to discover and verify microRNAs (miRNAs) differentially expressed in patients who later developed pre-eclampsia. Sera collected from 73 pre-eclampsia cases and 139 controls between 17 and 28 weeks gestational age (GA), divided into separate discovery and verification cohorts, are analyzed by small RNA-seq. Discovery and verification of univariate and bivariate miRNA biomarkers reveal that bivariate biomarkers verify at a markedly higher rate than univariate biomarkers. The majority of verified biomarkers contain miR-155-5p, which has been reported to mediate the pre-eclampsia-associated repression of endothelial nitric oxide synthase (eNOS) by tumor necrosis factor alpha (TNF-α). Deconvolution analysis reveals that several verified miRNA biomarkers come from the placenta and are likely carried by placenta-specific extracellular vesicles.
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