Intracerebroventricular transplantation of human iPSC-derived neural stem cells (hiPSC-NSCs) into neonatal mice.

Human neural stem cells (hNSCs) hold great promises for the development of cell-based therapies for neurodegenerative diseases, given their capability to provide immunomodulatory and trophic support and to replace, to a limited extent, damaged, or lost cells. Human NSCs are under clinical evaluation for the treatment of several neurodegenerative diseases. Still, issues related to the large-scale production of clinical-grade fetal hNSCs and their allogeneic nature-requiring immunosuppressive regimens-have hampered their full exploitation as therapeutics. NSCs derived from human induced pluripotent stem cells (hiPSCs) provide a valuable alternative to fetal hNSCs since they can be generated from autologous or HLA-matched donors expanded for large-scale clinical-grade production, and are amenable for gene addition/gene editing strategies, thus potentially addressing CNS diseases of genetic origin. The prospective use of hiPSC-derived NSCs (hiPSC-NSCs) for CNS-directed therapies demands a careful evaluation of the efficacy and safety of these cell populations in animal models. Here, we describe a protocol for the transplantation and phenotypical characterization of hiPSC-NSCs in neonatal immunodeficient mice. This protocol is relevant to assessing the safety and the efficacy of hiPSC-NSC transplantation to target early-onset neurodegenerative or demyelinating CNS diseases.

Generation of neuronal/glial mixed cultures from human induced pluripotent stem cells (hiPSCs).

For a long time, the understanding of neurological diseases has been limited by the lack of representative experimental models able to recapitulate essential features of the human pathologies. Human induced pluripotent stem cells (hiPSCs) have emerged as a powerful tool for disease modeling, drug screening, and the development of novel cell and gene therapies. A critical issue for the prospective use of hiPSCs in basic and translational research for central nervous system (CNS) disorders is to validate robust protocols able to efficiently differentiate pluripotent cells into neurons and glial cells of interest, specifically those that are most affected in pathological conditions. We describe here a three-step differentiation protocol optimized for feeder-free hiPSCs. The protocol includes a first step of neural induction mediated by dual SMAD inhibition to generate homogeneous populations of neural progenitor cells (NPCs), a second step of NPCs expansion, and a third phase of NPCs differentiation into a mixed culture of neurons, oligodendrocytes, and astrocytes. This experimental platform is relevant to recapitulate the neural induction of hiPSCs and to monitor NPC lineage specification and neuronal/glial differentiation in physiological conditions as well as in the context of CNS diseases. The protocol allows monitoring early pathological hallmarks in the different CNS cell types, also offering a simplified in vitro model to study the neuronal-glial crosstalk.

Therapeutic advantages of combined gene/cell therapy strategies in a murine model of GM2 gangliosidosis.

Genetic deficiency of ß-N-acetylhexosaminidase (Hex) functionality leads to accumulation of GM2 ganglioside in Tay-Sachs disease and Sandhoff disease (SD), which presently lack approved therapies. Current experimental gene therapy (GT) approaches with adeno-associated viral vectors (AAVs) still pose safety and efficacy issues, supporting the search for alternative therapeutic strategies. Here we leveraged the lentiviral vector (LV)-mediated intracerebral (IC) GT platform to deliver Hex genes to the CNS and combined this strategy with bone marrow transplantation (BMT) to provide a timely, pervasive, and long-lasting source of the Hex enzyme in the CNS and periphery of SD mice. Combined therapy outperformed individual treatments in terms of lifespan extension and normalization of the neuroinflammatory/neurodegenerative phenotypes of SD mice. These benefits correlated with a time-dependent increase in Hex activity and a remarkable reduction in GM2 storage in brain tissues that single treatments failed to achieve. Our results highlight the synergic mode of action of LV-mediated IC GT and BMT, clarify the contribution of treatments to the therapeutic outcome, and inform on the realistic threshold of corrective enzymatic activity. These results have important implications for interpretation of ongoing experimental therapies and for design of more effective treatment strategies for GM2 gangliosidosis.

DNA damage contributes to neurotoxic inflammation in Aicardi-Goutières syndrome astrocytes.

Aberrant induction of type I IFN is a hallmark of the inherited encephalopathy Aicardi-Goutières syndrome (AGS), but the mechanisms triggering disease in the human central nervous system (CNS) remain elusive. Here, we generated human models of AGS using genetically modified and patient-derived pluripotent stem cells harboring TREX1 or RNASEH2B loss-of-function alleles. Genome-wide transcriptomic analysis reveals that spontaneous proinflammatory activation in AGS astrocytes initiates signaling cascades impacting multiple CNS cell subsets analyzed at the single-cell level. We identify accumulating DNA damage, with elevated R-loop and micronuclei formation, as a driver of STING- and NLRP3-related inflammatory responses leading to the secretion of neurotoxic mediators. Importantly, pharmacological inhibition of proapoptotic or inflammatory cascades in AGS astrocytes prevents neurotoxicity without apparent impact on their increased type I IFN responses. Together, our work identifies DNA damage as a major driver of neurotoxic inflammation in AGS astrocytes, suggests a role for AGS gene products in R-loop homeostasis, and identifies common denominators of disease that can be targeted to prevent astrocyte-mediated neurotoxicity in AGS.

Isolation and Culture of Neural Stem/Progenitor Cells from the Postnatal Periventricular Region.

Due to the complexity of the neural stem cell (NSC) niche organization, the lack of specific NSC markers, and the difficulty of long-term tracking these cells and their progeny in vivo, the functional properties of the endogenous NSCs remain largely unexplored. These limitations have led to the development of methodologies to efficiently isolate, expand, and differentiate NSCs ex vivo. We describe here the peculiarities of the neurosphere assay (NSA) as a methodology that allows to efficiently isolate, expand, and differentiate somatic NSCs derived from the postnatal and adult forebrain periventricular region while preserving proliferation, self-renewal, and multipotency, the main attributes that provide their functional identification.

Delivery Platforms for CRISPR/Cas9 Genome Editing of Glial Cells in the Central Nervous System.

Glial cells (astrocytes, oligodendrocytes, and microglia) are emerging as key players in several physiological and pathological processes of the central nervous system (CNS). Astrocytes and oligodendrocytes are not only supportive cells that release trophic factors or regulate energy metabolism, but they also actively modulate critical neuronal processes and functions in the tripartite synapse. Microglia are defined as CNS-resident cells that provide immune surveillance; however, they also actively contribute to shaping the neuronal microenvironment by scavenging cell debris or regulating synaptogenesis and pruning. Given the many interconnected processes coordinated by glial cells, it is not surprising that both acute and chronic CNS insults not only cause neuronal damage but also trigger complex multifaceted responses, including neuroinflammation, which can critically contribute to the disease progression and worsening of symptoms in several neurodegenerative diseases. Overall, this makes glial cells excellent candidates for targeted therapies to treat CNS disorders. In recent years, the application of gene editing technologies has redefined therapeutic strategies to treat genetic and age-related neurological diseases. In this review, we discuss the advantages and limitations of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based gene editing in the treatment of neurodegenerative disorders, focusing on the development of viral- and nanoparticle-based delivery methods for in vivo glial cell targeting.

Human iPSC-based neurodevelopmental models of globoid cell leukodystrophy uncover patient- and cell type-specific disease phenotypes.

Globoid cell leukodystrophy (GLD) is a rare neurodegenerative lysosomal storage disease caused by an inherited deficiency of ß-galactocerebrosidase (GALC). GLD pathogenesis and therapeutic correction have been poorly studied in patient neural cells. Here, we investigated the impact of GALC deficiency and lentiviral vector-mediated GALC rescue/overexpression in induced pluripotent stem cell (iPSC)-derived neural progenitors and neuronal/glial progeny obtained from two GLD patients. GLD neural progeny displayed progressive psychosine storage, oligodendroglial and neuronal defects, unbalanced lipid composition, and early activation of cellular senescence, depending on the disease-causing mutation. The partial rescue of the neural differentiation program upon GALC reconstitution and psychosine clearance suggests multiple mechanisms contributing to neural pathology in GLD. Also, the pathological phenotype associated to supraphysiological GALC levels highlights the need of regulated GALC expression for proper human neural commitment/differentiation. These data have important implications for establishing safe therapeutic strategies to enhance disease correction of GLD.

In vivo Gene Therapy to the Liver and Nervous System: Promises and Challenges.

In vivo genetic engineering has recently shown remarkable potential as a novel effective treatment for an ever-growing number of diseases, as also witnessed by the recent marketing authorization of several in vivo gene therapy products. In vivo genetic engineering comprises both viral vector-mediated gene transfer and the more recently developed genome/epigenome editing strategies, as long as they are directly administered to patients. Here we first review the most advanced in vivo gene therapies that are commercially available or in clinical development. We then highlight the major challenges to be overcome to fully and broadly exploit in vivo gene therapies as novel medicines, discussing some of the approaches that are being taken to address them, with a focus on the nervous system and liver taken as paradigmatic examples.

Human iPSC-Based Models for the Development of Therapeutics Targeting Neurodegenerative Lysosomal Storage Diseases.

Lysosomal storage diseases (LSDs) are a group of rare genetic conditions. The absence or deficiency of lysosomal proteins leads to excessive storage of undigested materials and drives secondary pathological mechanisms including autophagy, calcium homeostasis, ER stress, and mitochondrial abnormalities. A large number of LSDs display mild to severe central nervous system (CNS) involvement. Animal disease models and post-mortem tissues partially recapitulate the disease or represent the final stage of CNS pathology, respectively. In the last decades, human models based on induced pluripotent stem cells (hiPSCs) have been extensively applied to investigate LSD pathology in several tissues and organs, including the CNS. Neural stem/progenitor cells (NSCs) derived from patient-specific hiPSCs (hiPS-NSCs) are a promising tool to define the effects of the pathological storage on neurodevelopment, survival and function of neurons and glial cells in neurodegenerative LSDs. Additionally, the development of novel 2D co-culture systems and 3D hiPSC-based models is fostering the investigation of neuron-glia functional and dysfunctional interactions, also contributing to define the role of neurodevelopment and neuroinflammation in the onset and progression of the disease, with important implications in terms of timing and efficacy of treatments. Here, we discuss the advantages and limits of the application of hiPS-NSC-based models in the study and treatment of CNS pathology in different LSDs. Additionally, we review the state-of-the-art and the prospective applications of NSC-based therapy, highlighting the potential exploitation of hiPS-NSCs for gene and cell therapy approaches in the treatment of neurodegenerative LSDs.

In vitro Validation of Chimeric ß-Galactosylceramidase Enzymes With Improved Enzymatic Activity and Increased Secretion.

Globoid Cell Leukodystrophy (GLD) is a lysosomal storage disease (LSD) caused by inherited defects of the ß-galactosylceramidase (GALC) gene. The infantile forms display a rapid and aggressive central and peripheral nervous system (CNS and PNS) dysfunction. No treatments are available for GLD patients. Effective gene therapy (GT) strategies for GLD require a safe and widespread delivery of the functional GALC enzyme to all affected tissues/organs, and particularly to the CNS. The use of chimeric lysosomal enzymes with increased secretion and enhanced transport across the blood-brain barrier (BBB) that boost the efficacy of GT approaches in pre-clinical models of similar neurodegenerative LSDs may benefit GLD as well. Here, we tested the safety and biological efficacy of chimeric GALC enzymes engineered to express an alternative signal peptide (iduronate-2-sulfatase - IDSsp) and the low-density lipoprotein receptor (LDLr)-binding domain from the Apolipoprotein E II (ApoE II) in GLD murine neural and hematopoietic stem/progenitor cells and progeny, which are relevant cells types in the context of in vivo and ex vivo GT platforms. We show that the lentiviral vector-mediated expression of the chimeric GALC enzymes is safe and leads to supranormal enzymatic activity in both neural and hematopoietic cells. The IDSsp.GALC shows enhanced expression and secretion in comparison to the unmodified GALC. The chimeric GALC enzymes produced by LV-transduced cells reduce intracellular galactosylceramide (GalCer) storage and effectively cross-correct GLD murine neurons and glial cells, indicating that the transgenic enzymes are delivered to lysosomes, efficiently secreted, and functional. Of note, the expression of LDLr and LDLr-related proteins in GLD neurons and glial cells supports the exploitation of this system to enhance the GALC supply in affected CNS cells and tissues. These in vitro studies support the use of chimeric GALC enzymes to develop novel and more effective GT approaches for GLD.

Novel bicistronic lentiviral vectors correct ß-Hexosaminidase deficiency in neural and hematopoietic stem cells and progeny: implications for in vivo and ex vivo gene therapy of GM2 gangliosidosis.

The favorable outcome of in vivo and ex vivo gene therapy approaches in several Lysosomal Storage Diseases suggests that these treatment strategies might equally benefit GM2 gangliosidosis. Tay-Sachs and Sandhoff disease (the main forms of GM2 gangliosidosis) result from mutations in either the HEXA or HEXB genes encoding, respectively, the α- or ß-subunits of the lysosomal ß-Hexosaminidase enzyme. In physiological conditions, α- and ß-subunits combine to generate ß-Hexosaminidase A (HexA, αß) and ß-Hexosaminidase B (HexB, ßß). A major impairment to establishing in vivo or ex vivo gene therapy for GM2 gangliosidosis is the need to synthesize the α- and ß-subunits at high levels and with the correct stoichiometric ratio, and to safely deliver the therapeutic products to all affected tissues/organs. Here, we report the generation and in vitro validation of novel bicistronic lentiviral vectors (LVs) encoding for both the murine and human codon optimized Hexa and Hexb genes. We show that these LVs drive the safe and coordinate expression of the α- and ß-subunits, leading to supranormal levels of ß-Hexosaminidase activity with prevalent formation of a functional HexA in SD murine neurons and glia, murine bone marrow-derived hematopoietic stem/progenitor cells (HSPCs), and human SD fibroblasts. The restoration/overexpression of ß-Hexosaminidase leads to the reduction of intracellular GM2 ganglioside storage in transduced and in cross-corrected SD murine neural progeny, indicating that the transgenic enzyme is secreted and functional. Importantly, bicistronic LVs safely and efficiently transduce human neurons/glia and CD34+ HSPCs, which are target and effector cells, respectively, in prospective in vivo and ex vivo GT approaches. We anticipate that these bicistronic LVs may overcome the current requirement of two vectors co-delivering the α- or ß-subunits genes. Careful assessment of the safety and therapeutic potential of these bicistronic LVs in the SD murine model will pave the way to the clinical development of LV-based gene therapy for GM2 gangliosidosis.

Polycystin-1 Regulates Actomyosin Contraction and the Cellular Response to Extracellular Stiffness.

Polycystin-1 (PC-1) and 2 (PC-2) are the products of the PKD1 and PKD2 genes, which are mutated in Autosomal Dominant Polycystic Kidney Disease (ADPKD). They form a receptor/channel complex that has been suggested to function as a mechanosensor, possibly activated by ciliary bending in the renal tubule, and resulting in calcium influx. This model has recently been challenged, leaving the question as to which mechanical stimuli activate the polycystins still open. Here, we used a SILAC/Mass-Spec approach to identify intracellular binding partners of tagged-endogenous PC-1 whereby we detected a class of interactors mediating regulation of cellular actomyosin contraction. Accordingly, using gain and loss-of-function cellular systems we found that PC-1 negatively regulates cellular contraction and YAP activation in response to extracellular stiffness. Thus, PC-1 enables cells to sense the rigidity of the extracellular milieu and to respond appropriately. Of note, in an orthologous murine model of PKD we found evidence of increased actomyosin contraction, leading to enhanced YAP nuclear translocation and transcriptional activity. Finally, we show that inhibition of ROCK-dependent actomyosin contraction by Fasudil reversed YAP activation and significantly improved disease progression, in line with recent studies. Our data suggest a possible direct role of PC-1 as a mechanosensor of extracellular stiffness.

Multicenter, randomised, open-label, non-comparative phase 2 trial on the efficacy and safety of the combination of bevacizumab and trabectedin with or without carboplatin in women with partially platinum-sensitive recurrent ovarian cancer.

BACKGROUND: Trabectedin, in addition to its antiproliferative effect, can modify the tumour microenvironment and this could be synergistic with bevacizumab. The efficacy and safety of trabectedin and bevacizumab ± carboplatin have never been investigated. METHODS: In this phase 2 study, women progressing between 6 and 12 months since their last platinum-based therapy were randomised to Arm BT: bevacizumab, trabectedin every 21 days, or Arm BT+C: bevacizumab, trabectedin and carboplatin every 28 days, from cycles 1 to 6, then trabectedin and bevacizumab as in Arm BT. Primary endpoints were progression-free survival rate (PFS-6) and severe toxicity rate (ST-6) at 6 months, assuming a PFS-6 ≤35% for BT and ≤40% for BT+C as not of therapeutic interest and, for both arms, a ST-6 ≥ 30% as unacceptable. RESULTS: BT+C (21 patients) did not meet the safety criteria for the second stage (ST-6 45%; 95%CI: 23%-69%) but PFS-6 was 85% (95%CI: 62%-97%). BT (50 patients) had 75% PFS-6 (95%CI: 60%-87%) and 16% ST-6 (95%CI 7%-30%). CONCLUSIONS: BT compared favourably with other platinum- and non-platinum-based regimens. The combination with carboplatin needs to be assessed further in a re-modulated safer schedule to confirm its apparent strong activity. CLINICAL TRIAL REGISTRATION: NCT01735071 (Clinicaltrials.gov).

Integrated Computational Analysis Highlights unique miRNA Signatures in the Subventricular Zone and Striatum of GM2 Gangliosidosis Animal Models.

This work explores for the first time the potential contribution of microRNAs (miRNAs) to the pathophysiology of the GM2 gangliosidosis, a group of Lysosomal Storage Diseases. In spite of the genetic origin of GM2 gangliosidosis, the cascade of events leading from the gene/protein defects to the cell dysfunction and death is not fully elucidated. At present, there is no cure for patients. Taking advantage of the animal models of two forms of GM2 gangliosidosis, Tay-Sachs (TSD) and Sandhoff (SD) diseases, we performed a microRNA screening in the brain subventricular zone (SVZ) and striatum (STR), which feature the neurogenesis and neurodegeneration states, respectively, in adult mutant mice. We found abnormal expression of a panel of miRNAs involved in lipid metabolism, CNS development and homeostasis, and neuropathological processes, highlighting region- and disease-specific profiles of miRNA expression. Moreover, by using a computational analysis approach, we identified a unique disease- (SD or TSD) and brain region-specific (SVZ vs. STR) miRNAs signatures of predicted networks potentially related to the pathogenesis of the diseases. These results may contribute to the understanding of GM2 gangliosidosis pathophysiology, with the aim of developing effective treatments.

Human iPSC-based models highlight defective glial and neuronal differentiation from neural progenitor cells in metachromatic leukodystrophy.

The pathological cascade leading from primary storage to neural cell dysfunction and death in metachromatic leukodystrophy (MLD) has been poorly elucidated in human-derived neural cell systems. In the present study, we have modeled the progression of pathological events during the differentiation of patient-specific iPSCs to neuroepithelial progenitor cells (iPSC-NPCs) and mature neurons, astrocytes, and oligodendrocytes at the morphological, molecular, and biochemical level. We showed significant sulfatide accumulation and altered sulfatide composition during the differentiation of MLD iPSC-NPCs into neuronal and glial cells. Changes in sulfatide levels and composition were accompanied by the expansion of the lysosomal compartment, oxidative stress, and apoptosis. The neuronal and glial differentiation capacity of MLD iPSC-NPCs was significantly impaired. We showed delayed appearance and/or reduced levels of oligodendroglial and astroglial markers as well as reduced number of neurons and disorganized neuronal network. Restoration of a functional Arylsulfatase A (ARSA) enzyme in MLD cells using lentiviral-mediated gene transfer normalized sulfatide levels and composition, globally rescuing the pathological phenotype. Our study points to MLD iPSC-derived neural progeny as a useful in vitro model to assess the impact of ARSA deficiency along NPC differentiation into neurons and glial cells. In addition, iPSC-derived neural cultures allowed testing the impact of ARSA reconstitution/overexpression on disease correction and, importantly, on the biology and functional features of human NPCs, with important therapeutic implications.

Generation of Human Induced Pluripotent Stem Cell-Derived Bona Fide Neural Stem Cells for Ex Vivo Gene Therapy of Metachromatic Leukodystrophy.

Allogeneic fetal-derived human neural stem cells (hfNSCs) that are under clinical evaluation for several neurodegenerative diseases display a favorable safety profile, but require immunosuppression upon transplantation in patients. Neural progenitors derived from patient-specific induced pluripotent stem cells (iPSCs) may be relevant for autologous ex vivo gene-therapy applications to treat genetic diseases with unmet medical need. In this scenario, obtaining iPSC-derived neural stem cells (NSCs) showing a reliable "NSC signature" is mandatory. Here, we generated human iPSC (hiPSC) clones via reprogramming of skin fibroblasts derived from normal donors and patients affected by metachromatic leukodystrophy (MLD), a fatal neurodegenerative lysosomal storage disease caused by genetic defects of the arylsulfatase A (ARSA) enzyme. We differentiated hiPSCs into NSCs (hiPS-NSCs) sharing molecular, phenotypic, and functional identity with hfNSCs, which we used as a "gold standard" in a side-by-side comparison when validating the phenotype of hiPS-NSCs and predicting their performance after intracerebral transplantation. Using lentiviral vectors, we efficiently transduced MLD hiPSCs, achieving supraphysiological ARSA activity that further increased upon neural differentiation. Intracerebral transplantation of hiPS-NSCs into neonatal and adult immunodeficient MLD mice stably restored ARSA activity in the whole central nervous system. Importantly, we observed a significant decrease of sulfatide storage when ARSA-overexpressing cells were used, with a clear advantage in those mice receiving neonatal as compared with adult intervention. Thus, we generated a renewable source of ARSA-overexpressing iPSC-derived bona fide hNSCs with improved features compared with clinically approved hfNSCs. Patient-specific ARSA-overexpressing hiPS-NSCs may be used in autologous ex vivo gene therapy protocols to provide long-lasting enzymatic supply in MLD-affected brains. Stem Cells Translational Medicine 2017;6:352-368.

Two factor-based reprogramming of rodent and human fibroblasts into Schwann cells.

Schwann cells (SCs) generate the myelin wrapping of peripheral nerve axons and are promising candidates for cell therapy. However, to date a renewable source of SCs is lacking. In this study, we show the conversion of skin fibroblasts into induced Schwann cells (iSCs) by driving the expression of two transcription factors, Sox10 and Egr2. iSCs resembled primary SCs in global gene expression profiling and PNS identity. In vitro, iSCs wrapped axons generating compact myelin sheaths with regular nodal structures. Conversely, iSCs from Twitcher mice showed a severe loss in their myelinogenic potential, demonstrating that iSCs can be an attractive system for in vitro modelling of PNS diseases. The same two factors were sufficient to convert human fibroblasts into iSCs as defined by distinctive molecular and functional traits. Generating iSCs through direct conversion of somatic cells offers opportunities for in vitro disease modelling and regenerative therapies.

Oligophrenin-1 regulates number, morphology and synaptic properties of adult-born inhibitory interneurons in the olfactory bulb.

Among the X-linked genes associated with intellectual disability, Oligophrenin-1 (OPHN1) encodes for a Rho GTPase-activating protein, a key regulator of several developmental processes, such as dendrite and spine formation and synaptic activity. Inhibitory interneurons play a key role in the development and function of neuronal circuits. Whether a mutation of OPHN1 can affect morphology and synaptic properties of inhibitory interneurons remains poorly understood. To address these open questions, we studied in a well-established mouse model of X-linked intellectual disability, i.e. a line of mice carrying a null mutation of OPHN1, the development and function of adult generated inhibitory interneurons in the olfactory bulb. Combining quantitative morphological analysis and electrophysiological recordings we found that the adult generated inhibitory interneurons were dramatically reduced in number and exhibited a higher proportion of filopodia-like spines, with the consequences on their synaptic function, in OPHN1 ko mice. Furthermore, we found that olfactory behaviour was perturbed in OPHN1 ko mice. Chronic treatment with a Rho kinase inhibitor rescued most of the defects of the newly generated neurons. Altogether, our data indicated that OPHN1 plays a key role in regulating the number, morphology and function of adult-born inhibitory interneurons and contributed to identify potential therapeutic targets.

Perspective on innovative therapies for globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD), or Krabbe's disease, is a lysosomal storage disorder resulting from deficiency of the lysosomal hydrolase galactosylceramidase. The infantile forms are characterized by a unique relentless and aggressive progression with a wide range of neurological symptoms and complications. Here we review and discuss the basic concepts and the novel mechanisms identified as key contributors to the peculiar GLD pathology, highlighting their therapeutic implications. Then, we evaluate evidence from extensive experimental studies on GLD animal models that have highlighted fundamental requirements to obtain substantial therapeutic benefit, including early and timely intervention, high levels of enzymatic reconstitution, and global targeting of affected tissues. Continuous efforts in understanding GLD pathophysiology, the interplay between various therapies, and the mechanisms of disease correction upon intervention may allow advancing research with innovative approaches and prioritizing treatment strategies to develop more efficacious treatments. © 2016 Wiley Periodicals, Inc.