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

Ornaghi, Francesca; Sala, Davide; Tedeschi, Fabiana; Maffia, M.; Bazzucchi, Martina; Morena, Francesco; Valsecchi, Manuela; Aureli, Massimo; Martino, Sabata; Gritti, Angela

doi:10.1016/j.nbd.2019.104667

Cited by 23 publications

(30 citation statements)

References 81 publications

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“…Recently, Ornaghi et al, 2020 used lentiviral vectors carrying both α- and β-subunits to transduce HSC isolated from healthy donors, as well as neural stem cells and murine HSC, showing an increase of up to 2-fold in the total Hex activity [ 115 ]. In addition, physiological functions of these stem cells, such as proliferation, self-renewal, or multipotency, remain unchanged, suggesting that ex vivo gene therapy could be an interesting option for the treatment of GM2 gangliosidoses [ 115 ]. Nevertheless, a common challenge in allogeneic HSCT is the graft-versus-host disease (GVHD) [ 116 ].…”

Section: Current Proposals For the Treatment Of Gm2 Gangliosidosesmentioning

confidence: 99%

“…These lentiviral vectors led to a Hex activity up to 5-fold higher than wild-type levels in murine neurons and human stem progenitor cells, as well as a 30–60% reduction of GM2 storage. Similarly, in SD fibroblasts the total Hex and HexA activity increased in an 8:1 ratio, respectively [ 115 ].…”

Section: Current Proposals For the Treatment Of Gm2 Gangliosidosesmentioning

confidence: 99%

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GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies

Leal

Benincore-Flórez

Solano-Galarza

et al. 2020

IJMS

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GM2 gangliosidoses are a group of pathologies characterized by GM2 ganglioside accumulation into the lysosome due to mutations on the genes encoding for the β-hexosaminidases subunits or the GM2 activator protein. Three GM2 gangliosidoses have been described: Tay–Sachs disease, Sandhoff disease, and the AB variant. Central nervous system dysfunction is the main characteristic of GM2 gangliosidoses patients that include neurodevelopment alterations, neuroinflammation, and neuronal apoptosis. Currently, there is not approved therapy for GM2 gangliosidoses, but different therapeutic strategies have been studied including hematopoietic stem cell transplantation, enzyme replacement therapy, substrate reduction therapy, pharmacological chaperones, and gene therapy. The blood–brain barrier represents a challenge for the development of therapeutic agents for these disorders. In this sense, alternative routes of administration (e.g., intrathecal or intracerebroventricular) have been evaluated, as well as the design of fusion peptides that allow the protein transport from the brain capillaries to the central nervous system. In this review, we outline the current knowledge about clinical and physiopathological findings of GM2 gangliosidoses, as well as the ongoing proposals to overcome some limitations of the traditional alternatives by using novel strategies such as molecular Trojan horses or advanced tools of genome editing.

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Section: Current Proposals For the Treatment Of Gm2 Gangliosidosesmentioning

confidence: 99%

Section: Current Proposals For the Treatment Of Gm2 Gangliosidosesmentioning

confidence: 99%

GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies

Leal

Benincore-Flórez

Solano-Galarza

et al. 2020

IJMS

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“…Briefly, gene therapy can be achieved through the use of advanced viral vectors (e.g., lentiviral, adeno-associated, adenoviral, retroviral [80][81][82][83][84][85]) or non-viral vector systems (e.g., liposomes systems) [86] that drive the therapeutic gene to the host cells [87][88][89][90][91]. The safety and efficacy of these strategies may be evaluated in engineered stem cell models, that are suitable for the treatment validation and for the screening of potential mutational insertions caused by the integration of the target gene in the host DNA [92][93][94][95]. Thus, even if the use of animal models is still essential before these treatments reach the clinical phase, stem cells are an alternative models enable to test the effectiveness of treatments in a highly efficient, reproducible and fast manner [84][85][86]90,91,96].…”

Section: Ex Vivo Stem Cell-based Modeling Systemsmentioning

confidence: 99%

Harnessing the Potential of Stem Cells for Disease Modeling: Progress and Promises

Argentati

Tortorella

Bazzucchi

et al. 2020

JPM

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Ex vivo cell/tissue-based models are an essential step in the workflow of pathophysiology studies, assay development, disease modeling, drug discovery, and development of personalized therapeutic strategies. For these purposes, both scientific and pharmaceutical research have adopted ex vivo stem cell models because of their better predictive power. As matter of a fact, the advancing in isolation and in vitro expansion protocols for culturing autologous human stem cells, and the standardization of methods for generating patient-derived induced pluripotent stem cells has made feasible to generate and investigate human cellular disease models with even greater speed and efficiency. Furthermore, the potential of stem cells on generating more complex systems, such as scaffold-cell models, organoids, or organ-on-a-chip, allowed to overcome the limitations of the two-dimensional culture systems as well as to better mimic tissues structures and functions. Finally, the advent of genome-editing/gene therapy technologies had a great impact on the generation of more proficient stem cell-disease models and on establishing an effective therapeutic treatment. In this review, we discuss important breakthroughs of stem cell-based models highlighting current directions, advantages, and limitations and point out the need to combine experimental biology with computational tools able to describe complex biological systems and deliver results or predictions in the context of personalized medicine.Since their establishment, cell cultures have been proven to be the first and most powerful tool for the in vitro investigation of cell biology, from basic research to more complex translational approaches [6]. Classical two-dimensional (2D)-cultures usually consist of a unique type of cells (primary or continuous cultures) that grow in tissue culture polystyrene as adherent monolayers or in floating suspension, both in culture media that provide essential nutrients and growth factors. 2D-strategies are widely used to obtain a large number of cells, thus allowing the in vitro study of molecular mechanisms and development of metabolic assays [7-9]. However, due to their inability to recreate the in vivo complexity of the biological environment, they are not suitable for accurate disease modeling. These limitations have been overcome by the development of three-dimensional (3D)-cell cultures systems [10]. The rationale design of 3D-cell cultures is to harvest cells in microstructures that resemble tissues or organs shape and organization, thus allowing better cell-to-cell/cell-environment contacts and signaling crosstalk [11][12][13][14][15], essential events for tissues correct development and functioning [14,15]. The 3D-cell culture strategy is articulated in many different methods and techniques that can be grouped in scaffold-based or non-scaffold based platforms, each with particular advantages and disadvantages that make them useful for different applications [10,[16][17][18][19]. 3D-cell culture strategies are supported by the in silico...

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“…Lineage negative HSPCs were isolated from TWI and WT adult mice (4-8 weeks) and plated (100,000 cells/cm 2 ) in StemSpan serum-free medium (Stemcell) supplemented with cytokines in the presence of LVs, as previously described (Gentner et al, 2010). After LV transduction (MOI 50 and 100 for 12 h), HSPCs were washed, counted and plated for the CFC assay (4,000 cells/ml in Methocult -StemCell) or to obtain liquid cultures (LC), as previously described (Gentner et al, 2010;Ornaghi et al, 2020). After 14 days, we counted the number of colonies (CFC assay) and evaluated the VCN and enzymatic activity in LC.…”

Section: Differentiation Of Npcs Into Neurons/gliamentioning

confidence: 99%

“…Immunofluorescence analysis of cultured neural cells and brain tissues was performed as previously described (Meneghini et al, 2017;Ornaghi et al, 2020). Primary and secondary antibodies used are listed in Supplementary Table 1.…”

Section: Immunofluorescence (If) Analysismentioning

confidence: 99%

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

Ricca

Cascino

Morena

et al. 2020

Front. Mol. Biosci.

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

Cited by 23 publications

References 81 publications

GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies

GM2 Gangliosidoses: Clinical Features, Pathophysiological Aspects, and Current Therapies

Harnessing the Potential of Stem Cells for Disease Modeling: Progress and Promises

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

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