scholarly journals In Vivo Neuroregeneration to Treat Ischemic Stroke in Adult Non-Human Primate Brains through NeuroD1 AAV-based Gene Therapy

2019 ◽  
Author(s):  
Long-Jiao Ge ◽  
Fu-Han Yang ◽  
Jie Feng ◽  
Nan-Hui Chen ◽  
Min Jiang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Ischemic Stroke ◽  
High Efficiency ◽  
Ectopic Expression ◽  
Reactive Astrocytes ◽  
Neural Regeneration ◽  
Cortical Astrocyte ◽  
Cell Conversion ◽  
Human Primate

ABSTRACTStroke is a leading cause of death and disability but most of the clinical trials have failed in the past, despite our increasing understanding of the molecular and pathological mechanisms underlying stroke. While many signaling pathways have been identified in the aftermath of stroke, the majority of current approaches are focusing on neural protection rather than neuroregeneration. In this study, we report an in vivo neural regeneration approach to convert brain internal reactive astrocytes into neurons through ectopic expression of a neural transcription factor NeuroD1 in adult non-human primate (NHP) brains following ischemic stroke. We demonstrate that NeuroD1 AAV-based gene therapy can convert reactive astrocytes into neurons with high efficiency (90%), but astrocytes are never depleted in the NeuroD1-expressed areas, consistent with the proliferative capability of astrocytes. The NeuroD1-mediated in vivo astrocyte-to-neuron (AtN) conversion in monkey cortex following ischemic stroke increased local neuronal density, reduced reactive microglia, and surprisingly protected parvalbumin interneurons in the converted areas. The NeuroD1 gene therapy showed a broad time window, from 10 days to 30 days following ischemic stroke, in terms of exerting its neuroregenerative and neuroprotective effects. The cortical astrocyte-converted neurons also showed Tbr1+ cortical neuron identity, similar to our earlier findings in rodent animal models. Unexpectedly, NeuroD1 expression in converted neurons showed a significant decrease after 6 months of viral infection, suggesting a potential self-regulatory mechanism of NeuroD1 in adult mature neurons of NHPs. These results suggest that in vivo cell conversion through NeuroD1-based gene therapy may be an effective approach to regenerate new neurons in adult primate brains for tissue repair.

scholarly journals Comment on “Rapid and efficient in vivo astrocyte-to-neuron conversion with regional identity and connectivity?”

2020 ◽  
Author(s):  
Gong Chen ◽  
Wen Li ◽  
Zongqin Xiang ◽  
Liang Xu ◽  
Minhui Liu ◽  
...  
Keyword(s):  
Glial Cells ◽  
Viral Vectors ◽  
Ectopic Expression ◽  
Regional Identity ◽  
Neuronal Loss ◽  
Critical Factor ◽  
Dose Finding ◽  
Toxic Dose ◽  
Cell Conversion

ABSTRACTRegenerating functional new neurons in the adult mammalian central nervous system (CNS) has been proven to be very challenging due to the inability of neurons to divide and repopulate themselves after neuronal loss. In contrast, glial cells in the CNS can divide and repopulate themselves under injury or disease conditions. Therefore, many groups around the world have been able to utilize internal glial cells to directly convert them into neurons for neural repair. We have previously demonstrated that ectopic expression of NeuroD1 in dividing glial cells can directly convert reactive glial cells into neurons. However, Wang et al. recently posted an article in bioRxiv challenging the entire field of in vivo glia-to-neuron conversion after using one single highly toxic dose of AAV (2×1013 gc/ml, 1 μl) in the mouse cortex, producing artifacts that are very difficult to interpret. We present data here that reducing AAV dosage to safe level will avoid artifacts caused by toxic dosage. We also demonstrate with Aldh1l1-CreERT2 and Ai14 reporter mice that lineage-traced astrocytes can be successfully converted into NeuN+ neurons after infected by AAV5 GFAP::NeuroD1. Retroviral expression of NeuroD1 further confirms our previous findings that dividing glial cells can be converted into neurons. Together, the incidence of Wang et al. sends an alarming signal to the entire in vivo reprogramming field that the dosage of viral vectors is a critical factor to consider when designing proper experiments. For AAV, we recommend a relatively safe dose of 1×1010 - 1×1012 gc/ml (~1 μl) in the rodent brain for cell conversion experiments addressing basic science questions. For therapeutic purpose under injury or diseased conditions, AAV dosage needs to be adjusted through a series of dose finding experiments. Moreover, we recommend that the AAV results are further verified with retroviruses that mainly express transgenes in dividing glial cells in order to draw solid conclusions.

scholarly journals In vivo Neuroregeneration to Treat Ischemic Stroke Through NeuroD1 AAV-Based Gene Therapy in Adult Non-human Primates

2020 ◽  
Vol 8 ◽  
Author(s):  
Long-Jiao Ge ◽  
Fu-Han Yang ◽  
Wen Li ◽  
Tao Wang ◽  
Yu Lin ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Ischemic Stroke

scholarly journals Construction of a bicistronic proangiogenic expression vector and its application in experimental angiogenesis in vivo.

2003 ◽  
Vol 50 (3) ◽  
pp. 875-882 ◽  
Author(s):  
Maciej Małecki ◽  
Małgorzata Przybyszewska ◽  
Przemysław Janik
Keyword(s):  
Gene Therapy ◽  
Plasmid Dna ◽  
High Efficiency ◽  
Single Gene ◽  
Encephalomyocarditis Virus ◽  
Clinical State ◽  
Entry Site ◽  
Internal Ribosome Entry

Manipulation of angiogenesis in vivo is an example of successful gene therapy strategies. Overexpression of angiogenic genes like VEGF, FGF or PDGF causes new vessel formation and improves the clinical state of patients. Gene therapy is a very promising procedure but requires large amounts of pharmaceutical-grade plasmid DNA. In this regard we have constructed a bicistronic plasmid DNA vector encoding two proangiogenic factors, VEGF165 and FGF-2. The construct (pVIF) contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV) which permits both genes to be translated from a single bicistronic mRNA. The IRES sequence allows for a high efficiency of gene expression in vivo. The pVIF vector was characterized in vitro and in vivo. In vivo angiogenesis studies showed that the bicistronic vector encoding two proangiogenic factors induces the formation of new vessels significantly more than pVEGF165 or pFGF-2 alone. In our opinion the combined proangiogenic approach with VEGF165 and FGF-2 is more powerful and efficient than single gene therapy. We also postulate that IRES sequence can serve as a useful device improving efficiency of gene therapy.

scholarly journals Reactive astrocytes prevent maladaptive plasticity after ischemic stroke

2021 ◽  
Author(s):  
Markus Aswendt ◽  
Ulrika Wilhelmsson ◽  
Frederique Wieters ◽  
Anna Stokowska ◽  
Felix Johannes Schmitt ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Functional Connectivity ◽  
Neural Plasticity ◽  
Infarct Volume ◽  
Optimal Recovery ◽  
Reactive Astrocytes ◽  
Sensorimotor Function ◽  
Functional Connections ◽  
Maladaptive Plasticity

Restoration of functional connectivity is a major contributor to functional recovery after stroke. We investigated the role of reactive astrocytes in functional connectivity and recovery after photothrombotic stroke in mice with attenuated reactive gliosis (GFAP−/−Vim−/−). Infarct volume and longitudinal functional connectivity changes were determined by in vivo T2-weighted MRI and resting-state functional MRI. Sensorimotor function was assessed with behavioral tests, and glial and neural plasticity responses were quantified in the peri-infarct region. Four weeks after stroke, GFAP−/−Vim−/− mice showed impaired recovery of sensorimotor function and aberrant restoration of global neuronal connectivity. These mice also exhibited maladaptive plasticity responses, shown by higher number of lost and newly formed functional connections between primary and secondary targets of cortical stroke regions and increased peri-infarct expression of the axonal plasticity marker Gap43. We conclude that reactive astrocytes are required for optimal recovery-promoting plasticity responses after ischemic stroke.

scholarly journals Development of a Single Construct System for Site-Directed RNA Editing Using MS2-ADAR

2020 ◽  
Vol 21 (14) ◽  
pp. 4943
Author(s):  
Tetsuto Tohama ◽  
Matomo Sakari ◽  
Toshifumi Tsukahara
Keyword(s):  
Gene Therapy ◽  
Rna Editing ◽  
Target Genes ◽  
High Efficiency ◽  
Cultured Cells ◽  
Point Mutations ◽  
Gene Repair ◽  
Guide Rnas ◽  
Single Construct

Site-directed RNA editing (SDRE) technologies have great potential for treating genetic diseases caused by point mutations. Our group and other researchers have developed SDRE methods utilizing adenosine deaminases acting on RNA (ADARs) and guide RNAs recruiting ADARs to target RNAs bearing point mutations. In general, efficient SDRE relies on introducing numerous guide RNAs relative to target genes. However, achieving a large ratio is not possible for gene therapy applications. In order to achieve a realistic ratio, we herein developed a system that can introduce an equal number of genes and guide RNAs into cultured cells using a fusion protein comprising an ADAR fragment and a plasmid vector containing one copy of each gene on a single construct. We transfected the single construct into HEK293T cells and achieved relatively high efficiency (up to 42%). The results demonstrate that efficient SDRE is possible when the copy number is similar for all three factors (target gene, guide RNA, and ADAR enzyme). This method is expected to be capable of highly efficient gene repair in vivo, making it applicable for gene therapy.

GMP Scale up for a Clinical Gene Therapy Trial - High Efficiency Human T Cell Expansion and Transduction in a Closed Culture System Utilizing Serumfree Medium and Low IL-2 Concentrations.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5250-5250
Author(s):  
Gerhard Bauer ◽  
Jon E. Walker ◽  
Julie Ritchey ◽  
Jan A. Nolta ◽  
John F. DiPersio
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Cell Expansion ◽  
Magnetic Beads ◽  
High Efficiency ◽  
Scale Up ◽  
Low Concentrations ◽  
T Cell Expansion

Abstract Translational research is an ever increasing focus in the field of cellular and gene therapy. Here we demonstrate the scale-up process for a gene therapy clinical trial aimed at abrogation of Graft versus Host Disease (GvHD) using a suicide gene fused to a truncated CD34 receptor as a selectable marker in a Donor Lymphocyte Infusion setting to treat relapsed hematologic malignancies after allogeneic bone marrow transplantation. Transduced T cells can be purified to over 95% in a magnetic cell sorter (CliniMacs, Miltenyi, Auburn, CA) using the CD34 receptor, normally not present on T cells. After infusion, these T cells produce a Graft versus Leukemia effect, but can be eliminated by administration of Ganciclovir if GvHD develops. Donor T Cells were cultured in a closed system using 2 types of serumfree media: X-VIVO 15 (Cambrex, Walkersville, MD) and the newly developed GMP grade Stemline T Cell Expansion Medium (Sigma, St. Louis, MO). Stimulation was done using clinical grade magnetic beads coated with antibodies to CD3 and CD28 (Xcyte, Seattle, WA). High concentrations of IL-2 (500 U/ml) impair the in vivo functionality of cultured T cells, as shown in our NOD SCID/B2 M deficient mouse model of T cell expansion. To maintain in vivo functionality of transduced T cells, it was imperative to lower the IL-2 concentration to 50 U/ml during culture. At least a 2 fold T cell expansion with maintenance of a physiological CD4 and CD8 compartment is necessary, since transduction using a Moloney leukemia virus based retroviral vector depends on cell division. It is also important to transduce both CD4 and CD8 cells in the same ratio for normal function of the infused T cell population, and expression of the selectable surface marker for subsequent enrichment of the transduced cells in both fractions. Requirements stipulated by regulatory agencies demanded not to introduce more than 1–2 vector copies per cell to keep the risk for insertional mutagenesis to a minimum. We demonstrated that a transduction frequency of 20–30% obtained at an MOI of 1–2 is required to generate this copy number (Rettig et al., 2003). After 48 hours of pre-stimulation, the cells were transduced 2 times, medium was replaced by spinning the culture bags, removing 2/3 of the supernatant and replacing it with fresh vector containing medium. Cells were harvested on day 5 by disrupting bead/cell clumps and bead removal in a magnetic field. With X-VIVO 15, strong variability in expansion and viability at the end of culture could be seen using low concentrations of IL-2. However, the consistency of cell expansion increased dramatically when the concentration of IL-2 was increased to 1000U/ml. In contrast, the newly formulated Stemline serumfree medium consistently worked equally well in high and low concentrations of IL-2. At 50 U/ml of IL-2, a 3 fold expansion of T cells with a 30% transduction efficiency, equally well distributed in the CD4 and CD8 compartment, was observed. CD4/CD8 ratio was maintained at input ratio, with greater than 95% cell viability. Removal of magnetic beads after culture was consistently more than 2 logs. The transduced cells could be purified by magnetic cell sorting using the selectable marker, which was highly expressed. In summary, a new serumfree media formulation allows for reproducible, high efficiency expansion and transduction of T cells in low IL-2 concentrations to optimize in vivo T cell functionality for human gene therapy trials.

scholarly journals Regeneration of dorsal spinal cord neurons after injury via in situ NeuroD1-mediated astrocyte-to-neuron conversion

2019 ◽  
Author(s):  
Brendan Puls ◽  
Yan Ding ◽  
Fengyu Zhang ◽  
Mengjie Pan ◽  
Zhuofan Lei ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Dorsal Horn ◽  
Cell Fate ◽  
High Efficiency ◽  
Reactive Astrocytes ◽  
Dorsal Spinal Cord ◽  
Spinal Cord Neurons ◽  
Cord Injury ◽  
Dorsal Spinal

AbstractSpinal cord injury (SCI) often leads to impaired motor and sensory functions, partially because the injury-induced neuronal loss cannot be easily replenished through endogenous mechanisms. In vivo neuronal reprogramming has emerged as a novel technology to regenerate neurons from endogenous glial cells by forced expression of neurogenic transcription factors. We have previously demonstrated successful astrocyte-to-neuron conversion in mouse brains with injury or Alzheimer’s disease by overexpressing a single neural transcription factor NeuroD1 via retroviruses. Here we demonstrate regeneration of dorsal spinal cord neurons from reactive astrocytes after SCI via adeno-associated virus (AAV), a more clinically relevant gene delivery system. We find that NeuroD1 converts reactive astrocytes into neurons in the dorsal horn of stab-injured spinal cord with high efficiency (∼95%). Interestingly, NeuroD1-converted neurons in the dorsal horn mostly acquire glutamatergic neuronal subtype, expressing spinal cord-specific markers such as Tlx3 but not brain-specific markers such as Tbr1, suggesting that the astrocytic lineage and local microenvironment affect the cell fate of conversion. Electrophysiological recordings show that the NeuroD1-converted neurons can functionally mature and integrate into local spinal cord circuitry by displaying repetitive action potentials and spontaneous synaptic responses. We further show that NeuroD1-mediated neuronal conversion can occur in the contusive SCI model, allowing future studies of evaluating this reprogramming technology for functional recovery after SCI. In conclusion, this study may suggest a paradigm shift for spinal cord repair using in vivo astrocyte-to-neuron conversion technology to generate functional neurons in the grey matter.

scholarly journals Lentivirus- or AAV-mediated gene therapy interventions in ischemic stroke: A systematic review of preclinical in vivo studies

2021 ◽  
pp. 0271678X2110399
Author(s):  
Laura Skukan ◽  
Matea Brezak ◽  
Rok Ister ◽  
Lars Klimaschewski ◽  
Aleksandar Vojta ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Ischemic Stroke ◽  
Viral Vector ◽  
Infarct Volume ◽  
Meta Analysis ◽  
Artery Occlusion ◽  
In Vivo Studies ◽  
Specific Cell ◽  
Systematic Analysis

Due to the limited therapeutic options after ischemic stroke, gene therapy has emerged as a promising choice, especially with recent advances in viral vector delivery systems. Therefore, we aimed to provide the current state of the art of lentivirus (LV) and adeno-associated virus (AAV) mediated gene interventions in preclinical ischemic stroke models. A systematic analysis including qualitative and quantitative syntheses of studies published until December 2020 was performed. Most of the 87 selected publications used adult male rodents and the preferred stroke model was transient middle cerebral artery occlusion. LV and AAV vectors were equally used for transgene delivery, however loads of AAVs were higher than LVs. Serotypes having broad cell tropism, the use of constitutive promoters, and virus delivery before the stroke induction via stereotaxic injection in the cortex and striatum were preferred in the analyzed studies. The meta-analysis based on infarct volume as the primary outcome confirmed the efficacy of the preclinical interventions. The quality assessment exposed publication bias and setbacks in regard to risks of bias and study relevance. The translational potential could increase by using specific cell targeting, post-stroke interventions, non-invasive systematic delivery, and use of large animals.

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