ABSTRACT
Huntington's disease (HD) is caused by Huntingtin (Htt) gene mutation resulting in the loss of striatal GABAergic neurons and motor functional deficits. We report here an in vivo cell conversion technology to reprogram striatal astrocytes into GABAergic neurons in both R6/2 and YAC128 HD mouse models through AAV-mediated ectopic expression of NeuroD1 and Dlx2 transcription factors. We found that the astrocyte-to-neuron (AtN) conversion rate reached 80% in the striatum and >50% of the converted neurons were DARPP32+ medium spiny neurons. The striatal astrocyte-converted neurons showed action potentials and synaptic events, and projected their axons to the targeted globus pallidus and substantia nigra in a time-dependent manner. Behavioral analyses found that NeuroD1 and Dlx2-treated R6/2 mice showed a significant extension of life span and improvement of motor functions. This study demonstrates that in vivo AtN conversion may be a disease-modifying gene therapy to treat HD and other neurodegenerative disorders.
Subject(s)
Astrocytes/physiology , Cellular Reprogramming Techniques/methods , Corpus Striatum/pathology , GABAergic Neurons/physiology , Genetic Therapy/methods , Huntington Disease/therapy , Action Potentials/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors , Behavior Observation Techniques , Behavior, Animal , Corpus Striatum/cytology , Dependovirus/genetics , Disease Models, Animal , Ectopic Gene Expression , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , HEK293 Cells , Homeodomain Proteins , Humans , Huntingtin Protein/genetics , Huntington Disease/genetics , Huntington Disease/pathology , Longevity , Mice , Mice, Transgenic , Patch-Clamp Techniques , Stereotaxic Techniques , Transcription FactorsABSTRACT
Adult mammalian brains have largely lost neuroregeneration capability except for a few niches. Previous studies have converted glial cells into neurons, but the total number of neurons generated is limited and the therapeutic potential is unclear. Here, we demonstrate that NeuroD1-mediated in situ astrocyte-to-neuron conversion can regenerate a large number of functional new neurons after ischemic injury. Specifically, using NeuroD1 adeno-associated virus (AAV)-based gene therapy, we were able to regenerate one third of the total lost neurons caused by ischemic injury and simultaneously protect another one third of injured neurons, leading to a significant neuronal recovery. RNA sequencing and immunostaining confirmed neuronal recovery after cell conversion at both the mRNA level and protein level. Brain slice recordings found that the astrocyte-converted neurons showed robust action potentials and synaptic responses at 2 months after NeuroD1 expression. Anterograde and retrograde tracing revealed long-range axonal projections from astrocyte-converted neurons to their target regions in a time-dependent manner. Behavioral analyses showed a significant improvement of both motor and cognitive functions after cell conversion. Together, these results demonstrate that in vivo cell conversion technology through NeuroD1-based gene therapy can regenerate a large number of functional new neurons to restore lost neuronal functions after injury.
Subject(s)
Astrocytes/metabolism , Basic Helix-Loop-Helix Transcription Factors/genetics , Brain Ischemia/therapy , Cellular Reprogramming/genetics , Genetic Therapy/methods , Neurons/metabolism , Action Potentials , Animals , Basic Helix-Loop-Helix Transcription Factors/metabolism , Dependovirus/genetics , Disease Models, Animal , Male , Mice , Mice, Transgenic , Nerve Degeneration/therapy , Neuroglia/metabolism , Rats , Rats, Sprague-Dawley , Treatment OutcomeABSTRACT
Virotherapy with oncolytic viruses is a highly promising approach for cancer therapy. To improve further the therapeutic effect of oncolytic viruses, therapeutic genes have been incorporated into these types of vectors. In this study, we have inserted hTRAIL (approved gene symbol TNFSF10) into the ZD55 vector, which was based on deletion of the adenoviral E1B 55-kDa gene and could replicate in and lyse p53-deficient tumors. Our data shows that infection of colorectal carcinoma cells with ZD55-hTRAIL resulted in tumor cell death that was much greater than that induced by ZD55 vector or replication-defective adenovirus expressing hTRAIL. In contrast to these, ZD55-hTRAIL did not induce any cytopathic effect in normal cells. Treatment of established tumor with ZD55-hTRAIL resulted in dramatic inhibition of tumor growth in an animal model of colorectal carcinoma. However, when the established tumors were treated by coadministration of ZD55-hTRAIL and Ad-k5, we observed complete eradication of the established tumors in all animals treated with the combined therapy. This strong anti-tumor activity was due to the fact that two genes may act with compensative (or synergic) effect through different mechanisms to kill tumors. Therefore, targeting dual gene-virotherapy may be one of the best strategies for cancer therapy if two suitable genes are chosen.