CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe.
Following spinal cord injury (SCI) there are drastic changes that occur in the spinal microvasculature, including ischemia, hemorrhage, endothelial cell death and blood-spinal cord barrier disruption. Vascular endothelial growth factor-A (VEGF-A) is a pleiotropic factor recognized for its pro-angiogenic properties; however, VEGF has recently been shown to provide neuroprotection. We hypothesized that delivery of AdV-ZFP-VEGF--an adenovirally delivered bio-engineered zinc-finger transcription factor that promotes endogenous VEGF-A expression--would result in angiogenesis, neuroprotection and functional recovery following SCI. This novel VEGF gene therapy induces the endogenous production of multiple VEGF-A isoforms; a critical factor for proper vascular development and repair. Briefly, female Wistar rats--under cyclosporin immunosuppression--received a 35 g clip-compression injury and were administered AdV-ZFP-VEGF or AdV-eGFP at 24 hours post-SCI. qRT-PCR and Western Blot analysis of VEGF-A mRNA and protein, showed significant increases in VEGF-A expression in AdV-ZFP-VEGF treated animals (p<0.001 and p<0.05, respectively). Analysis of NF200, TUNEL, and RECA-1 indicated that AdV-ZFP-VEGF increased axonal preservation (p<0.05), reduced cell death (p<0.01), and increased blood vessels (p<0.01), respectively. Moreover, AdV-ZFP-VEGF resulted in a 10% increase in blood vessel proliferation (p<0.001). Catwalk™ analysis showed AdV-ZFP-VEGF treatment dramatically improves hindlimb weight support (p<0.05) and increases hindlimb swing speed (p<0.02) when compared to control animals. Finally, AdV-ZFP-VEGF administration provided a significant reduction in allodynia (p<0.01). Overall, the results of this study indicate that AdV-ZFP-VEGF administration can be delivered in a clinically relevant time-window following SCI (24 hours) and provide significant molecular and functional benefits.
Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4(+) T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating >10(10) CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection.
Glial proliferation and activation are associated with disease progression in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia. In this study, we describe a unique platform to address the question of cell autonomy in transactive response DNA-binding protein (TDP-43) proteinopathies. We generated functional astroglia from human induced pluripotent stem cells carrying an ALS-causing TDP-43 mutation and show that mutant astrocytes exhibit increased levels of TDP-43, subcellular mislocalization of TDP-43, and decreased cell survival. We then performed coculture experiments to evaluate the effects of M337V astrocytes on the survival of wild-type and M337V TDP-43 motor neurons, showing that mutant TDP-43 astrocytes do not adversely affect survival of cocultured neurons. These observations reveal a significant and previously unrecognized glial cell-autonomous pathological phenotype associated with a pathogenic mutation in TDP-43 and show that TDP-43 proteinopathies do not display an astrocyte non-cell-autonomous component in cell culture, as previously described for SOD1 ALS. This study highlights the utility of induced pluripotent stem cell-based in vitro disease models to investigate mechanisms of disease in ALS and other TDP-43 proteinopathies.
Recent studies have identified anti-apoptotic functions for vascular endothelial growth factor (VEGF) in the central nervous system (CNS). However, VEGF therapy has been hampered by a tendency to promote vascular permeability, edema, and inflammation. Recently, engineered zinc finger proteins (ZFPs) that upregulate multiple forms of VEGF in their natural biological ratios, have been developed to overcome these negative side effects. We used retinal trauma and ischemia models, and a cortical pial strip ischemia model to determine if VEGF upregulating ZFPs are neuroprotective in the adult CNS. Optic nerve transection and ophthalmic artery ligation lead to the apoptotic degeneration of retinal ganglion cells (RGCs) and are, respectively, two highly reproducible models for CNS trauma or ischemia. Adeno-associated vectors (AAV) vectors encoding VEGF-ZFPs (AAV-VEGF-ZFP) significantly increased RGC survival by ?twofold at 14 days after optic nerve transection or ophthalmic artery ligation. Furthermore, AAV-VEGF-ZFP enhanced recovery of the pupillary light reflex. RECA-1 immunostaining demonstrated no appreciable differences between retinas treated with AAV-VEGF-ZFP and controls, suggesting that AAV-VEGF-ZFP treatment did not affect retinal vasculature. Following pial strip of the forelimb motor cortex, brains treated with an adenovirus encoding VEGF ZFPs (AdV-ZFP) showed higher neuronal survival, accelerated wound contraction, and reduced lesion volume between 1 and 6 weeks after injury. Behavioral testing using the cylinder test for vertical exploration showed that AdV-VEGF-ZFP treatment enhanced contralateral forelimb function within the first 2 weeks after injury. Our results indicate that VEGF ZFP therapy is neuroprotective following traumatic injury or stroke in the adult mammalian CNS.
Spinal cord injury (SCI) leads to local vascular disruption and progressive ischemia, which contribute to secondary degeneration. Enhancing angiogenesis through the induction of vascular endothelial growth factor (VEGF)-A expression therefore constitutes an attractive therapeutic approach. Moreover, emerging evidence suggests that VEGF-A may also exhibit neurotrophic, neuroprotective, and neuroproliferative effects. Building on this previous work, we seek to examine the potential therapeutic benefits of an engineered zinc finger protein (ZFP) transcription factor designed to activate expression of all isoforms of endogenous VEGF-A (ZFP-VEGF). Administration of ZFP-VEGF resulted in increased VEGF-A mRNA and protein levels, an attenuation of axonal degradation, a significant increase in vascularity and decreased levels of apoptosis. Furthermore, ZFP-VEGF treated animals showed significant improvements in tissue preservation and neurobehavioural outcomes. These data suggest that activation of VEGF-A via the administration of an engineered ZFP transcription factor holds promise as a therapy for SCI and potentially other forms of neurotrauma.
Vascular endothelial growth factor (VEGF) plays a role in angiogenesis and has been shown to be neuroprotective following central nervous system trauma. In the present study we evaluated the pro-angiogenic and neuroprotective effects of an engineered zinc-finger protein transcription factor transactivator targeting the vascular endothelial growth factor A (VEGF-ZFP). We used two virus delivery systems, adeno-virus and adeno-associated virus, to examine the effects of early and delayed VEGF-A upregulation after brain trauma, respectively. Male Sprague-Dawley rats were subject to a unilateral fluid percussion injury (FPI) of moderate severity (2.2-2.5?atm) followed by intracerebral microinjection of either adenovirus vector (Adv) or an adeno-associated vector (AAV) carrying the VEGF-ZFP construct. Adv-VEGF-ZFP-treated animals had significantly fewer TUNEL positive cells in the injured penumbra of the cortex (p<0.001) and hippocampus (p=0.001) relative to untreated rats at 72?h post-injury. Adv-VEGF-ZFP treatment significantly improved fEPSP values (p=0.007) in the CA1 region relative to injury alone. Treatment with AAV2-VEGF-ZFP resulted in improved post-injury microvascular diameter and improved functional recovery on the balance beam and rotarod task at 30 days post-injury. Collectively, the results provide supportive evidence for the concept of acute and delayed treatment following TBI using VEGF-ZFP to induce angiogenesis, reduce cell death, and enhance functional recovery.
Related JoVE Video
Journal of Visualized Experiments
What is Visualize?
JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.
How does it work?
We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.
Video X seems to be unrelated to Abstract Y...
In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.