Virus vector-mediated genetic modification of brain tumor stromal cells after intravenous delivery.

The malignant primary brain tumor, glioblastoma (GBM) is generally incurable. New approaches are desperately needed. Adeno-associated virus (AAV) vector-mediated delivery of anti-tumor transgenes is a promising strategy, however direct injection leads to focal transgene spread in tumor and rapid tumor division dilutes out the extra-chromosomal AAV genome, limiting duration of transgene expression. Intravenous (IV) injection gives widespread distribution of AAV in normal brain, however poor transgene expression in tumor, and high expression in non-target cells which may lead to ineffective therapy and high toxicity, respectively. Delivery of transgenes encoding secreted, anti-tumor proteins to tumor stromal cells may provide a more stable and localized reservoir of therapy as they are more differentiated than fast-dividing tumor cells. Reactive astrocytes and tumor-associated macrophage/microglia (TAMs) are stromal cells that comprise a large portion of the tumor mass and are associated with tumorigenesis. In mouse models of GBM, we used IV delivery of exosome-associated AAV vectors driving green fluorescent protein expression by specific promoters (NF-κB-responsive promoter and a truncated glial fibrillary acidic protein promoter), to obtain targeted transduction of TAMs and reactive astrocytes, respectively, while avoiding transgene expression in the periphery. We used our approach to express the potent, yet toxic anti-tumor cytokine, interferon beta, in tumor stroma of a mouse model of GBM, and achieved a modest, yet significant enhancement in survival compared to controls. Noninvasive genetic modification of tumor microenvironment represents a promising approach for therapy against cancers. Additionally, the vectors described here may facilitate basic research in the study of tumor stromal cells in situ.

Intracranial AAV-IFN-ß gene therapy eliminates invasive xenograft glioblastoma and improves survival in orthotopic syngeneic murine model.

The highly invasive property of glioblastoma (GBM) cells and genetic heterogeneity are largely responsible for tumor recurrence after the current standard-of-care treatment and thus a direct cause of death. Previously, we have shown that intracranial interferon-beta (IFN-ß) gene therapy by locally administered adeno-associated viral vectors (AAV) successfully treats noninvasive orthotopic glioblastoma models. Here, we extend these findings by testing this approach in invasive human GBM xenograft and syngeneic mouse models. First, we show that a single intracranial injection of AAV encoding human IFN-ß eliminates invasive human GBM8 tumors and promotes long-term survival. Next, we screened five AAV-IFN-ß vectors with different promoters to drive safe expression of mouse IFN-ß in the brain in the context of syngeneic GL261 tumors. Two AAV-IFN-ß vectors were excluded due to safety concerns, but therapeutic studies with the other three vectors showed extensive tumor cell death, activation of microglia surrounding the tumors, and a 56% increase in median survival of the animals treated with AAV/P2-Int-mIFN-ß vector. We also assessed the therapeutic effect of combining AAV-IFN-ß therapy with temozolomide (TMZ). As TMZ affects DNA replication, an event that is crucial for second-strand DNA synthesis of single-stranded AAV vectors before active transcription, we tested two TMZ treatment regimens. Treatment with TMZ prior to AAV-IFN-ß abrogated any benefit from the latter, while the reverse order of treatment doubled the median survival compared to controls. These studies demonstrate the therapeutic potential of intracranial AAV-IFN-ß therapy in a highly migratory GBM model as well as in a syngeneic mouse model and that combination with TMZ is likely to enhance its antitumor potency.

Direct Intracranial Injection of AAVrh8 Encoding Monkey ß-N-Acetylhexosaminidase Causes Neurotoxicity in the Primate Brain.

GM2 gangliosidoses, including Tay-Sachs disease and Sandhoff disease, are lysosomal storage disorders caused by deficiencies in ß-N-acetylhexosaminidase (Hex). Patients are afflicted primarily with progressive central nervous system (CNS) dysfunction. Studies in mice, cats, and sheep have indicated safety and widespread distribution of Hex in the CNS after intracranial vector infusion of AAVrh8 vectors encoding species-specific Hex α- or ß-subunits at a 1:1 ratio. Here, a safety study was conducted in cynomolgus macaques (cm), modeling previous animal studies, with bilateral infusion in the thalamus as well as in left lateral ventricle of AAVrh8 vectors encoding cm Hex α- and ß-subunits. Three doses (3.2 × 1012 vg [n = 3]; 3.2 × 1011 vg [n = 2]; or 1.1 × 1011 vg [n = 2]) were tested, with controls infused with vehicle (n = 1) or transgene empty AAVrh8 vector at the highest dose (n = 2). Most monkeys receiving AAVrh8-cmHexα/ß developed dyskinesias, ataxia, and loss of dexterity, with higher dose animals eventually becoming apathetic. Time to onset of symptoms was dose dependent, with the highest-dose cohort producing symptoms within a month of infusion. One monkey in the lowest-dose cohort was behaviorally asymptomatic but had magnetic resonance imaging abnormalities in the thalami. Histopathology was similar in all monkeys injected with AAVrh8-cmHexα/ß, showing severe white and gray matter necrosis along the injection track, reactive vasculature, and the presence of neurons with granular eosinophilic material. Lesions were minimal to absent in both control cohorts. Despite cellular loss, a dramatic increase in Hex activity was measured in the thalamus, and none of the animals presented with antibody titers against Hex. The high overexpression of Hex protein is likely to blame for this negative outcome, and this study demonstrates the variations in safety profiles of AAVrh8-Hexα/ß intracranial injection among different species, despite encoding for self-proteins.

Systemic AAV9-IFNß gene delivery treats highly invasive glioblastoma.

BACKGROUND: Complete surgical removal of all glioblastoma (GBM) cells is impossible due to extensive infiltration into brain parenchyma that ultimately leads to tumor recurrence. The current standard of care affords modest improvements in survival. New therapeutic interventions are needed to prevent recurrence. Local AAV-hIFNß gene delivery to the brain was previously shown to eradicate noninvasive orthotopic U87 tumors in mice. However, widespread CNS gene delivery may be necessary to treat invasive GBMs. Here we investigated the therapeutic effectiveness of systemically infused AAV9-hIFNß against an invasive orthotopic GBM8 model. METHODS: Mice bearing human GBM8 brain tumors expressing firefly luciferase (Fluc) were treated systemically with different doses of scAAV9-hIFNß vector. Therapeutic efficacy was assessed by sequential bioluminescence imaging of tumor Fluc activity and animal survival. Brains were analyzed post mortem for the presence and appearance of tumors. Two transcriptionally restricted AAV vectors were used to assess the therapeutic contribution of peripheral hIFNß. RESULTS: Systemic infusion of scAAV9-hIFNß vector induced complete regression of established GBM8 tumors in a dose-dependent manner. The efficacy of this approach was also dependent on the stage of tumor growth at the time of treatment. We also showed that peripherally produced hIFNß contributed considerably to the therapeutic effect of scAAV9-hIFNß. A comparative study of systemic and unilateral intracranial delivery of scAAV9-hIFNß in a bilateral GBM8 tumor model showed the systemic route to be the most effective approach for treating widely dispersed tumors. CONCLUSIONS: Systemic delivery of AAV9-IFNß is an attractive approach for invasive and multifocal GBM treatment.

Multimodal electrophoresis of gold nanoparticles: a real time approach.

A real time multimodal data acquisition imaging setup is developed for the electrophoretic movement of plasmonic nanoparticles. Movement of the nanoparticles is recorded by time-lapse digital imaging at 8-megapixel resolutions. The analysis of the moving nanoparticle band is performed using threshold image and color extraction at respective time frames. The migration dynamics is sensitive to size, nature and the extent of the surface conjugation to the nanoparticle. The dynamics of color intensity of the nanoparticles is shown to be dependent on the extent of stabilization of nanoparticle (by a given agent). The stability of nanoparticle is determined by stationary nature and also the relative proportions of pixel intensities in respective color planes (R, G and B). Detergents stabilize nanoparticles in a concentration dependent fashion. In case of neutral polymers the extent of stabilization depends on the relative proportion of the polymer and also on the nature of the same, e.g., PEG (polyethylene glycol) at low concentration imparts higher stability as compared to PVP (polyvinylpyrilidone). The ascending or declining temporal dynamics of color profiles observed in case of citrate stabilized or amino acid conjugated nanoparticles, represent enrichment of plasmonic particles, or their diffusion resulting from loss of charge during migration. The higher dimensional imaging technique thus can be exploited for discriminating the nanoparticles on the basis of their migration behavior and their stability as reflected from color dynamics. The technique is applicable to other nano-sized colored objects, e.g. proteins like hemoglobin where the protein color has important clinical value.