Secreted Luciferase for In Vivo Evaluation of Systemic Protein Delivery in Mice
Molecular and Cell Therapy Program, Division of Experimental Hematology & Cancer Biology, Cancer and Blood Diseases Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.Molecular Biotechnology (Impact Factor: 1.88). 03/2012; 53(1). DOI: 10.1007/s12033-012-9519-6
A naturally secreted Gaussia luciferase (Gluc) has been utilized as a reporter for bioluminescence imaging (BLI) evaluation. However, the potential application of Gluc for in vivo monitoring of systemic protein delivery, as well as its natural biodistribution, has not been studied. To examine Gluc secretion and uptake profile, we injected Gluc-encoding plasmids into mice by hydrodynamic tail-vein injection. Whole-body BLI showed that imaging quantification obtained at pawpad was directly correlated to blood Gluc activities. When gene expression was restricted to the liver by the use of a hepatic promoter, in vivo Gluc biodistribution analysis revealed the kidney/bladder, stomach/intestine, and lung as the major uptake organs. Three-dimensional BLI identified liver/stomach and lung as the main internal luminescent sources, demonstrating the feasibility of detecting major uptake organs in live animals by 3D BLI with high-background signals in circulation. Notably, Gluc levels in capillary-depleted brain samples from Gluc-injected mice were comparable to controls, suggesting that Gluc may not cross the blood-brain barrier. Gluc uptake kinetics and intracellular half-life were assessed in various types of cell lines, implicating the involvement of non-specific pinocytosis. These results suggest that Gluc-based system may provide a useful tool for in vivo evaluation of protein/agent biodistribution following systemic delivery.
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ABSTRACT: To realize the potential of large molecular weight substances to treat neurological disorders, novel approaches are required to surmount the blood-brain barrier (BBB). We investigated whether fusion of a receptor-binding peptide from apolipoprotein E (apoE) with a potentially therapeutic protein can bind to LDL receptors on the BBB and be transcytosed into the CNS. A lysosomal enzyme, α-L-iduronidase (IDUA), was used for biological and therapeutic evaluation in a mouse model of mucopolysaccharidosis (MPS) type I, one of the most common lysosomal storage disorders with CNS deficits. We identified two fusion candidates, IDUAe1 and IDUAe2, by in vitro screening, that exhibited desirable receptor-mediated binding, endocytosis, and transendothelial transport as well as appropriate lysosomal enzyme trafficking and biological function. Robust peripheral IDUAe1 or IDUAe2 generated by transient hepatic expression led to elevated enzyme levels in capillary-depleted, enzyme-deficient brain tissues and protein delivery into nonendothelium perivascular cells, neurons, and astrocytes within 2 d of treatment. Moreover, 5 mo after long-term delivery of moderate levels of IDUAe1 derived from maturing red blood cells, 2% to 3% of normal brain IDUA activities were obtained in MPS I mice, and IDUAe1 protein was detected in neurons and astrocytes throughout the brain. The therapeutic potential was demonstrated by normalization of brain glycosaminoglycan and β-hexosaminidase in MPS I mice 5 mo after moderate yet sustained delivery of IDUAe1. These findings provide a noninvasive and BBB-targeted procedure for the delivery of large-molecule therapeutic agents to treat neurological lysosomal storage disorders and potentially other diseases that involve the brain.Proceedings of the National Academy of Sciences 02/2013; 110(8). DOI:10.1073/pnas.1222742110 · 9.67 Impact Factor
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ABSTRACT: Focused ultrasound (FUS) exposure in the presence of microbubbles can temporally open the blood-brain barrier (BBB) and is an emerging technique for non-invasive brain therapeutic agent delivery. Given the potential to deliver large molecules into the CNS via this technique, we propose a reliable strategy to synergistically apply FUS-BBB opening for the non-invasive and targeted delivery of non-viral genes into the CNS for therapeutic purpose. In this study, we developed a gene-liposome system, in which the liposomes are designed to carry plasmid DNA (pDNA, containing luciferase reporter gene) to form a liposomal-plasmid DNA (LpDNA) complex. Pulsed FUS exposure was delivered to induce BBB opening (500-kHz, burst length = 10 ms, 1% duty cycle, PRF = 1 Hz). The longitudinal expression of luciferase was quantitated via an in vivo imaging system (IVIS). The reporter gene expression level was confirmed via immunoblotting, and histological staining was used to identify transfected cells via fluorescent microscopy. In a comparison of gene transduction efficiency, the LpDNA system showed better cell transduction than the pDNA system. With longitudinal observation of IVIS monitoring, animals with FUS treatment showed significant promotion of LpDNA release into the CNS and demonstrated enhanced expression of genes upon sonication with FUS-BBB opening, while both the luciferase and GDNF protein expression were successfully measured via Western blotting. The gene expression peak was observed at day 2, and the gene expression level was up to 5-fold higher than that in the untreated hemisphere (compared to a 1-fold increase in the direct-inject positive-control group). The transfection efficiency was also found to be LpDNA dose-dependent, where higher payloads of pDNA resulted in a higher transfection rate. Immunoblotting and histological staining confirmed the expression of reporter genes in glial cells as well as astrocytes. This study suggests that IV administration of LpDNA in combination with FUS-BBB opening can provide effective gene delivery and expression in the CNS, demonstrating the potential to achieve non-invasive and targeted gene delivery for treatment of CNS diseases. Copyright © 2015. Published by Elsevier B.V.Journal of Controlled Release 06/2015; 212. DOI:10.1016/j.jconrel.2015.06.010 · 7.71 Impact Factor
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ABSTRACT: Introduction: Mesenchymal stem cells (MSCs) are adult multipotent stem cells that possess regenerative and immunomodulatory properties. They have been widely investigated as therapeutic agents for a variety of disease conditions, including tissue repair, inflammation, autoimmunity, and organ transplantation. Importantly, systemically infused MSCs selectively home to primary and metastatic tumors, though the molecular mechanisms of tumor tropism of MSCs remain incompletely understood. We have exploited the active and selective MSCs homing to cancer microenvironments to develop a rapid and selective blood test for the presence of cancer. Methods: We tested the concept of using transplanted MSCs as the basis for a simple cancer blood test. MSCs were engineered to express humanized Gaussia luciferase (hGluc). In a minimally invasive fashion, hGluc secreted by MSCs into circulation as a reporter for cancer presence, was assayed to probe whether MSCs co-localize with and persist in cancerous tissue. Results: In vitro, hGluc secreted by engineered MSCs was detected stably over a period of days in the presence of serum. In vivo imaging showed that MSCs homed to breast cancer lung metastases and persisted longer in tumor-bearing mice than in tumor-free mice (P < 0.05). hGluc activity in blood of tumor-bearing mice was significantly higher than in their tumor-free counterparts (P < 0.05). Conclusions: Both in vitro and in vivo data show that MSCs expressing hGluc can identify and report small tumors or metastases in a simple blood test format. Our novel and simple stem cell-based blood test can potentially be used to screen, detect, and monitor cancer and metastasis at early stages and during treatment.Stem Cell Research & Therapy 09/2015; 6(1):181. DOI:10.1186/s13287-015-0151-9 · 3.37 Impact Factor
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