Intracerebral lentiviral ABCD1 gene therapy in an early disease onset ALD mouse model.

X-linked adrenoleukodystrophy (ALD) is a genetic disorder of the ABCD1 gene. We aimed to treat ALD via direct intracerebral injection of lentiviral ABCD1 (LV.ABCD1). Lentiviral vectors (LVs) were injected into the brain of wild type mice to access toxicities and biodistribution. Confocal microscopy illustrated supraphysiological ABCD1 expression surrounding the injection sites, and LVs were also detected in the opposite site of the unilaterally injected brain. In multi-site bilateral injections (4, 6, 8, and 9 sites), LV.ABCD1 transduced most brain regions including the cerebellum. Investigation of neuronal loss, astrogliosis and microglia activation did not detect abnormality. For efficacy evaluation, a novel ALD knockout (KO) mouse model was established by deleting exons 3 to 9 of the ABCD1 gene based on CRISPR/Cas9 gene editing. The KO mice showed behavioral deficit in open-field test (OFT) and reduced locomotor activities in rotarod test at 6 and 7 months of age, respectively. We treated 3-month-old KO mice with bilateral LV.ABCD1 injections into the external capsule and thalamus. ABCD1 expression was detected 15 days later, and the impaired motor ability was gradually alleviated. Our studies established an early onset ALD model and illustrated neurological improvement after LV.ABCD1 intracerebral injection without immunopathological toxicity.

Transduction of modified factor VIII gene improves lentiviral gene therapy efficacy for hemophilia A.

Hemophilia A (HA) is a bleeding disorder caused by deficiency of the coagulation factor VIII (F8). F8 replacement is standard of care, whereas gene therapy (F8 gene) for HA is an attractive investigational approach. However, the large size of the F8 gene and the immunogenicity of the product present challenges in development of the F8 gene therapy. To resolve these problems, we synthesized a shortened F8 gene (F8-BDD) and cloned it into a lentiviral vector (LV). The F8-BDD produced mainly short cleaved inactive products in LV-transduced cells. To improve F8 functionality, we designed two novel F8-BDD genes, one with an insertion of eight specific N-glycosylation sites (F8-N8) and another which restored all N-glycosylation sites (F8-299) in the B domain. Although the overall protein expression was reduced, high coagulation activity (>100-fold) was detected in the supernatants of LV-F8-N8- and LV-F8-299-transduced cells. Protein analysis of F8 and the procoagulation cofactor, von Willebrand Factor, showed enhanced interaction after restoration of B domain glycosylation using F8-299. HA mouse hematopoietic stem cell transplantation studies illustrated that the bleeding phenotype was corrected after LV-F8-N8 or -299 gene transfer into the hematopoietic stem cells. Importantly, the F8-299 modification markedly reduced immunogenicity of the F8 protein in these HA mice. In conclusion, the modified F8-299 gene could be efficiently packaged into LV and, although with reduced expression, produced highly stable and functional F8 protein that corrected the bleeding phenotype without inhibitory immunogenicity. We anticipate that these results will be beneficial in the development of gene therapies against HA.

Hemophilia Gene Therapy: New Development from Bench to Bed Side.

Novel gene therapy strategies have changed the prognosis of many inherited diseases in recent years. New development in genetic tools and study models has brought us closer to a complete cure for hemophilia. This review will address the latest gene therapy research in hemophilia A and B including gene therapy tools, genetic strategies and animal models. It also summarizes the results of recent clinical trials. Potential solutions are discussed regarding the current barriers in gene therapy for hemophilia.

Dextran-coated iron oxide nanoparticle-improved therapeutic effects of human mesenchymal stem cells in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is a prevalent neurodegenerative disease characterized by the loss of dopaminergic (DA) neurons. With their migration capacity toward the sites of diseased DA neurons in the PD brain, mesenchymal stem cells (MSCs) have the potential to differentiate to DA neurons for the replacement of damaged neurons and to secrete neurotrophic factors for the protection and regeneration of diseased DA neurons; therefore MSCs show promise for the treatment of PD. In this study, for the first time, we demonstrate that dextran-coated iron oxide nanoparticles (Dex-IO NPs) can improve the therapeutic efficacy of human MSCs (hMSCs) in a mouse model of PD induced by a local injection of 6-hydroxydopamine (6-OHDA). In situ examinations not only show that Dex-IO NPs can improve the rescue effect of hMSCs on the loss of host DA neurons but also demonstrate that Dex-IO NPs can promote the migration capacity of hMSCs toward lesioned DA neurons and induce the differentiation of hMSCs to DA-like neurons at the diseased sites. We prove that in vitro Dex-IO NPs can enhance the migration of hMSCs toward 6-OHDA-damaged SH-SY5Y-derived DA-like cells, induce hMSCs to differentiate to DA-like neurons in the conditioned media derived from 6-OHDA-damaged SH-SY5Y-derived DA-like cells and promote the protection/regeneration effects of hMSCs on 6-OHDA-damaged SH-SY5Y-derived DA-like cells. We confirm the potential of MSCs for cell-based therapy for PD. Dex-IO NPs can be used as a tool to accelerate and optimize MSC therapeutics for PD applicable clinically.

The Affinity of Elongated Membrane-Tethered Ligands Determines Potency of T Cell Receptor Triggering.

T lymphocytes are important mediators of adoptive immunity but the mechanism of T cell receptor (TCR) triggering remains uncertain. The interspatial distance between engaged T cells and antigen-presenting cells (APCs) is believed to be important for topological rearrangement of membrane tyrosine phosphatases and initiation of TCR signaling. We investigated the relationship between ligand topology and affinity by generating a series of artificial APCs that express membrane-tethered anti-CD3 scFv with different affinities (OKT3, BC3, and 2C11) in addition to recombinant class I and II pMHC molecules. The dimensions of membrane-tethered anti-CD3 and pMHC molecules were progressively increased by insertion of different extracellular domains. In agreement with previous studies, elongation of pMHC molecules or low-affinity anti-CD3 scFv caused progressive loss of T cell activation. However, elongation of high-affinity ligands (BC3 and OKT3 scFv) did not abolish TCR phosphorylation and T cell activation. Mutation of key amino acids in OKT3 to reduce binding affinity to CD3 resulted in restoration of topological dependence on T cell activation. Our results show that high-affinity TCR ligands can effectively induce TCR triggering even at large interspatial distances between T cells and APCs.

Iron oxide nanoparticle-induced epidermal growth factor receptor expression in human stem cells for tumor therapy.

Superparamagnetic iron oxide (SPIO) nanoparticles show promise as labels for cellular magnetic resonance imaging (MRI) in the application of stem cell-based therapy. However, the unaddressed concerns about the impact of SPIO nanoparticles on stem cell attributes make the feasibility of SPIO labeling uncertain. Here, we show that the labeling of human mesenchymal stem cells (hMSCs) with ferucarbotran can induce epidermal growth factor receptor (EGFR) overexpression. Labeled hMSCs with their overexpressed EGFR were attracted by tumorous EGF and more effectively migrated toward tumor than unlabeled cells, resulting in more potent intrinsic antitumor activity. Moreover, the captured binding of tumorous EGF by overexpressed EGFR of labeled hMSCs blocked EGF/EGFR signaling-derived tumor growth, tumorous angiogenesis, and tumorous VEGF expression also responsible for tumor progression and development. Our results show that the impact of SPIO nanoparticles on stem cell attributes is not necessarily harmful but can be cleverly used to be beneficial to stem cell-based therapy.

YC-1 rescues cancer cachexia by affecting lipolysis and adipogenesis.

Loss of adipose tissue, primarily due to increased lipolysis but also to an impairment of adipogenesis, is a key feature of weight loss in cancer cachexia. Because of the myriad pathogenic signaling pathways essential for atrophy of adipose tissue, effective therapeutic agents for cachectic adipose loss are lacking and urgently needed. The authors evaluated the effects of YC-1 on adipogenesis of 3T3-L1 preadipocytes, TNF-α- and tumor-cell-induced lipolysis in 3T3-L1 adipocytes, and cachectic weight loss in colon-26 adenocarcinoma-bearing mice because YC-1 has been shown to possess versatile pharmacological actions, including anticancer activity. It was found that YC-1 promotes the differentiation of 3T3-L1 preadipocytes into adipocytes through activation of Akt and extracellular signal-regulated kinase (ERK) signaling pathways as well as activation of several adipogenic mediators, such as peroxisome proliferator-activated receptor γ (PPARγ), insulin receptor α (IRα), insulin receptor substrate-3 (IRS-3) and glucose transporter-4 (GLUT-4). In the in vitro lipolysis models, YC-1 attenuates TNF-α-induced lipolysis of adipocytes by antagonizing TNF-α-mediated activation of ERK and downregulation of perilipin (PLIN). It was also found that YC-1 inhibits colon-26 adenocarcinoma cell-induced lipolysis of 3T3-L1 adipocytes. Moreover, YC-1 effectively rescues cachectic weight loss in colon-26 adenocarcinoma-bearing mice by blocking lipolysis, involving insulin. Taken together the results show that YC-1 with its anticancer and anticachexia talents is highly worth developing as a novel agent for cancer therapy.

Mesoporous silica nanoparticles as a delivery system of gadolinium for effective human stem cell tracking.

The progress of using gadolinium (Gd)-based nanoparticles in cellular tracking lags behind that of superparamagnetic iron oxide (SPIO) nanoparticles in magnetic resonance imaging (MRI). Here, dual functional Gd-fluorescein isothiocyanate mesoporous silica nanoparticles (Gd-Dye@MSN) that possess green fluorescence and paramagnetism are developed in order to evaluate their potential as effective T1-enhancing trackers for human mesenchymal stem cells (hMSCs). hMSCs are labeled efficiently with Gd-Dye@MSN via endocytosis. Labeled hMSCs are unaffected in their viability, proliferation, and differentiation capacities into adipocytes, osteocytes, and chondrocytes, which can still be readily MRI detected. Imaging, with a clinical 1.5-T MRI system and a low incubation dosage of Gd, low detection cell numbers, and short incubation times is demonstrated on both loaded cells and hMSC-injected mouse brains. This study shows that the advantages of biocompatibility, durability, high internalizing efficiency, and pore architecture make MSNs an ideal vector of T1-agent for stem-cell tracking with MRI.

Internalization of mesoporous silica nanoparticles induces transient but not sufficient osteogenic signals in human mesenchymal stem cells.

The biocompatibility of nanoparticles is the prerequisite for their applications in biomedicine but can be misleading due to the absence of criteria for evaluating the safety and toxicity of those nanomaterials. Recent studies indicate that mesoporous silica nanoparticles (MSNs) can easily internalize into human mesenchymal stem cells (hMSCs) without apparent deleterious effects on cellular growth or differentiation, and hence are emerging as an ideal stem cell labeling agent. The objective of this study was to thoroughly investigate the effect of MSNs on osteogenesis induction and to examine their biocompatibility in hMSCs. Uptake of MSNs into hMSCs did not affect the cell viability, proliferation and regular osteogenic differentiation of the cells. However, the internalization of MSNs indeed induced actin polymerization and activated the small GTP-bound protein RhoA. The MSN-induced cellular protein responses as believed to cause osteogenesis of hMSCs did not result in promotion of regular osteogenic differentiation as analyzed by cytochemical stain and protein activity assay of alkaline phosphatase (ALP). When the effect of MSNs on ALP gene expression was further examined by reverse transcriptase polymerase chain reaction, MSN-treated hMSCs were shown to have significantly higher mRNA expression than control cells after 1-hour osteogenic induction. The induction of ALP gene expression by MSNs, however, was absent in cells after 1-day incubation with osteogenic differentiation. Together our results show that the internalization of MSNs had a significant effect on the transient protein response and osteogenic signal in hMSCs, thereby suggesting that the effects of nanoparticles on diverse aspects of cellular activities should be carefully evaluated even though the nanoparticles are generally considered as biocompatible at present.

The effect of surface charge on the uptake and biological function of mesoporous silica nanoparticles in 3T3-L1 cells and human mesenchymal stem cells.

Cellular uptake of nanoparticles for stem cell labeling/tracking is considered as the most promising method. Recently mesoporous silica nanoparticles (MSNs) are emerging as an idea agent for efficient stem cell labeling. The objective of this study was to evaluate the effect of surface charge on the highly efficient cellular uptake and in vitro cytotoxicity of MSNs in human mesenchymal stem cells (hMSCs). The surface charge was varied by the degree of surface modification with N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride and the uptake of MSNs was detected by flow cytometry. 3T3-L1 cells were also used to compare the uptake behavior of MSNs between cell types. A clear correlation of positive surface charge and the number of fluorescence-labeled cells was mainly observed in 3T3-L1 cells. In both cells, uptake of unmodified MSNs was inhibited by phenylarsine oxide (PAO) and cytochalasin D (Cyt D) suggesting a clathrin- and an actin-dependent endocytosis were involved. With strongly positive-charged MSNs, the inhibitory effects were observed in 3T3-L1 cells but not in hMSCs. Without regard to the surface charge, uptake of MSNs into both cells did not affect their viability, proliferation, and differentiation. Our results show that MSNs uptake by hMSCs can be regulated by a threshold of positive surface charge but also imply that the modulation of surface charge on MSNs uptake is specific to cell type.

Bifunctional magnetic silica nanoparticles for highly efficient human stem cell labeling.

A superparamagnetic iron oxide (SPIO) nanoparticle is emerging as an ideal probe for noninvasive cell tracking. However, its low intracellular labeling efficiency has limited the potential usage and has evoked great interest in developing new labeling strategies. We have developed fluorescein isothiocyanate (FITC)-incorporated silica-coated core-shell SPIO nanoparticles, SPIO@SiO2(FITC), with diameters of 50 nm, as a bifunctionally magnetic vector that can efficiently label human mesenchymal stem cells (hMSCs), via clathrin- and actin-dependent endocytosis with subsequent intracellular localization in late endosomes/lysosomes. The uptake process displays a time- and dose-dependent behavior. In our system, SPIO@SiO2(FITC) nanoparticles induce sufficient cell MRI contrast at an incubation dosage as low as 0.5 microg of iron/mL of culture medium with 1.2x105 hMSCs, and the in vitro detection threshold of cell number is about 1x104 cells. Furthermore, 1.2x105 labeled cells can also be MRI-detected in a subcutaneous model in vivo. Labeled hMSCs are unaffected in their viability, proliferation, and differentiation capacities into adipocytes and osteocytes which can still be readily MRI detected. This is the first report that hMSCs can be efficiently labeled with MRI contrast nanoparticles and can be monitored in vitro and in vivo with a clinical 1.5-T MRI imager under low incubation concentration of iron oxide, short incubation time, and low detection cell numbers at the same time.