Hematopoietic stem cell gene therapy for the treatment of SYNGAP1-related non-specific intellectual disability.

BACKGROUND: Synaptic Ras GTPase activating protein 1 (SYNGAP1)-related non-specific intellectual disability is a neurodevelopmental disorder caused by an insufficient level of SynGAP1 resulting in a dysfunction of neuronal synapses and presenting with a wide array of clinical phenotypes. Hematopoietic stem cell gene therapy has the potential to deliver therapeutic levels of functional SynGAP1 to affected neurons upon transduction of hematopoietic stem and progenitor cells with a lentiviral vector. METHODS: As a novel approach toward the treatment of SYNGAP1, we have generated a lentiviral vector expressing a modified form of SynGAP1 for transduction of human CD34+ hematopoietic stem and progenitor cells. The gene-modified cells were then transplanted into adult immunodeficient SYNGAP1+/- heterozygous mice and evaluated for improvement of SYNGAP1-related clinical phenotypes. Expression of SynGAP1 was also evaluated in the brain tissue of transplanted mice. RESULTS: In our proof-of-concept study, we have demonstrated significant improvement of SYNGAP1-related phenotypes including an improvement in motor abilities observed in mice transplanted with the vector transduced cells because they displayed decreased hyperactivity in an open field assay and an increased latency to fall in a rotarod assay. An increased level of SynGAP1 was also detected in the brains of these mice. CONCLUSIONS: These early-stage results highlight the potential of this stem cell gene therapy approach as a treatment strategy for SYNGAP1.

Highly Efficient Differentiation of Endothelial Cells from Pluripotent Stem Cells Requires the MAPK and the PI3K Pathways.

Pluripotent stem cells are a promising source of endothelial cells (ECs) for the treatment of vascular diseases. We have developed a robust protocol to differentiate human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) into ECs with high purities (94%-97% CD31+ and 78%-83% VE-cadherin+ ) in 8 days without cell sorting. Passaging of these cells yielded a nearly pure population of ECs (99% of CD31+ and 96.8% VE-cadherin+ ). These ECs also expressed other endothelial markers vWF, Tie2, NOS3, and exhibited functions of ECs such as uptake of Dil-acetylated low-density lipoprotein and formation of tubes in vitro or vessels in vivo on matrigel. We found that FGF2, VEGF, and BMP4 synergistically induced early vascular progenitors (VPs) from hiPSC-derived mesodermal cells. The MAPK and PI3K pathways are crucial not only for the initial commitment to vascular lineages but also for the differentiation of vascular progenitors to ECs, most likely through regulation of the ETS family transcription factors, ERG and FLI1. We revealed novel roles of the p38 and JNK MAPK pathways on EC differentiation. Furthermore, inhibition of the ERK pathway markedly promoted the differentiation of smooth muscle cells. Finally, we demonstrate that pluripotent stem cell-derived ECs are capable of forming patent blood vessels that were connected to the host vasculature in the ischemic limbs of immune deficient mice. Thus, we demonstrate that ECs can be efficiently derived from hiPSCs and hESCs, and have great potential for vascular therapy as well as for mechanistic studies of EC differentiation. Stem Cells 2017;35:909-919.

Preclinical evaluation of mesenchymal stem cells overexpressing VEGF to treat critical limb ischemia.

Numerous clinical trials are utilizing mesenchymal stem cells (MSC) to treat critical limb ischemia, primarily for their ability to secrete signals that promote revascularization. These cells have demonstrated clinical safety, but their efficacy has been limited, possibly because these paracrine signals are secreted at subtherapeutic levels. In these studies the combination of cell and gene therapy was evaluated by engineering MSC with a lentivirus to overexpress vascular endothelial growth factor (VEGF). To achieve clinical compliance, the number of viral insertions was limited to 1-2 copies/cell and a constitutive promoter with demonstrated clinical safety was used. MSC/VEGF showed statistically significant increases in blood flow restoration as compared with sham controls, and more consistent improvements as compared with nontransduced MSC. Safety of MSC/VEGF was assessed in terms of genomic stability, rule-out tumorigenicity, and absence of edema or hemangiomas in vivo. In terms of retention, injected MSC/VEGF showed a steady decline over time, with a very small fraction of MSC/VEGF remaining for up to 4.5 months. Additional safety studies completed include absence of replication competent lentivirus, sterility tests, and absence of VSV-G viral envelope coding plasmid. These preclinical studies are directed toward a planned phase 1 clinical trial to treat critical limb ischemia.

Hypoxic pre-conditioning increases the infiltration of endothelial cells into scaffolds for dermal regeneration pre-seeded with mesenchymal stem cells.

Many therapies using mesenchymal stem cells (MSC) rely on their ability to produce and release paracrine signals with chemotactic and pro-angiogenic activity. These characteristics, however, are mostly studied under standard in vitro culture conditions. In contrast, various novel cell-based therapies imply pre-seeding MSC into bio-artificial scaffolds. Here we describe human bone marrow-derived MSC seeded in Integra matrices, a common type of scaffold for dermal regeneration (SDR). We show and measured the distribution of MSC within the SDR, where cells clearly establish physical interactions with the scaffold, exhibiting constant metabolic activity for at least 15 days. In the SDR, MSC secrete VEGF and SDF-1α and induce transwell migration of CD34(+) hematopoietic/endothelial progenitor cells, which is inhibited in the presence of a CXCR4/SDF-1α antagonist. MSC in SDR respond to hypoxia by altering levels of angiogenic signals such as Angiogenin, Serpin-1, uPA, and IL-8. Finally, we show that MSC-containing SDR that have been pre-incubated in hypoxia show higher infiltration of endothelial cells after implantation into immune deficient mice. Our data show that MSC are fully functional ex vivo when implanted into SDR. In addition, our results strongly support the notion of hypoxic pre-conditioning MSC-containing SDR, in order to promote angiogenesis in the wounds.

In vivo validation and 3D visualization of broadband ultrasound molecular imaging.

Ultrasound can selectively and specifically visualize upregulated vascular receptors through the detection of bound microbubbles. However, most current ultrasound molecular imaging methods incur delays that result in longer acquisition times and reduced frame rates. These delays occur for two main reasons: 1) multi-pulse imaging techniques are used to differentiate microbubbles from tissue and 2) acquisition occurs after free bubble clearance (>6 minutes) in order to differentiate bound from freely circulating microbubbles. In this paper, we validate tumor imaging with a broadband single pulse molecular imaging method that is faster than the multi-pulse methods typically implemented on commercial scanners. We also combine the single pulse method with interframe filtering to selectively image targeted microbubbles without waiting for unbound bubble clearance, thereby reducing acquisition time from 10 to 2 minutes. The single pulse imaging method leverages non-linear bubble behavior by transmitting at low and receiving at high frequencies (TLRH). We implemented TLRH imaging and visualized the accumulation of intravenously administrated integrin-targeted microbubbles in a phantom and a Met-1 mouse tumor model. We found that the TLRH contrast imaging has a ~2-fold resolution improvement over standard contrast pulse sequencing (CPS) imaging. By using interframe filtering, the tumor contrast was 24.8±1.6 dB higher after the injection of integrin-targeted microbubbles than non-targeted control MBs, while echoes from regions lacking the target integrin were suppressed by 26.2±2.1 dB as compared with tumor echoes. Since real-time three-dimensional (3D) molecular imaging provides a more comprehensive view of receptor distribution, we generated 3D images of tumors to estimate their volume, and these measurements correlated well with expected tumor sizes. We conclude that TLRH combined with interframe filtering is a feasible method for 3D targeted ultrasound imaging that is faster than current multi-pulse strategies.

Insonation of targeted microbubbles produces regions of reduced blood flow within tumor vasculature.

OBJECTIVES: In ultrasound molecular imaging, a sequence of high-pressure ultrasound pulses is frequently applied to destroy bound targeted microbubbles, to quantify accumulated microbubbles or to prepare for successive microbubble injections; however, the potential for biological effects from such a strategy has not been fully investigated. Here, we investigate the effect of high-pressure insonation of bound microbubbles and the potential for thrombogenic effects. MATERIALS AND METHODS: A total of 114 mice carrying either Met-1 or neu deletion mutant (NDL) tumors was insonified (Siemens Sequoia system, 15L8 transducer, 5-MHz color-Doppler pulses, 4 or 2 MPa peak-negative pressure, 8.1-millisecond pulse repetition period, 6-cycle pulse length, and 900-millisecond insonation). Microbubbles conjugated with cyclic-arginine-glycine-aspartic acid (cRGD) or cyclic-aspartic-acid-glycine-tyrosine (3-NO)-glycine-hydroxyproline-asparagine (LXY-3) peptides or control (no peptide) microbubbles were injected, and contrast pulse sequencing was used to visualize the flowing and bound microbubbles. An anti-CD41 antibody was injected in a subset of animals to block potential thrombogenic effects. RESULTS: After the accumulation of targeted microbubbles and high-pressure (4 MPa) insonation, reduced blood flow, as demonstrated by a reduction in echoes from flowing microbubbles, was observed in 20 Met-1 mice (71%) and 4 NDL mice (40%). The area of low image intensity increased from 22 ± 13% to 63 ± 17% of the observed plane in the Met-1 model (P < 0.01) and from 16 ± 3% to 45 ± 24% in the NDL model (P < 0.05). Repeated microbubble destruction at 4 MPa increased the area of low image intensity to 76.7 ± 13.4% (P < 0.05). The fragmentation of bound microbubbles with a lower peak-negative pressure (2 MPa) reduced the occurrence of the blood flow alteration to 28% (5/18 Met-1 tumor mice). The persistence of the observed blood flow change was approximately 30 minutes after the microbubble destruction event. Dilated vessels and enhanced extravasation of 150 kDa fluorescein-isothiocyanate (FITC)-dextran were observed by histology and confocal microscopy. Preinjection of an anti-CD41 antibody blocked the reduction of tumor blood flow, where a reduction in blood flow was observed in only 1 of 26 animals. CONCLUSION: High-pressure fragmentation of microbubbles bound to tumor endothelial receptors reduced blood flow within 2 syngeneic mouse tumor models for ∼30 minutes. Platelet activation, likely resulting from the injury of small numbers of endothelial cells, was the apparent mechanism for the flow reduction.