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BackgroundNeuroblastoma is the second most common extracranial cancer in children. Current therapies for neuroblastoma, which use a combination of chemotherapy drugs, have limitations for high-risk subtypes and can cause significant long-term adverse effects in young patients. Therefore, a new therapy is needed. In this study, we investigated the transcription factor MXD3 as a potential therapeutic target in neuroblastoma.MethodsMXD3 expression was analyzed in five neuroblastoma cell lines by immunocytochemistry and quantitative real-time reverse transcription PCR, and in 18 primary patient tumor samples by immunohistochemistry. We developed nanocomplexes using siRNA and superparamagnetic iron oxide nanoparticles to target MXD3 in neuroblastoma cell lines in vitro as a single-agent therapeutic and in combination with doxorubicin, vincristine, cisplatin, or maphosphamide - common drugs used in current neuroblastoma treatment.ResultsMXD3 was highly expressed in neuroblastoma cell lines and in patient tumors that had high-risk features. Neuroblastoma cells treated in vitro with the MXD3 siRNA nanocomplexes showed MXD3 protein knockdown and resulted in cell apoptosis. Furthermore, on combining MXD3 siRNA nanocomplexes with each of the four drugs, all showed additive efficacy.ConclusionThese results indicate that MXD3 is a potential new target and that the use of MXD3 siRNA nanocomplexes is a novel therapeutic approach for neuroblastoma.

Background Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the Factor VIII (FVIII) gene leading to deficient blood coagulation. As a monogenic disorder, HA is an ideal target for cell-based gene therapy, but successful treatment has been hampered by insufficient engraftment of potential therapeutic cells. Methods In this study, we sought to determine whether co-transplantation of endothelial colony-forming cells (ECFCs) and placenta-derived mesenchymal stromal cells (PMSCs) can achieve long-term engraftment and FVIII expression. ECFCs and PMSCs were transduced with a B domain deleted factor VIII (BDD-FVIII) expressing lentiviral vector and luciferase, green fluorescent protein or Td-Tomato containing lentiviral tracking vectors. They were transplanted intramuscularly into neonatal or adult immunodeficient mice. Results In vivo bioluminescence imaging showed that the ECFC only and the co-transplantation groups but not the PMSCs only group achieved long-term engraftment for at least 26 weeks, and the co-transplantation group showed a higher engraftment than the ECFC only group at 16 and 20 weeks post-transplantation. In addition, cell transplantation at the neonatal age achieved higher engraftment than at the adult age. Immunohistochemical analyses further showed that the engrafted ECFCs expressed FVIII, maintained endothelial phenotype, and generated functional vasculature. Next, co-transplantation of ECFCs and PMSCs into F8 knock-out HA mice reduced the blood loss volume from 562.13 ± 19.84 μl to 155.78 ± 44.93 μl in a tail-clip assay. Conclusions This work demonstrated that co-transplantation of ECFCs with PMSCs at the neonatal age is a potential strategy to achieve stable, long-term engraftment, and thus holds great promise for cell-based treatment of HA. Electronic supplementary material The online version of this article (10.1186/s13287-019-1138-8) contains supplementary material, which is available to authorized users.

The use of cell transplantation for spinal cord injury is a rapidly evolving field in regenerative medicine. Numerous animal models are currently being used. However, translation to human patients is still a challenging step. Dogs are of increasing importance as a translational model for human disease since there is a greater awareness of the need to increase the quality of preclinical data. The use of dogs ultimately brings benefit to both human and veterinary medicine. In this review we analyze experimental and clinical studies using cell transplantation for canine spinal cord injury. Overall, in experimental studies, transplantation groups showed improvement over control groups. Improvements were measured at the functional, electrophysiological, histological, RNA and protein levels. Most clinical studies support beneficial effects of cell transplantation despite the fact that methodological limitations preclude definitive conclusions. However, the mechanisms of action and underlying the behavior of transplanted cells in the injured spinal cord remain unclear. Overall, we conclude here that stem cell interventions are a promising avenue for the treatment of spinal cord injury. Canines are a promising model that may help bridge the gap between translational research and human clinical trials.

A clinical trial of retroviral-mediated transfer of the adenosine deaminase (ADA) gene into umbilical cord blood CD34(+) cells was started in 1993. ADA-containing peripheral blood mononuclear cells (PBMCs) have persisted in patients from this trial, with T lymphocytes showing the highest prevalence of gene marking. To gain a greater understanding of the nature and number of the transduced cells that were engrafted, we used linear amplification-mediated PCR (LAM-PCR) to identify clonal vector proviral integrants. In one patient, a single vector integrant was predominant in T lymphocytes at a stable level over most of the eight-year time span analyzed and was also detected in some myeloid samples. T-cell clones with the predominant integrant, isolated after eight years, showed multiple patterns of T-cell receptor (TCR) gene rearrangement, indicating that a single pre-thymic stem or progenitor cell served as the source of the majority of the gene-marked cells over an extended period of time. It is important to distinguish the stable pattern of monoclonal gene marking that we observed here from the progressive increase of a T-cell clone with monoclonal gene marking that results from leukemic transformation, as observed in two subjects in a clinical trial of gene therapy for X-linked severe combined immunodeficiency (SCID).

Recent evidence suggests that expression of CD34 on the cell membrane does not always correlate with stem cell activity. In the mouse, there is a highly quiescent population of stem cells that lacks CD34 expression, but has full reconstituting capacity. The current review addresses the discovery of a similar population of dormant CD34-negative human hematopoietic stem cells. This information casts some uncertainty on the benefits of CD34+ cell isolation for stem cell transplantation, until more is known about the novel CD34-negative stem cell population. Methods designed to achieve removal of specific mature blood cell lineages might prove to be most advantageous in the future.

The lack of human B lymphocyte development in beige/nude/XID (bnx) mice is in sharp contrast to the robust development observed in another immune deficient strain, the NOD/SCID mouse. The ability to generate human B lymphocytes in the NOD/SCID, but not bnx mouse has been hypothesized to be caused by differences in the microenvironments or systemic cytokine concentrations. In the current studies we report that the differences in development can be primarily attributed to the source of the progenitors transplanted into the mice. The prior studies in bnx mice used cultured pediatric or adult bone marrow (BM) as the source of the CD34+ cells, whereas the NOD/SCID studies have predominantly used fresh or cultured umbilical cord blood (UCB). We have analyzed BM and UCB for the number of human CD34+/CD38- cells capable of in vitro B lymphocyte development, and have found a lower frequency of B lymphocyte generation in BM. The individual B lymphocyte clones that developed from bone marrow produced 100-fold fewer cells than the UCB-derived clones. In agreement with the in vitro studies, human B lymphocytes developed in bnx mice from both CD34+ and CD34+/CD38- cells isolated from human umbilical cord blood, but not from equivalent numbers of CD34+ and CD34+/CD38- progenitors from bone marrow. Therefore, the lower generative capacity, and frequency of B lymphocyte precursors in human marrow may be responsible for the previous results that showed a lack of B lymphocyte development in bnx mice.

Pluripotent hematopoietic stem cells (HSC) are the ideal targets for gene transfer because they can repopulate a sublethally irradiated recipient, giving rise to all lineages of blood cells. Thus, introduction of a corrected gene into HSC (stem cell gene therapy) should ensure persistent transmission of the gene. To date, the most efficient mode of gene delivery is via Moloney murine leukemia virus (MoMuLV)-based retroviral vectors which stably integrate into the genome of the target cell. The quiescent nature of HSC and the fact that MoMuLV-based retroviral vectors can only integrate into dividing cells are major obstacles in gene therapy. While increasing efforts have been directed toward identifying growth factors which facilitate division of primary hematopoietic progenitor and stem cells, little is known about the molecular mechanisms which these cells use to enter cell cycle. In this review, we will discuss the correlation between the hematopoietic inhibitory and growth factors and their impact on the regulation of the cell cycle components.

Haematopoietic stem cells in umbilical cord blood are an attractive target for gene therapy of inborn errors of metabolism. Three neonates with severe combined immunodeficiency were treated by retroviral-mediated transduction of the CD34+ cells from their umbilical cord blood with a normal human adenosine deaminase complementary DNA followed by autologous transplantation. The continued presence and expression of the introduced gene in leukocytes from bone marrow and peripheral blood for 18 months demonstrates that umbilical cord blood cells may be genetically modified with retroviral vectors and engrafted in neonates for gene therapy.

One of the greatest challenges facing biomedical research since the sequencing of the human genome has been to understand the role of genetic variation in human disease. Many genetic variants have been associated with common diseases. However, determining the functional consequences of these variants has been hard. Several variants are often inherited together in tightly linked blocks, making it difficult to determine the causative variant. People have millions of other genetic differences, making it difficult to correlate cellular phenotypes with a particular variant. Different gene sets are expressed in different cells, but it is difficult to extract disease-relevant cells from large numbers of patients. We describe a method with the potential to revolutionize the functional analysis of genetic variation, using custom nucleases to genetically modify individual variants in induced pluripotent stem cells. This process would provide unprecedented analytical power, present the first general method to determine if a variant is causative, and analyze function disease-relevant cell types. We will focus on variants at the 9p21 region of the genome that have been associated with coronary artery disease (CAD). The methods should provide a new way to unlock the wealth of data from genome-wide association studies, and to probe the genetic architecture of common diseases. We will describe our improved methods for inexpensive and rapid construction of highly active zinc finger and TALE nucleases to examine the functional role of polymorphisms at the 9p21 CAD risk locus.

Mesenchymal stem cells (MSC) implanted into the brain have shown strong neurorestorative effects in animal models of Huntington's disease (HD) and other neurodegenerative disorders. In the brain tissue, MSC reduce inflammation, neutralize reactive oxygen species, and diminish apoptosis to delay the loss of neurons. For the past 20-plus years, our group has studied the biosafety of human MSC engineered to produce cytokines and other factors in vivo, and we have shown sustained expression in many tissues for at least 18 months. However, low numbers of MSC cross the blood-brain barrier in our chronic disease models. Intracranial (IC) implantation of MSC and brain-derived neurotrophic factor (BDNF)–modified MSC have been shown to be safe and effective in rodent HD models. Clinical trials of MSC infusion into the brains of humans with ALS and traumatic brain injury have been done without adverse events, but therapies using this IC delivery route for MSC have not yet been initiated in the U.S.A. through FDA-approved trials. Our ongoing studies are evaluating the biosafety of MSC and then inducible BDNF-modified MSC implantation into the brains of rodents and non-human primates to generate data for the FDA in support of an initial planned phase 1 clinical trial to treat HD. We will next validate the biosafety of our patented approach to use MSC to directly deliver small interfering RNA (siRNA) into damaged and at-risk neurons for the second planned trial. SiRNA directed against mutant huntingtin mRNA are effective at ameliorating the neurodegenerative effects seen in HD and other disorders, since the mutant protein is no longer made. But it is very difficult to get siRNA across the blood-brain barrier in human patients, which has thwarted effective clincial use. We have shown that MSC can move nimbly through tissues and deliver their contents directly into target cells. Thus, for the second planned trial, we will use MSC to continually deliver anti-mutant htt siRNA directly into damaged striatal neurons, to reduce levels of mutant htt RNA and protein. Since there are currently no cures or highly effective treatments for HD, there is a high potential benefit-to-risk ratio for these studies.

Bone marrow transplant (BMT) is followed by a period of profound immune deficiency, which is a major problem in clinical BMT and is likely to be important for gene therapy directed at immunologic diseases, e.g., SCID or AIDS. We have previously shown that human IL-7 (huIL-7) induces early thymic reconstitution in syngeneic murine BMT recipients (C57/B6). Another approach to achieve the same effect as multiple IL-7 injections would be to co-transplant bone marrow stroma engineered to produce high amounts of IL-7. Murine bone marrow stromal cultures were transduced with the retroviral vector JZEN hIL-7/tk neo to express huIL-7 (>1000pg/106 cells/24hours). Mice received T cell depleted BMT (TCD-BMT) and 5×106 stromal cells transduced with the human IL-7 gene (IL-7STR) via tail vein injection. Control animals received the same TCD-BMT but were co-transplanted with stromal cells transduced with the LNCX vector, which expresses only the neomycin resistance gene (LN-STR). Co-transplantation of the IL-7STR resulted in enhanced thymic reconstitution by day 28. The mean thymic cell number was 110 ± 7.5×106 in normal mice, 88 ± 21×106 in IL-7STR mice, and 9.8 ± 6.7×106 for the LN-STR mice. Thymic subsets in IL-7STR and normal mice were comparable, but LN-STR mice had a block in early thymic maturation. T and B cell function was tested using the T cell mitogen ConA and the T-dependent antibody response to sheep RBC. Both proliferative responses to ConA and anti-SRBC titers developed sooner and were of greater magnitude in IL-7STR than in LN-STR mice. Transduced bone marrow stromal cells producing huIL-7 (1068-968 pg/106 transduced stromal cells/24hours) could be cultured from the thymus on D28 post-BMT. No toxic effects of the IL-7 STR have been observed in recipient mice, with follow up of 1 year. Thus, bone marrow stroma transduced to overexpress IL-7 has the same effects on post-BMT thymopoiesis as IL-7 injections. Clinical strategies using IL-7STR gene therapy may be useful in enhancing thymopoiesis in BMT, SCID gene therapy, or AIDS patients.