Mucopolysaccharidosis type III (Sanfilippo) is comprised of four phenotypically similar lysosomal storage disorders (MPS IIIA-D) caused by the deficiency of enzymes that catabolise heparan sulphate (HS). Progressive accumulation of HS results in abnormal behaviour, progressive cognitive and motor impairment and death in mid-teens. There are currently no treatments for MPS III. To assess the effect of novel therapeutics in the mouse models of MPS III it is necessary to examine the effect on primary storage of HS, secondary storage and behaviour. The reported behaviour of MPS IIIA and B mice is conflicting therefore we developed a one-hour open field test, performed at the same time of day during a period of hyperactivity observed in a previous circadian rhythm study of MPS IIIB mice. At 8 months of age MPS IIIB mice were hyperactive, with increased rapid exploratory behaviour and a reduction in immobility time. The MPS IIIA mice presented with the same behavioural phenotype as the MPS IIIB mice and were significantly hyperactive at 4 and 6 months of age and also displayed a reduced sense of danger. The hyperactivity and reduced sense of danger observed in the mice is consistent with the patient phenotype. Whilst haematopoietic stem cell transplant (HSCT) is the standard therapy used to treat the similar HS storage disorder MPS I Hurler, it is ineffectual in MPS IIIA. We hypothesise that HSCT failure in MPS IIIA is due to insufficient enzyme production in the brain by donor-derived microglial cells. By increasing expression of N-sulphoglucosamine sulphohydrolase (SGSH) we may be able to treat MPS IIIA. Therefore we compared the effect of HSCT using normal haematopoietic stem cells (WT-HSCT) to lentiviral overexpression of SGSH in normal cells (LV-WT-HSCT) or MPS IIIA cells (LV-IIIA-HSCT) in MPS IIIA mice, using the behavioural tests developed.SGSH activity in the brain of MPS IIIA recipients was not significantly increased by WT-HSCT, but was significantly increased by LV-IIIA-HSCT and LV-WT-HSCT. HS was significantly reduced by all transplants but the best treatment was LV-WT-HSCT. Neuroinflammation, indicated by the number of microglia in the brain, was significantly reduced by all treatments but remains significantly elevated. GM2 gangliosides were significantly reduced by WT-HSCT and LV-WT-HSCT and were no longer significantly elevated, but LV-IIIA-HSCT had no significant effect. Critically LV-WT-HSCT corrected the behaviour at 4 and 6 months of age whilst the other treatments had no significant effect. LV-WT-HSCT and WT-HSCT reduced GM2 gangliosides and neuroinflammation equally but only LV-WT-HSCT corrected behaviour and primary HS storage, suggesting they are the important factors in MPS IIIA pathology. LV-WT-HSCT corrects the neurological phenotype in MPS IIIA mice and is a clinically viable approach to treat MPS IIIA and other neuropathic lysosomal storage disorders.

Human cytomegalovirus (HCMV, Herpesviridae) can cause severe complications in the infected individuals undergoing hematopoietic stem cell transplantation. Nowadays, these patients are treated using antivirotics or HCMV-specific T cells derived from the seropositive graft donor. This study explored the possibility of redirecting HCMV-non-specific T cells from a seronegative donor towards HCMV-infected cells via chimeric antigen receptor (CAR), i.e. artificially designed T cell receptor. Viral glycoprotein B (gB) has been selected as a target for this receptor. Published sequence of a single chain variable fragment of a human antibody was used for the design of the CAR against gB (gBCAR). After the verification of production and surface localization in cell lines, gBCAR was being introduced into human T cells via lentiviral vectors. Human fetal lung fibroblasts (LEP) infected with HCMV were used as target cells after the expression of gB at their surface was demonstrated. gBCAR functionality was evaluated by the incubation of modified T cells with infected cells and subsequent analysis of media for IFNγ concentration, which was significantly higher in the setting of gBCAR T cells incubated with HCMV-LEP than in the control incubations. The results obtained show the specificity of gBCAR against...

Background: The CD19 chimeric antigen receptor (CAR) adoptive T-cell therapy for B-cell leukemia is a promising treatment for relapsed or refractory malignities. The overall response rate of CD19 CAR-T cells in clinical trials was greater than 80% for patients with B-cell acute lymphoblastic leukemia (B-ALL) and non-Hodgkin's lymphoma (NHL). However, CAR-T cell therapy of leukemias and solid tumors has been limited by a lot of factors such as antigen loss of tumor escape variants, reduced proliferation, persistence and tumor-infiltration of CAR-T cells in vivo, immunosuppressive tumor environment, absence of ideal antigens and on-target, off-tumor toxicities. Therefore, new strategies improving the safety and efficacy of CAR-T cells, including further T-cell modification to overcome the immune suppression, are tested. Aims: (i) Bispecific CARs designed to express two antigen-binding domains prevent of antigen escape. (ii) T-cells were genetically modified to express CAR along with an inducible IL-21 gene cassette driven by NFAT-responsive promoter. IL-21 directly enhances CAR-T cell activity and anti-tumor effects. (iii) Applying suicide epitope modification in CAR enables significantly increasing the therapeutic safety of CAR-T cells. Methods: CARs were constructed by using molecular biology...

Improving the repopulation potential of human umbilical cord blood (UCB) haemopoietic stem cells (HSCs) remains a paramount goal in HSC transplantation (HSCT) therapy. This implies enhancing the homing and engraftment potential of UCB-CD34+CD133+ cells to the bone marrow (BM). Although an array of molecules continues to be identified as ‘key’ homing molecules, the molecular mechanisms controlling HSC homing are still not fully understood. The regulatory implications of hypoxia in the BM, with the concomitant stabilisation of hypoxia inducible transcription factor-1α (HIF-1α), are becoming more apparent, yet at the commencement of this thesis no study had explored whether hypoxia induced signalling can be adopted to regulate the homing and engraftment of transplanted HSCs. The aim of this DPhil project was thus to investigate whether hypoxic conditions as detected in the BM influence the adhesion of UBC-CD133+ cells to osteoblasts, BM stromal cells and BM endothelial cells-60 (BMEC-60), as well as their transmigration towards chemokine SDF-1α across BMEC-60. Increasing the exposure of UCB-CD133+ cells to 1.5% O2 doubled the percentage of transmigrating cells (p<0.05), and while hypoxia stimulated UCB-CD133+ cells preferentially adhered to IL-1β stimulated BMEC-60, their adhesion to non-stimulated (BMEC-60) was significantly improved (p<0.001). To help unravel the underlying molecular mechanisms, we attempted to examine the potential involvement of hypoxia regulated scaffolding protein HEF-1/NEDD9/Cas-L (HEF-1) in the increased percentage of migrating UCB-CD133+ cells after hypoxia pre-conditioning. The role of HEF-1 in HSCs is unexplored, and its multifunctional contribution in a variety of processes including cell migration, attachment and invasion make HEF-1 a prime candidate as a contributing homing molecule. After identifying a suitable short-hairpin RNA (shRNA) sequence to knockdown HEF-1, generating lentiviral (LV)-particles in house and optimising transduction protocols, HEF-1 knockdown was achieved in haemopoietic model cell lines KG-1 and KG-1A (KG-1/KG-1A–HEF1). Significantly decreased KG-1A–HEF1 cell adhesion to non-stimulated BMEC-60 was detected. Together, these studies provide a promising platform to further explore the role of HEF-1 in hypoxia induced UCB-CD133+ cell transmigration towards the key homing molecule SDF-1α.

Indiana University-Purdue University Indianapolis (IUPUI) / Immunotherapy for cancer has held much promise as a potent modality of cancer treatment. The ability to selectively destroy diseased cells and leave healthy cells unharmed has been the goal of cancer immunotherapy for the past thirty years. However, the full capabilities of cancer immunotherapies have been elusive. Cancer immunotherapies have been consistently hampered by limited immune reactivity, a diminishing immune response over time, and a failure to overcome self-tolerance. Many of these deficiencies have been borne-out by immunotherapies that have focused on the adoptive transfer of activated or genetically modified mature CD8+ T cells. The limitations inherent in therapies involving terminally differentiated mature lymphocytes include limited duration, lack of involvement of other components of the immune system, and limited clinical efficacy. We sought to overcome these limitations by altering and enhancing long-term host immunity by genetically modifying then transplanting HSCs. To study these questions and test the efficiency of gene transfer, we cloned a tumor reactive HLA-DR4-restricted CD4+ TCR specific for the melanocyte differentiation antigen TRP-1, then constructed both a high expression lentiviral delivery system and a TCR Tg expressing the same TCR genes. We demonstrate with both mouse and human HSCs durable, high-efficiency TCR gene transfer, following long-term transplantation. We demonstrate the induction of spontaneous autoimmune vitiligo and a TCR-specific TH1 polarized memory effector CD4+ T cell population. Most importantly, we demonstrate the destruction of subcutaneous melanoma without the aid of vaccination, immune modulation, or cytokine administration. Overall, these results demonstrate the creation of a novel translational model of durable lentiviral gene transfer, the induction of spontaneous CD4+ T cell immunity, the breaking of self-tolerance, and the induction of anti-tumor immunity.

Neurological disorders affect more than 14% of the population worldwide and together with traumatic brain and spinal cord injuries represent major health, public and economic burden of the society. Incidence of inherited and idiopathic neurodegenerative disorders and acute CNS injuries is growing globally while neuroscience society is being challenged by numerous unanswered questions. Therefore, research of the CNS disorders is essential. Since animal models of the CNS diseases and injuries represent the key step in the conversion of the basic research to the clinics, we focused our work on generation of new animal models and on their use in pre-clinical research. We generated and characterized transgenic minipig model of Huntington's disease (HD) which represents the only successful establishment of a transgenic model of HD in minipig which should be valuable for testing of long term safety of HD therapeutics. Next, we crossed the well characterized R6/2 mouse HD model with the gad mouse model which lacks the expression of UCHL1 which led to results that support the theory of "protective" role of mutant huntingtin aggregates and suggest that UCHL1 function(s) may be affected in HD disturbing certain branches of Ubiquitin Proteasome System. Traumatic spinal cord injury and Amyotrophic Lateral...