Gene therapeutic approaches for the treatment of vascular pathologies driven by Transforming Growth Factor-Beta

Coronary heart disease is a leading cause of death in Scotland. Although lifestyle changes and prescription drugs can help treat coronary heart disease, revascularisation surgeries such as percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG) are sometimes necessary. The development of intimal hyperplasia after PCI or CABG significantly reduces their success rates, resulting in risky and costly repeat interventions. The cytokine transforming growth factor-beta (TGFß) promotes intimal hyperplasia and represents a promising target for gene therapy. However TGFß regulates many important cell and tissue processes, which means that a targeted and selective approach to modulate TGFß is essential. Additionally, recent studies have identified a novel TGFß signalling pathway that is activated during neointima formation in rodent coronary heart disease models. My work is focused on improving our understanding of how TGFß signalling regulates the development of intimal hyperplasia. We are also interested in generating gene therapy strategies targeting the TGFß pathway that could potentially be used to prevent the development of intimal hyperplasia after PCI or CABG, thereby ameliorating the outcome of these two revascularisation surgeries.

Publications

1. Bradshaw AC and Baker AH. Gene therapy for cardiovascular disease: perspectives and progress. Vascular Pharmacology 2012 Nov7. [Epub ahead of print]
2. Bradshaw AC, Coughlan L, Miller AM, van Rooijen N, Nicklin SA, Baker AH. Biodistribution and inflammatory profiles of novel FX-ablated serotype 5 adenoviruses. Journal of Controlled Release 2012;164:394-402.
3. Coughlan L, Bradshaw AC, Parker AL, Robinson H, White K, Campton J, Custers J, Goudsmit J, van Rooijen N, Barouch D, Nicklin SA, Baker AH. exon hypervariable region substitutions affect viral biodistribution, toxicity and inflammatory responses in vivo.  Mol Ther. 2012;20:2268-2281
4. Coughlan L, Alba RA, Parker AL, Bradshaw AC, McNeish IA, Nicklin SA, Baker AH – Tropism-modification strategies for targeted gene delivery using adenoviral vectors. Viruses 2012;2:2290-355.
5. Bradshaw AC, Parker AL, Duffy MR, Coughlan L, van Rooijen N, Kahari VM, Nicklin SA, Baker AH Requirements for receptor engagement during infection by adenovirus complexed with blood coagulation factor X.PLoS Pathogens 2010;6:e1001142
6. Pardali E, Goumans MJ, ten Dijke P – Signaling by members of the transforming growth factor-beta family in vascular morphogenesis and disease Trends Cell. Biol. 2010; 20:556-67

Development of stem cell based technologies and regenerative medicines

Stem cells have remarkable properties, unlike mature cells that play an important bodily function eg. Liver cells or muscle cells, all stem cells are unspecialised and can keep proliferating and increasing in number indefinitely. Additionally, pluripotent stem cells (PSC) have the capacity to mature into all cell types of the body. Therefore, these PSC have great potential for the development of model systems to study different cell types affected by disease and also present the opportunity to develop cellular or regenerative therapies.
We are interesting in finding new reagents and techniques to maintain these important cells in the laboratory and to efficiently induce them to differentiate into the mature cells that we want to work with. Our specific interests are in blood and blood vessel development, disease and therapies.

Progress towards the cGMP production of hESC derived RBCs

Blood Transfusion has become a mainstay of modern medical practice. However problems persist both nationally and internationally in maintaining adequacy of supply, managing the risk of transmission of infectious agents and immune incompatibility between donor and recipient. Human embryonic and induced pluripotent stem cells (hESCs & iPSC) have unique properties in that they can be maintained indefinitely in culture in an undifferentiated state and yet retain the ability to form all the cells and tissues within the body. They therefore offer a potentially limitless source from which to generate red cells (RBCs) for use in clinical transfusion. Initially in vitro RBCs may have particular utility for patients who receive regular transfusions such as those with haemoglobinopathies.  Within the project we have generated hESC to cGMP grade (Roslin Cells Ltd) in compliance withUK regulatory requirements for eventual clinical use. We are able to differentiate these to form haematopoietic progenitor cells (HPC) resulting in ≥95% conversion to erythroid cells with high expansion in cell numbers, in a stromal free, suspension based culture system. We have also demonstrated that this methodology is similarly effective for iPSC.  

Funded by the Wellcome Trust, Scottish Funding Council and SNBTS

Generation of vascular endothelial cells from pluripotent stem cells.

Our work is based on the refinement and application of vascular endothelial cell production for regenerative medicine within the context of ischemic cardiovascular diseases. Our principal focus is on the basic biology of differentiation to vascular endothelium and the application of this to in vivo peripheral and myocardial vasculogenesis. This area has received significantly less attention than the production of cardiomyocytes and yet offers the potential for therapy for a large population of patients if effectively harnessed. Our proposed programme of work is founded on collective data demonstrating our ability to induce differentiation of hES cells to vascular endothelial cells in a highly efficient feeder-free, serum-free manner with potential for scale-up, our ability to reprogramme somatic cells to iPS cells from patients with cardiovascular disease.  Our aim is to fully dissect key signalling events, transcriptional networks and epigenetic changes that lead to effective hESC and iPSC-derived endothelial cells with assessment of vasculogenesis in vivo and, ultimately, in patients.  

Funded by the British Heart Foundation, MRC and EPSRC.

Publications

1. Kaupisch A, Kennedy L, Stelmanis V, Tye B, Kane NM, Mountford JC, Courtney A, Baker AH. Derivation of Vascular Endothelial Cells from Human Embryonic Stem Cells Under GMP-Compliant Conditions: Towards Clinical Studies in Ischaemic Disease. J Cardiovasc Transl Res. Oct;5(5):605-17. 2012
2. Kane NM, Howard L, Descamps B, Meloni M, McClure JD, Lu R, McCahill A, Breen C, Mackenzie RM, Delles C, Mountford JC, Milligan G, Emanueli C, Baker AH. A Role for microRNAs 99b, 181a and 181b in the Differentiation to Vascular Endothelial Cells from Human Embryonic Stem Cells. Stem Cells. Apr 30(4):643-54. 2012
3. Marenah L, Allan EK, Mountford JC, Holyoake TL, Jørgensen HG, Elliott MA. Investigation into omacetaxine solution stability for in vitro study. Biomed Chromatogr. May;26(5):545-7. 2012
4. Yung S, Ledran M, Moreno-Gimeno I, Conesa A, Montaner D, Dopazo J, Dimmick I, Slater NJ, Marenah L, Real PJ, Paraskevopoulou I, Bisbal V, Burks D, Santibanez-Koref M, Moreno R, Mountford J, Menendez P, Armstrong L, Lako M. Large-scale transcriptional profiling and functional assays reveal important roles for Rho-GTPase signalling and SCL during haematopoietic differentiation of human embryonic stem cells. Hum Mol Genet. Dec 20;(24):4932-46. 2011
5. Kane NM, Nowrouzi A, Mukherjee S, Blundell MP, Greig JA, Lee WK, Houslay MD, Milligan G, Mountford JC, von Kalle C, Schmidt M, Thrasher AJ, Baker AH. Lentivirus-mediated reprogramming of somatic cells in the absence of transgenic transcription factors. Mol Ther. Dec18;(12):2139-45. 2010
6. Burton P, Adams DR, Abraham A, Allcock RW, Jiang Z, McCahill A, Gilmour J, McAbney J, Kaupisch A, Kane NM, Baillie GS, Baker AH, Milligan G, Houslay MD, Mountford JC. Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) blocks differentiation and maintains the expression of pluripotency markers in human embryonic stem cells. Biochem J. Nov 25;432(3):575-84. 2010
7. Andrews PD, Becroft M, Aspegren A, Gilmour J, James MJ, McRae S, Kime R, Allcock RW, Abraham A, Jiang Z, Strehl R, Mountford JC, Milligan G, Houslay MD, Adams DR, Frearson JA. High-content screening of feeder-free human embryonic stem cells to identify pro-survival small molecules. Biochem J. Oct 25;432(1):21-33. 2010
8. Kane NM, Meloni M, Spencer HL, Craig MA, Strehl R, Milligan G, Houslay MD, Mountford JC, Emanueli C, Baker AH. Derivation of endothelial cells from human embryonic stem cells by directed differentiation: analysis of microRNA and angiogenesis in vitro and in vivo. Arterioscler Thromb Vasc Biol. Jul;30(7):1389-97. 2010
9. Khanim FL, Hayden RE, Birtwistle J, Lodi A, Tiziani S, Davies NJ, Ride JP, Viant MR, Gunther UL, Mountford JC, Schrewe H, Green RM, Murray JA, Drayson MT, Bunce CM. Combined bezafibrate and medroxyprogesterone acetate: potential novel terapy for acute myeloid leukaemia. PLoS One, Dec 7;4(12):e8147. 2009
10. Pellicano F, Copland M, Jorgensen HJ, Mountford JC, Leber B and Holyoake TL. BMS-214662 induces mitochondrial apoptosis in CML stem/progenitor cells, including CD34+38- cells, through activation of protein kinase C . Blood, 114(19):4186-96. 2009
11. Davies A, Jordanides NE, Giannoudis A, Lucas CM, Hatziieremia S, Harris RJ, Jørgensen HG, Holyoake TL, Pirmohamed M, Clark RE and Mountford JC. Mountford JC. Nilotinib concentration in cell lines and primary CD34+ chronic myeloid leukemia cells is not mediated by active uptake or efflux by major drug transporters. Leukaemia, 23(11):1999-2006. 2009
12. Hatziieremia S, Jordanides NE, Holyoake TL, Jørgensen HG and Mountford JC. Inhibition of MDR1 does not sensitise primitive chronic myeloid leukaemia CD34+ cells to imatinib. Exp Hematol. 7(6):692-700. 2009
13. Myssina S, Serrels A, Jørgensen HG, Bhatia R, Modi H, Helgason GV, Mountford JC. Hamilton A, Schemionek M, Koschmieder S, Brunton V, Holyoake TL. Combined BCR-ABL inhibition with lentiviral-delivered shRNA and dasatinib augments induction of apoptosis in Philadelphia positive cells. Exp Hematol. 37(2):206-14. 2009

Reviews
1. Mountford JC, Turner M. In vitro production of red blood cells. Transfus Apher Sci. Aug 45(1):85-9. 2011
2. Burton P, Adams DR, Abraham A, Allcock RW, Jiang Z, McCahill A, Gilmour J, McAbney J, Kane NM, Baillie GS, McKenzie FR, Baker AH, Houslay MD, Mountford JC, Milligan G. Identification and characterization of small-molecule ligands that maintain pluripotency of human embryonic stem cells. Biochem Soc Trans. Aug;38(4):1058-61. 2010
3. Mountford J, Olivier E, Turner M. Prospects for the manufacture of red cells for transfusion. Br J Haematol. 149 (1):22-34. 2010
4. Mountford JC, Olivier E, Jordanides NE, de Sousa P, Turner ML. Red blood cells from pluripotent stem cells for use in transfusion. Regen Med. May;5(3):411-23. 2010