Gene and cell therapy approaches for treatment of cardiovascular disease

We are interested in developing novel therapies for patients with heart disease. Specifically, patients who undergo coronary artery bypass grafting or undergo implantation of stents. Both these procedures have the potential complication of blood vessel blockage following the grafting or stent implantation and this limits the effectiveness of the therapy. Our strategies are to develop novel therapies that prevent this process and can be delivered at the same time as the procedure is performed, thus providing lasting benefit to patients. We test specific genes and new molecules called “microRNA” in this context and evaluate them in accepted models of disease and in human tissue.

Human embryonic stem cells and induced pluripotent stem cells (iPS) hold broad potential in regenerative medicine. Such cells can generate all cell types upon stimulation to differentiate along defined cell commitment pathways. We are interested in the mechanisms that govern stimulation of these cells into vascular endothelial cells and their subsequent application to regenerative medicine. When cells are stimulated in this way certain “pathways” are activated and new genes are transcribed from DNA into RNA and then to protein. We are particularly interested in finding out what controls these steps and in this project we will address this important issue and, importantly, manipulate them to refine and optimise cardiac and vascular endothelium commitment.

Gene therapy vector development

(Baker, Nicklin)

Research within the virology theme focuses primarily upon the development of genetically manipulated adenoviral vectors (primarily based on Ad5) for gene therapy applications in cardiovascular disease. Our work characterises the interactions occurring between virus and host proteins and cells that define adenoviral tropism, toxicity and fate following intravenous administration. Of particular importance to our work has been defining and precluding the high affinity interaction between the Ad5 hexon protein and blood clotting factor X (FX, see figure 1 below). This interaction underpins the profound liver tropism of Ad5 based vectors following intravenous administration by retargeting the Ad5: FX complex to a particularly sulphated form of heparan sulphate proteoglygcan receptor, expressed abundantly upon liver hepatocytes. Ongoing research within our group seeks to build upon Ad5 based vectors genetically engineered to ablate FX binding to retarget them to alternative disease restricted receptors, as well as investigating the potential of new forms of adenoviruses for therapeutic applications.

Figure 1:  Computaional reconstruction of CryoEM images of Ad5 alone (cyan, resolved to 26Å, A&C), Ad5 in complex with FX (magenta, resolved to 23Å, B&D), and the composite image (E&F) demonstrating the FX binding to the hexon hypervariable regions (HVRs).

Gene Therapy Vector Development References

Vascular Remodeling & Therapy

(Baker, Nicklin, Berry)

The migration and proliferation of vascular smooth muscle cells are central to the development of neointima formation associated with late vein graft failure and in-stent restenosis (ISR). Over the past 10 years we have developed and evaluated, in relevant models and human tissues, effective strategies to prevent neointima formation by genetic manipulation of the vasculature leading to a potential clinical trial, based on adenovirus-mediated gene delivery of tissue inhibitors of metalloproteinase-3 (TIMP-3) to human saphenous vein. We are developing our detailed knowledge of adenoviral biology and engineering to construct and optimise the next generation of systems for vascular intervention studies. Previously studied mechanisms to manipulate the virus capsid in order to optimise vascular gene delivery and minimise dissemination and inflammation are integrated into single component viruses. We are pursuing both known (gene-based) and novel (miRNA-based) interventions to fully assess the safety and efficacy of such approaches in large animal and human tissue models of bypass graft failure and in-stent restenosis. 

Figure 2:  A.  First-generation Ad mediated expression of lacZ in porcine vein grafts, 7 days post grafting.  (George SJ et al.  Circulation 2000;101:269-304) B. Long term efficacy of First-generation Ad mediated over-expression of TIMP3 in porcine vein grafts. C. TIMP3 expression significantly reduced neointimal formation 3 months post grafting.  n=11-15 grafts/group.  George, Wan and Baker, unpublished

Vascular Gene Therapy References

Stem Cell Therapy

(Baker, Mountford, Milligan)

Revascularisation of underperfused tissues remains one of the biggest priorities in cardiovascular therapeutics. One focus has been the omnipotence of human embryonal stem cells (hESC) and their ability to form vascular endothelial cells (EC). Our published protocol for feeder- and serum-free monolayer hESC-EC-directed differentiation represents an optimal tool for the attainment of hESC-derived EC in a GMP-compliant manner for clinical trial purposes (Kane et al. 2010).

In collaboration with Roslin Cells Ltd., funded by the Technology Strategy Board for “the clinical translation of endothelial cells derived from human embryonic stem cells”, we were in the position to transfer aforementioned protocol and successfully differentiate GMP-derived Roslin hESC lines towards EC.

Figure 3: Stepwise development of hESC-derived endothelial cells.  Upper panel: morphological changes from d0 to d21 from left to right.  Middle and lower panel: time-matched immunofluorescent staining for endothelial markers VE-Cadherin and PECAM-1.

Stem Cell Therapy References

Publications

Gene Therapy Vector Development

• Chick HE, Nowrouzi A Fronza R, McDonald RA, Kane NM, Alba R, Delles C, Sessa WC, Schmidt M, Thrasher AJ, Baker AH. Integrase-deficient lentiviral vectors mediate efficient gene transfer to human vascular smooth muscle cells with minimal genotoxic risk.  Human Gene Therapy, 2012, 23: 1247-1257.
• Coughlan L, Bradshaw, AC, Parker AL, Robinson H, White K, Custers J,Goudsmit J, van Rooijen N,  Barouch DH, Nicklin SA, Baker AH. Ad5:Ad48 hexon hypervariable region substitutions affect toxicity and increase inflammatory responses following intravenous delivery.  Molecular Therapy, 2012 Aug, 20 (12): 2268-2281
• Alba R, Bradshaw AC, Coughlan L, Denby L, McDonald RA, Waddington SN, Buckley SM, Greig JA, Parker AL, Miller AM, Wang H, Lieber A, van Rooijen N, McVey JH, Nicklin SA, Baker AH.  Biodistribution and retargeting of FX-binding ablated adenovirus serotype 5 vectors. Blood 2010;116(15):2656-2664.
• 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 Pathog 2010;6(10):e1001142.
• Alba R, Bradshaw AC, Parker AL, Bhella D, Waddington SN, Nicklin SA, van Rooijen N, Custers J, Goudsmit J, Barouch DH, McVey JH, Baker AH. Identification of coagulation factor (F)X binding sites on the adenovirus serotype 5 hexon: Effect of mutagenesis on FX interactions and gene transfer. Blood 2009;114(5):965-71.
• Parker AL, Waddington SN, Buckley SMK, Custers J, Havenga MJ, van Rooijen N, Goudsmit J, McVey JH, Nicklin SA, Baker AH.  Effect of neutralizing sera on factor X-mediated adenovirus serotype 5 gene transfer.  J Virol 2009;83(1):479-83.
• Waddington SN, McVey JH, Bhella D, Parker AL, Barker K, Atoda H, Pink R, Buckley SM, Greig JA, Denby L, Custers J, Morita T, Francishetti IM, Monteiro RQ, Barouch DH, van Rooijen N, Napoli C, Havenga MJ, Nicklin SA, Baker AH.  Adenovirus serotype 5 hexon protein mediates liver gene transfer.  Cell 2008;132(3):397-409.
• Waddington SN, Parker AL, Havenga M, Nicklin SA, Buckley SM, McVey JH, Baker AH.  Targeting of adenovirus serotype 5 (Ad5) and 5/47 pseudotyped vectors in vivo: a fundamental involvement of coagulation factors and redundancy of CAR binding by Ad5. J Virol 2007;81(17):9568-71.
• Parker AL, McVey JH, Doctor JH, Lopez-Franco O, Waddington SN, Havenga MJ, Nicklin SA, Baker AH.  Influence of coagulation factor zymogens on the infectivity of adenoviruses pseudotyped with fibers from subgroup D. J Virol 2007;81(7):3627-31.
• Parker AL, Waddington SN, Nicol CG, Shayakhmetov DM, Buckley SM, Denby L, Kemball-Cook G, Ni S, Lieber A, McVey JH, Nicklin SA, Baker AH.  Multiple Vitamin K-dependent coagulation zymogens promote adenovirus-mediated gene delivery to hepatocytes.  Blood 2006;108(8):2554-61.
• Greig JA, Shirley R, Graham D, Denby L, Dominiczak AF, Work LM, Baker AH. Vascular-targeting anti-oxidant therapy in a model of hypertension and stroke. J Cardiovasc Pharmacol, 2010; 56: (6) 642-650.
• Duffy MR, Bradshaw AC, Parker AL, McVey JH and Baker AH. A cluster of basic amino acids in the factor X serine protease mediate surface attachment of adenovirus:FX complexes.  J. Virol., 2011, 85: 10914-10919.
• Bradshaw A, Coughlan L, Miller A, Alba R, van Rooijen N, Nicklin S, Baker AH.  Biodistribution and inflammatory profiles of novel penton and hexon double-mutant serotype 5 adenoviruses.  Journal of Controlled Release, 2012, Available online doi: 1016/j. jconrel.2012.05.025

Vascular Remodelling and Therapy

• Baker AH, Wilkinson GW, Hembry RM, Murphy G, Newby AC.  Development of recombinant adenoviruses that drive high level expression of the human metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 and -2 genes: characterization of their infection into rabbit smooth muscle cells and human MCF-7 adenocarcinoma cells. Matrix Biol 1996;15(6):383-395.
• Kranshofer A, Baker AH, George SJ, Newby A.  Expression of tissue inhibitor of metalloproteinase-1, -2, and -3 during neointima formation in organ cultures of human saphenous vein. Arterioscler Thromb Vasc Biol 1999;19(2):255-65.
• George SJ, Baker AH, Angelini GD, Newby AC. Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins. Gene Ther 1998;5(11):1552-1560.
• Baker AH, Zaltsman AB, George SJ, Newby AC. Divergent effects of tissue inhibitor of metalloproteinase-1, - 2, or -3 overexpression on rat vascular smooth muscle cell invasion, proliferation, and death in vitro. TIMP-3 promotes apoptosis.  J Clin Invest 1998;101(6):1478-1487.
• George SJ, Lloyd CT, Angelini GD, Newby AC, Baker AH. Inhibition of late vein graft neointima formation in human and porcine models by adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase-3. Circulation 2000;101(3):296-304.
• George SJ, Johnson JL, Angelini GD, Newby AC, Baker AH. Adenovirus-mediated gene transfer of the human TIMP-1 gene inhibits smooth muscle cell migration and neointima formation in human saphenous vein. Hum Gene Ther 1998;9:867-877.
• Bond M, Murphy G, Bennett MR, Amour A, Knauper V, Newby AC, Baker AH. Localization of the death domain of tissue inhibitor of metalloproteinase-3 to the N terminus. Metalloproteinase inhibition is associated with proapoptotic activity.  J Biol Chem 2000;275(52):41358-41363.
• Bond M, Murphy G, Bennett MR, Newby AC, Baker AH.  Tissue inhibitor of metalloproteinase-3 induces a Fas-associated death domain-dependent type II apoptotic pathway. J Biol Chem 2002;277(16):13787-13795.
• Qi JH, Ebrahem Q, Moore N, Murphy G, Claesson-Welsh L, Bond M, Baker A, Anand-Apte B.  A novel function for tissue inhibitor of metalloproteinases-3 (TIMP3): inhibition of angiogenesis by blockage of VEGF binding to VEGF receptor-2.  Nat Med 2003;9(4):407-415.
• George SJ, Angelini GD, Capogrossi MC, Baker AH.  Wild type p53 gene transfer inhibits neointima formation in human saphenous vein by modulation of smooth muscle cell migration and induction of apoptosis. Gene Therapy 2001;8(9):668-676.
• Kritz AB, Yu J, Wright PL, Wan S, George SJ, Halliday C, Kang N, Sessa WC, Baker AH.  In vivo modulation of Nogo-B attenuates neointima formation. Mol Ther 2008;16(11):1798-804.
• George SJ, Wan S, Hu J, McDonald R, Johnson JL, Baker AH.  Sustained reduction of Vein Graft Neointima Formation by ex vivo TIMP-3 Gene Therapy.  Circulation, 2011, 124: (11 Suppl) S135-142.
• Caruso P,  MacLean MR, Khanin R,  McClure J, Soon E, Southgate M,  McDonald RA, Greig JA, Robertson KE,  Masson R, Denby L, Dempsie Y, Long L, Morrell NW, Baker AH. Dynamic changes in lung microRNA profiles during the development of pulmonary hypertension due to chronic hypoxia and monocrotaline.  Arterioscler Thromb Vasc Biol 2010; 30:716-723. PMID: 20110569.
• Caruso P, Dempsie Y, Stevens H, McDonald RA, Long L, Lu R, White K, Mair K, McClure JD, Southwood M, Upton P, Xin M, van Rooij E, Olson E, Morrell NW, Maclean MR and Baker AH. A role for miR-145 in pulmonary arterial hypertension: Evidence from mouse models and patients samples.  Circulation Research, 2012, 111: 290-300. PMID: 22715469

Stem Cell Therapy

• 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 2010;30(7):1389-97.
• 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 2010;432(3):575-84.
• Kane MN, 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 2010;18(12):2139-45.
• Kane NM, Howard L, Descamps B, Meloni M, McClure J, 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, 2012 Apr; 30(4):643-54. PMID: 22232059
• 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.  Journal of Cardiovascular Translational Research, 2012, available on  line: DOI 10.1007/s12265-012-9379-2.