Transposons as non-viral vectors for gene therapy
Transposon-based, non-viral integrating vector systems represent a novel technology that opens up new possibilities for gene therapy. Due to stable chromosomal insertion, these systems can result in robust, long-term expression of the integrated transgene. The plasmid-based hyperactive SB100X transposon system () has become a popular tool for non-viral, therapeutic transgene delivery. The SB100X transposon has a favorable safety profile as compared to widely used retro/lentiviral approaches. In contrast to viruses, transposons have low intrinsic activity, and are self-regulated. Interactions with cellular host factors appear to allow wild type transposons to persist in the host without producing serious levels of genetic damage. Notably, the plasmid-based transposon vectors have reduced immune complications, and have no strict limitation of the size of expression cassettes. Therefore, the vector can tolerate larger and more complex therapeutic genes. SB transposition does not require active cell division to integrate and has a fairly random genomic insertion pattern. In a combination with a Zn-finger technology, it is possible to enrich the specificity of transposon integration. Further advantages of the SB system include its relative resistance to gene silencing when compared to retro/lentiviral vectors. These features of SB are particularly favorable attributes for stable, long-term expression in various primary and stem cells. As an important issue regarding the implementation of clinical trials, transposon vectors can be maintained and propagated as plasmid DNA, making them simple and inexpensive to manufacture (e.g. GMP vector production).
In order to fill the gap between the recent vector development and clinical trials, our strategies are the followings: (1) try the SB system in disease models that were already on clinical trials using retroviral vectors, but where safety was a serious issue; (2) try the SB system in disease models that were already on clinical trials, using non-viral approaches, but efficacy was a limiting factor - Age-related Macular Degeneration (AMD); Von Willebrand disease Type III ; (3) include models where the transposon-based regenerative technology has a potential - cell-based pacemaker [collaboration M. Morad (University of South Carolina)], dysferlinopathy [collaboration S. Spuler (ECRC), MyoGrad)]; (4) combine the plasmid-based integration vectors with the cutting-edge DNA delivery strategies;
Generation of cardiomyocytes with pacemaker activity from iPSC by the Sleeping Beauty transposon system
PhD student Angélica García-Pérez
The pacemaker cells of the heart, localized in the sino-atrial node (SAN), initiate and determine the rate and rhythm of the heartbeat. When these specialized cells are damage due to aging or genetic disease, the implantation of an electronic pacemaker is required. Several strategies that combine gene and cell-based approaches are being pursued to develop biological pacemakers as an alternative to battery driven electronic pacemakers. We propose a combined cell and gene therapy strategy, where the non-viral integrating transposon system Sleeping Beauty is used to overexpressed HCN4, the most abundant isoform of HCN in the SAN, in human induced pluripotent stem cells (hiPSC) as platform for differentiation of pacemaker cardiomyocytes (In collaboration with Martin Morad, Medical University of South Carolina, USA).
Transposon-based therapeutic strategies to treat Age-related Macular Degeneration
Dr. Huiqiang Cai, PhD Student Himanshu Bhusan Samal
Age-related Macular Degeneration (AMD), a neurodegenerative disease of the retina, is a major cause of blindness in elderly people. It presents two distinct forms, a slowly progressing nonvascular atrophic form (dry or avascular AMD) and a rapidly progressing blinding form (neovascular AMD). Currently there is no available treatment for avascular AMD. Treatment with VEGF inhibitors for neovascular AMD is effective in about 30% of patients, however the effect is limited in time. Since administration of PEDF, a natural antagonist of VEGF, to the subretinal space could inhibit choroidal neovascularization (CNV) in neovascular AMD, our collaborators and we are trying to develop a non-viral gene transfer system, Sleeping Beauty (SB) system, to treat AMD, with considerations of the efficiency of gene delivery, transgene expression and safe harbor integration, as well the potential risks. The whole project, called TargetAMD and sponsored by FP7, is an ongoing clinical trial project in which patients will be subretinally injected with genetically modified, patient-derived iris pigmented epithelium (IPE) or retinal pigmented epithelium (RPE) cells, by which it could overexpress PEDF to provide a long-lasting cure of AMD. Till now, we optimized the SB delivery system to improve its efficiency and biosafety profile via using SB100X mRNA as a source of transposase, adding insulators and loading PEDF-transposon in pFAR4. From the preclinical trials, it shows PEDF could be secreted stably in animal models and primary human RPE and IPE cells. Besides, we can also guarantee that the SB system is neutral and safe in human RPE cell lines. It is very hopeful that this SB system will be working well in clinical trial.
Non-autonomous gene expression: causes, consequences and lessons for gene therapy
PhD student Felix Lundberg
A recent technology called Thousands of Reporters Integrated in Parallel have facilitated the investigation of gene expression and the organisation of the genome (Akhtar et al. 2013).
By inserting thousands of identical transgenes, inside a Sleeping Beauty vector, into various locations in human induced-pluripotent stem cells (hiPSC), we aim to ask which inserts are affected by the neighbours and which aren’t. Conversely, we shall characterize why some inserts affect the neighbours but others don’t. Further, we can ask about the properties of these insulated domains. In this manner genomic safe harbours, areas where an inserted gene is correctly expressed yet do not impact its neighbours, can hopefully be discovered. Our choice of using hiPSCs is in part also motivated by a parallel approach: we are looking to see whether insulation to expression change over evolutionary time also predicts zones of transgene insulation. If so, then the evolutionary approach could be a short cut for knowing where in the genome a transgene should be inserted for any tissue/cell type of interest. We have RNASeq data for iPSC cells from across the primates thus rendering the test feasible. Moreover, knowing the expression level of the various areas of the genome is of great interest in and of itself. Position effects and domain-wide regulation are known to play a role in many diseases and are also a fundamental aspect of the genome and its evolution.
Sleeping Beauty and Chromatin 3D mapping
PhD student Himanshu Bhusan Samal
In this project the utilization of the Sleeping Beauty transposon as a novel tool for chromatin 3D mapping is examined. This chromatin mapping approach is based on the presumption that transposon local hopping is actually a spatial phenomenon and SB lands to spatially close chromosomal positions near the donor site. Thus, by defining the transposon reintegration sites, the neighboring chromatin regions of the donor site can be mapped. A great advantage of the transposon-based method would be that chromatin regions are signed under physiological conditions avoiding chemical fixation and limitations of cross-linking. Furthermore, in vivo application gives the possibility to analyze chromatin environment of a given locus in different tissues and various developmental stages in living organisms.
Deciphering the genetic background of hormone-induced breast cancer
PhD student Himanshu Bhusan Samal
Although numerous studies implicate an association between estrogens and the development of breast cancer, the molecular mechanisms through which estrogens induce or promote breast cancer development are not clear. The SB transposon is suitable for somatic mutagenesis and emerged as a new tool in cancer research. Transposon-based insertional mutagenesis screens are able to identify both oncogenes and tumor-suppressor genes. In this project, we utilize a rat model to study the genetics of the estrogen-induced mammary cancer applying the method of a SB transposon-based forward genetic screen. Unlike the situation in mouse, the development of mammary cancer in rat is similar to human as it is also estrogen-dependent. The transposon mutagenesis approach is expected to be a powerful tool to decipher gene regulatory networks cooperating in hormone dependent breast cancer development, progression and metastasis (collaboration with Prof. M. Bader (MDC), D. Largaespada (University of Minnesota), J. Shull (University of Wisconsin), Schneider).
Genome engineering of genetic defects of KCNQ1 to generate pancreatic progenitor In Vitro from neonatal diabetic patients for pathophysiological mechanisms elucidation
PhD student Zhimin Zhou
KATP channels and voltage-gated potassium (Kv) channels interact with voltage-dependent Ca2+ channels to trigger and maintain glucose-stimulated insulin secretion of pancreatic β cells. The patient’s KCNQ1 mutation causes permanent neonatal diabetes after genome-wide association studies. Our final purpose is to learn more about the mechanisms of permanent neonatal diabetes. There are two objectives to achieve our purpose: one is to reverse early pancreatic lineage with insulin producing from phiPSC after homology directed targeted modification by CRISPR-Cas9 system (pChiPSC-PL) and phiPSC (phiPSC-PL). As models, they will be used to explore pathological mechanisms and to elucidate much more the signal pathway regulation of insulin.