Project #4: Translating experience accumulated in TE research to cutting-edge technologies


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 (Molecule of the Year, 2009) 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) TargetAMD EU-sposored clinical trial; Von Willebrand disease Type III Transposmart, ERARE consortium; (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, University of South Carolina, USA).


Transposon-based therapeutic stategies to treat Age-related Macular Degeneration

PhD student Huiqiang Cai, PhD student Chuanbo Sun, PhD student Angelica Garcia-Pérez

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). Up to today 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 target site selection, as well the potential risks. The whole project, which is called TargetAMD and sponsored by FP7, is a 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, in addition of 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 also guarantte that the SB system is neutral and safe in human RPE cell lines. It will be very hopeful that this SB system will be working well in clinical trial.


A transposon-based TCR gene transfer for clinical use

PhD student Ilija Bilic

The overall goal of this project is to establish a clinically applicable, feasible protocol to express tumour specific T cells for personalized cancer immunotherapy. The engineered, clinical grade T cells will express TCRs identified and characterized by the BIH consortium. While, currently most clinical applications use γ-retro or lentiviral systems for gene transfer into T cells, we aim at establishing a non-viral strategy. To avoid the expense and manufacturing difficulty associated with transducing T cells with recombinant viral vectors, we shall use the Sleeping Beauty transposon-based, non-viral, integrating vector system (SB100X). Similarly to retroviruses, SB integrates into the chromosomes of host cells. However, compared to viral vectors, DNA-based transposon vectors are plasmid-based and are less expensive to manufacture (e.g. GMP vector production). Furthermore, they do not require reverse transcription, have no strict limitation of the size of expression cassettes and are less prone to rearrangements. The SB vector has particularly favourable attributes for stable, long-term expression in various cell types, including human primary cells (in collaboration with the BIH consortium).


A transposon-based gene delivery in muscle stem cells

PhD student Helena Escobar

We successfully altered gene expression in cultured human PAX7+ satellite cells with Sleeping Beauty transposon–mediated non-viral gene transfer, highlighting the potential of this system for use in gene therapy (press release).


Sleeping Beauty and Chromatin 3D mapping

Dr. EnikÅ‘ Éva Nagy

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 Shreevathsa Srinivasan Chilkunda

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).


Transposon mutagenesis in rat spermatogonial stem cells

Transposons can be harnessed as vehicles for introducing mutations into genes. Decades of exquisite phenotyping and detailed analysis of crosses of inbred rats have resulted in initial localization of hundreds of loci involved in complex diseases and quantitative phenotypes (QTLs). In a reverse genetic approach, transposons can be harnessed as vehicles for introducing genetic mutations into genomes. The genes inactivated by transposon insertion are "tagged" by the transposable element, which can be used for subsequent cloning of the mutated allele. By combining transposon mutagenesis with the innate potency of spermatogonia, comprehensive libraries of gene knockouts are cultivated in physiologically competent, rat germline stem cells, thereby paving the way for high-throughput functional genomics in the laboratory rat. The work describing the pilot screen of 100 KO rat mutants was published (Izsvak, 2010, Ivics, 2011). In order to upscale the KO technology, we have generated a KO library (KO of 2,000 genes – supported by FP7, EURATRANS). The project would generate a valuable KO source for rat that is the preferred model organism of cardiovascular, as well as toxicology and behavioral studies (collaboration Kent Hamra, UTSW, USA).