Izsvák Lab

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;

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

Huiqiang Cai*

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.

Genomic safety analysis of Sleeping Beauty transposon engineered ROR1 CAR-T cells

Tamás Rasko, Felix Lundberg

The goal of this project is to obtain pre-clinical proof-of-concept for the safety and antitumor efficacy of engineered T cells. The T cells are engineered using the non-viral Sleeping Beauty (SB)-mediated gene transfer to express a chimeric antigen receptor (CAR), specific for the tumor antigen ROR1 in breast and lung cancer (ROR1 CAR-T). Our specific task in the project is the genomic safety analysis of ROR1 CAR-T cells in a pre-clinical study. The analyses include the determination (i) of the stability of therapeutic gene expression from the transposon-base vector; (ii) transposon copy number and (iii) genomic integration profile in the T‐cell genome.

This project is part of the Helmholtz PoC consortium: Max Delbruck Center,

IZI, U of Wurzburg

Non-autonomous gene expression: causes, consequences and lessons for gene therapy

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, using the 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.

Targeting integration of Sleeping Beauty to specific genomic loci - Improving the safety of the non-viral Sleeping Beauty gene transfer system for therapeutic applications

Bertrand Tangu Teneng

Sleeping Beauty (SB) chromosome integration occurs in a close-to-random manner, with low, but not zero risks of insertional mutagenesis and/or transactivation of oncogenes around the integration site. Whether the close-to-random integration profile of SB is the result of the inherent ability of the transposase itself or assisted by host-encoded factors is yet to be clarified. Proof of principle exist that SB integrations can be targeted into predetermined safe chromosomal loci though at a low efficiency. To improve the targeting efficiency of SB, we are focusing on identifying and disrupting relevant SB/host-factor interactions. It is expected that by interfering with these interactions, SB integration might be targeted more efficiently towards the specified genomic loci, thereby improve the safety of the system for application in humans.

Deciphering the genetic background of hormone-induced breast cancer - see Project#5

Genome engineering of genetic defects of KCNQ1 to generate pancreatic progenitor In Vitro from neonatal diabetic patients for pathophysiological mechanisms elucidation

Zhimin Zhou

K_ATP 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.