Bringing the Sleeping Beauty transposon system to the clinic
Cell & Gene Therapy Insights 2023; 9(9), 1183–1189
What are you working on right now?
ZI: I am working on the Sleeping Beauty transposon system. There are two components of a transposon system—a DNA component and a protein component. We are trying to modify these components by genetically engineering biochemical aspects of transposition, thereby changing how the transposon works so that it is more fine-tuned towards medically relevant applications.
I am also working to push the system into medical applications through collaboration; teaming up with medical doctors and clinical scientists, showing them how promising the system is so that they can bring it into a clinically relevant environment.
The very first European clinical trial using this system, which was based on the most highly developed Sleeping Beauty components using a CAR-T application, was completed just two months ago. This study was conducted by a Horizon 2020-funded research consortium with the goal of utilizing Sleeping Beauty technology in multiple myeloma patients.
That was a really exciting development for the field, and the next clinical trials are already being planned. Eventually, this technology will be brought into the context of a drug product that a doctor can prescribe to a cancer patient, or indeed, any type of patient.
When we last spoke at the beginning of 2021 Ivics Z. Update on the Sleeping Beauty transposon system: current state-of-the-art & remaining challenges. Cell & Gene Therapy Insights 2021; 7(2), 99–105., we discussed some of the challenges in developing Sleeping Beauty transposons and applying them in human medicines—specifically, efficiency, toxicity, and avoiding a negative impact on the cell from the integration of the Sleeping Beauty transposon into the genome. Can you update us on what recent progress you have seen in these areas?
ZI: We have made very good progress in increasing both the efficiency and the safety of adding genes to cells. We have modified the transposase polypeptide (the Sleeping Beauty transposase) dramatically. We were able to switch a single amino acid in that polypeptide, shifting the genomic integration pattern of the transposon from a pattern that we had annotated and described in different cell types (including human cells).
We identified that Sleeping Beauty integrates relatively randomly in the human genome. Random integration is actually not too bad, considering that some viral vectors, including lentiviruses and retroviruses, do not have random distribution in the genome, but rather tend to favor transcriptionally active regions, transcription units, gene bodies, and transcriptionally relevant regulatory regions of genes for integration. These regions carry a certain risk in the context of a gene therapy application. By comparison, the random transposition of Sleeping Beauty is relatively safe, and we made it even safer by simply shifting around that single amino acid in the catalytic core domain of the transposase. We believe that the biochemical explanation for this is that after this change, the transposase can neither bend target DNA nor can it maintain a bent structure.
Bending DNA is an important structural feature of DNA integration machineries, including those employed by retroviruses and transposons. The inability of the generated mutant to undergo a bend reaction or to stabilize a bent structure is a defect, so to speak, in the transposase and therefore, it seeks out sites in the genome that are already highly bendable. These sites happen to have a biased base composition in that they have adenine-thymine (A-T)-rich DNA. These A-T repeated base pair sequences do not occur very frequently in exons and they are also typically depleted in the transcriptional regulatory regions of genes. The outcome of this is that when we implement Sleeping Beauty transposition in human cells with this particular transposase mutant, the integration sites tend to avoid coding sequences represented by exons, and also regulatory promoter elements and enhancer elements, which are probably the riskiest parts of the genome for foreign gene integration Miskey C, Kesselring L, Querques I, Abrusán G, Barabas O, Ivics Z. Engineered Sleeping Beauty transposase redirects transposon integration away from genes. Nucleic Acids Res. 2022; 50(5), 2807–2825.
This is very significant. By using this mutant transposase, gene delivery becomes safer because a lower percentage of the integrations will end up in these potentially dangerous sites of the genome. Again, a single amino acid change can do this.
In terms of improving efficiency, we again shifted a single amino acid in the Sleeping Beauty transposase, which allows for hyperactivity in the transposition reaction. This highlights that there is still potential in this polypeptide composed of 340 amino acids for further improvement from both safety and efficiency perspectives.
Finally, we have shown that we can combine these two features and make a double mutant transposase, shifting transposition sites away from the dangerous sites in the human genome and providing hyperactivity. By fusing these two features, we can make polypeptides that are highly efficient in moving genes into genomes, and at the same time reducing the relative risk of adverse genetic engineering in human cells.
How has the field progressed in terms of the capability of nucleic acid delivery technologies such as lipid nanoparticles (LNPs) to efficiently deliver naked nucleic acids into primary human cells?
ZI: The LNP field is blooming, mainly due to the COVID-19 pandemic and the successful application of LNPs for mRNA delivery in some of the vaccines. This provided a tremendous boost to the field in the context of using LNPs as nucleic acid carriers not only for vaccination, but also for gene therapy or for other therapeutic purposes that require moving nucleic acids to cells. Today, we are seeing LNP applied as a delivery technology across the nucleic acid space, whether it be mRNA, small interfering (si)RNA, or, in the context of CRISPR-Cas9 engineering, single guide RNAs. There is an array of new developments, new findings, and new technologies based on LNP-mediated RNA delivery in the context of gene therapy applications.
Despite this success, there are some limiting factors to LNPs that need to be addressed. For one thing, we are still finding it difficult to move DNA (particularly plasmid-sized DNA) molecules into cells. The chemical composition of an LNP plays a fundamental role in the fate of the nucleic acid. In order for DNA to be expressed, it needs to be localized in the nucleus of the cell where it can undergo transcription. This is quite different from simply moving RNA molecules into the cells. However, I am beginning to see abstracts at conferences and titles of presentations that promise that researchers may be breaking through these boundaries and tapping into the potential of using LNP technology with DNA.
I am really excited about this because it will allow us to fully explore the potential of the Sleeping Beauty transposon with non-viral technology, especially for in vivo applications. LNP technologies represent a highly promising option in this context because they have the potential to be tagged by different targeting proteins that may interact with certain antigens, or with other proteins either exposed on the cell surface or in the cell. In the future, a fully non-viral and synthetic in vivo gene therapy application that carries a certain nucleic acid into a specific type of cell or organ in the human body could be achieved using this method.
What are the pros and cons of transposon systems in the context of the full range of genome editing tool options that is currently available to advanced therapy developers?
ZI: Genome engineering provides many different opportunities to treat genetic diseases, allowing investigators and medical doctors to convert a disease phenotype. There is now an entire spectrum of genetic engineering tools that are available to accomplish this.
For example, if a single base change can fix a disease, one may tap into base editing. If just a few base pairs need to be edited, prime editing can be applied. If longer pieces of genetic information need to be added to a cell, one may consider using either a viral-based system or a transposon-based system because of the robustness of these particular gene vectors in inserting larger pieces of DNA.
That is the niche of the transposon: to efficiently move larger pieces of DNA into a target cell genome. Nothing else can do this as well as a transposon. Prime editing is suitable for inserting up to 200 base pairs into the genome, but that is the rough limitation of that particular reaction. Transposons circumvent that limitation since they typically utilize longer pieces of genetic information for their own propagation.
As we have already discussed, transposons do this integration relatively randomly, which, in this context, may present a disadvantage compared to other systems. Targeted gene editing systems such as CRISPR-Cas9 systems, base editing systems, and prime editing systems are site-specific, which allows them to very precisely introduce a genetic change in the genome. Transposons cannot be this precise currently, but we are working to engineer transposon systems to overcome this disadvantage so that they become safer for human applications.
As someone who works at the Paul-Ehrlich-Institut (PEI), an organization that represents the interface between science and regulation, what is your view of recent regulatory guidance development relevant to this space? And what might we expect to see in the way of further guidance moving forward?
ZI: In terms of recent regulatory guidance, German advanced therapy stakeholders and politicians recently launched a national strategy for gene and cell therapy. This came out of the realization that the pipeline for translating gene and cell therapy preclinical research findings into the clinical and, subsequently, into full drug development, was inefficient. This new strategy is specifically working toward helping sponsors and clinical trial applicants through regulatory procedures, expediting dialogue between regulators and developers, and standardizing regulatory procedures on a national level.
Right now, every sponsor and every applicant is communicating with the PEI on an individual basis. This will not change, however, what can change is that through an enhanced level of input and communication, it will be much simpler to spell out standardized expectations and requirements in order to troubleshoot an application.
Additionally, since the beginning of 2023, there has been a new European system that does not necessarily change regulatory guidance with respect to how gene and cell therapies need to go through clinical trials, but rather introduces a new web-based portal where clinical trial applications need to be submitted. This means that everything goes through a central portal, which will hopefully expedite the process of a developer gaining regulatory approval for a clinical trial application.
Looking to the future, transposon technology has gone through clinical tests and trials very quickly, and it will now be expected to turn this into a drug that is available to patients. However, regulatory guidance and the actual tests that need to be documented and carried out in the dossier that an investigator submits to the regulatory authorities in a clinical trial are still evolving alongside the science. For example, because of the potential risks that off-target CRISPR-Cas9 genome cleavage can introduce to a certain drug product, regulatory guidance for that application was based on annotating the sites where cleavage occurred. Of course, guide RNA design has drastically improved over the last couple of years and regulators have followed what that innovation produced with respect to assays and bioinformatics tools.
What is still missing, though, from the perspective of a regulator, is a sophisticated tool to predict potential risks associated with genetic engineering technologies. This concern is not necessarily limited to a CRISPR-Cas9, or any other gene editing system for that matter, but encompasses any kind of genetic change that we introduce into a cell including, of course, Sleeping Beauty transposition. We need to think about how to put together cell-based assays that a sponsor can employ preclinically with the actual components of genetic engineering (e.g., CRISPR-Cas9, base editing, prime editing, or a Sleeping Beauty transposon), and with a certain level of output (bioinformatic output, transcriptional output, or cell phenotype-based output), deliver strong data with respect to the safety of that particular genetic engineering step. Of course, this kind of tool would be mostly applicable to ex vivo genetic engineering and not an in vivo therapy, but if ex vivo applications were associated with a certain level of risk, that would help regulators and developers alike.
Can you highlight some of the key future applications and areas of development for transposon platforms that you expect to see moving forward?
ZI: In the coming years, transposon technology will be translationally applied to the genetic engineering of immune cells. That leads us to the engineering of T cells with either CARs or with a T cell receptor. This is a relatively straightforward and efficient genetic engineering technology mainly for ex vivo applications where T cells, for example, can be electroporated with plasmid constructs and mRNA encoding the transposase.
There are clear advantages to using Sleeping Beauty or another transposon (as opposed to using viral technology) in the genetic engineering of immune cells. Sleeping Beauty-engineered CAR-T cells are, biologically speaking, as potent as lentiviral-engineered CAR-T cells, but the actual application is easier. Regulatory approval is also more streamlined because there is no environmental risk assessment needed for a transposon vector, which is just a nucleic acid. There is no potential release of an infectious viral particle to the investigator or to other human beings. Furthermore, the costs associated with manufacturing nucleic acids at the GMP level are lower than those for viral engineering technology.
There are currently quite a number of CAR-T cells engineered with transposons in the early stages of clinical development, and I believe that number will continue to grow. Another area of application that we will likely soon see is Sleeping Beauty-engineered natural killer cells. Furthermore, I am hopeful that we will see additional immune system applications in the future, using cell types like macrophages, to treat not just cancer but potentially other human pathologies, too.
Another application that I am currently working on relates to tapping into hematopoietic stem cells (HSCs). This is really a different level of application, being not so focused on the cancer field but rather on the area of inherited monogenic diseases that affect one component of the blood system – either the immune system or other components of the blood. We are using electroporation techniques with Sleeping Beauty components, but instead of a human T cell, we are using HSCs. By resupplying or converting a missing factor in these HSCs throughout their differentiation into blood cells, we can phenotypically correct the cells to be without disease. This is not as advanced as the CAR-T cell applications – with the Sleeping Beauty transposon system, we are still at the level of preclinical research. However, the preclinical data looks very promising. We are in a situation now where we have robust technologies to genetically modify both mouse and human HSCs.
We are working on this while simultaneously pushing forward with the CAR-T cell applications (mainly in the context of cancer therapy) in the second wave of development. My expectation is that in the next wave of clinically relevant applications (hopefully within the next 2 years), the first clinical trials with Sleeping Beauty vectors will be initiated, widening the horizon, and allowing us to work on treating not only cancer but also rare genetic diseases with this particular non-viral genetic engineering technology.
1. Ivics Z. Update on the Sleeping Beauty transposon system: current state-of-the-art & remaining challenges. Cell & Gene Therapy Insights 2021; 7(2), 99–105. Crossref
2. Miskey C, Kesselring L, Querques I, Abrusán G, Barabas O, Ivics Z. Engineered Sleeping Beauty transposase redirects transposon integration away from genes. Nucleic Acids Res. 2022; 50(5), 2807–2825 Crossref
Zoltán Ivics received his PhD in Molecular Biology in 1994. After postdoctoral studies at the University of Minnesota in the USA and the Netherlands Cancer Institute, he was appointed as a Research Group Leader at the Max Delbrück Center for Molecular Medicine in Berlin, Germany. He was appointed as Head of Division at the Paul Ehrlich Institute in Langen, Germany, in 2011, where he is currently heading the Research Centre embedded in the Division of Hematology, Gene and Cell Therapy. Professor Ivics’ major scientific achievement is the molecular reconstruction of the Sleeping Beauty transposon and development of technologies based on Sleeping Beauty gene transfer for a wide array of applications involving genetic engineering of cells. Professor Ivics has published >150 papers in peer reviewed journals, with a total Impact Factor of >1200 and >8000 citations (h-index: 64 by Google Scholar), and is co-inventor on 12 issued patents. Since 2000, his research efforts were supported by 25 research grants from the German Research Foundation, the German Ministry of Education and Research, the European Commission, the Volkswagen Foundation and the German Consortium for Translational Cancer Research. He received recognition of the ‘Molecule of the Year’ in 2009 for developing a hyperactive Sleeping Beauty transposase that opened the door for clinical applications. He served as a member of the Board of the European Society of Gene and Cell Therapy (ESGCT) between 2012 and 2022. He is a current member of the Board of the German Society of Gene Therapy and member of the committee for “Clinical trials and regulatory affairs” of the German Stem Cell Network. Professor Ivics organized several international conferences, including the Annual Congress of the ESGCT in Berlin in 2017. He is an Elected Member of the Academia Europaea and the Hungarian Academy of Sciences. His current interests focus on establishing clinically relevant methods and protocols for non-viral gene therapy.
Professor Dr Zoltán Ivics
Head of Research,
Division of Hematology, Gene & Cell Therapy,
Paul Ehrlich Institute
Authorship & conflict of interest
Contributions: The named author takes responsibility for the integrity of the work as a whole, and has given his approval for this version to be published.
Disclosure and potential conflicts of interest: The author is the inventor of Sleeping Beauty patents, which can result in payment, if the technology is licensed.
Funding declaration: The author received no financial support for the research, authorship and/or publication of this article.
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Copyright: Published by Cell & Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0 which allows anyone to copy, distribute, and transmit the article provided it is properly attributed in the manner specified below. No commercial use without permission.
Attribution: Copyright © Ivics Z. Published by Cell & Gene Therapy Insights under Creative Commons License Deed CC BY NC ND 4.0.
Article source: Invited; externally peer reviewed.
Revised manuscript received: Oct 3, 2023 Publication date: Oct 18, 2023