Department of Cellular Immunotherapy

The future of cell and gene therapy will be virus-free, but it will not be enabled by only one technology. It requires the right combination of DNA design, gene-transfer strategy, cell therapy process development and translational manufacturing expertise. With our proven expertise in these areas, Fraunhofer IZI supports developers and companies worldwide whose product candidates are on the verge of translation.

To further advance non-viral cell and gene therapy approaches Fraunhofer IZI and PlasmidFactory GmbH have signed a Memorandum of Understanding. Together, the goal is to support next-generation virus-free engineering platforms for CAR T, TCR T, NK and other immune cell therapies. The collaboration builds on previous joint work in virus-free gene transfer and CAR T manufacturing. Since the signing of the MoU in September 2025, several pilot projects have already been initiated to further explore how these technologies can be brought together in practical development settings.

This direction is also highly relevant for broader European initiatives such as EASYGEN, where Minicircle DNA technology is being explored as a central basis for faster, virus-free CAR T manufacturing.

Clinical translation experience, including CAR T manufacturing projects such as LION-1 clinical trial, further underlines why the combination of DNA technology and advanced cell manufacturing know-how is so important.

Because the future of advanced cell therapies may not only depend on what we deliver. It may also depend on how we engineer them.

Virus-free CAR T engineering is gaining increasing attention as the field looks for scalable, flexible and potentially more cost-efficient alternatives to viral vector-based manufacturing.

But replacing the vector is only one part of the challenge. The real question is: How can a virus-free gene transfer approach be developed into a robust manufacturing protocol that supports GMP translation and clinical application?

Dr. Katrin Mestermann, Head of GMP Development Unit at Fraunhofer IZI discussed the development of a virus-free clinical CAR T manufacturing protocol at a webinar organized by PlasmidFactory GmbH.

The webinar took place on June 30, 2026 and addressed advancing practical, translationally relevant approaches for next-generation virus-free cell and gene therapies.

The recording of the webinar is available here.

Viral vectors have long been the dominant route for generating gene-modified cells. They are powerful, established and clinically proven. But as the field matures, we have to ask a broader question: Do we always need a viral vector – or do we need more flexible ways to engineer cells?

This is becoming relevant for CAR T, TCR T, CAR NK and related cell therapies as the field moves toward scalable, reproducible and clinically translatable manufacturing.

The collaboration with Fraunhofer IZI is important because it brings process development, GMP manufacturing expertise and clinical translation experience into one workflow. In that context, virus-free engineering becomes a strategic development direction. It can support faster development timelines, reduce dependence on complex viral vector manufacturing, lower process costs, and simplify CMC and QC aspects compared to LVV-based workflows. It also opens design space where integration profile, payload design, transient expression or non-integrating delivery become part of the therapeutic concept.

But virus-free engineering also depends on the right DNA architecture. Minicircle DNA is compact, supercoiled and free of functional bacterial backbone sequences such as antibiotic resistance genes. It is reduced to the functional expression cassette – the part actually needed for the therapeutic design. Especially when DNA is used in combination with transposon systems, CRISPR-based approaches, electroporation, nucleofection or emerging nanoparticle-based delivery concepts.

For us, Minicircle DNA is not a single-use format. It can support different virus-free engineering strategies – from transposon-mediated CAR integration to CRISPR donor templates and nanoparticle-based delivery. Minicircle DNA was not yet broadly available at GMP level, which limited its clinical adoption. With GMP-grade Minicircle DNA now accessible, this architecture has become a viable and scalable alternative for virus-free cell engineering.

At PlasmidFactory, we do not see Minicircle DNA as replacing everything. We see it as enabling workflows where unnecessary bacterial backbone sequences should not be part of the therapeutic design. Minicircle-based approaches are already part of clinical and translational programs, including CAR T and other gene-modified cell therapy applications. As delivery technologies evolve, virus-free engineering may increasingly move beyond ex vivo manufacturing toward targeted in vivo cell engineering concepts. DNA architecture will become even more important. What is encouraging today is where Minicircle DNA is becoming relevant: Between biological function, manufacturing feasibility, safety considerations and clinical translation. That is exactly where the future of advanced cell therapies will be decided.

Bringing a new CAR-T cell technology from the laboratory into a clinical trial, including the establishment of GMP-grade manufacturing, can take years. But where exactly is the time lost, and how can development timelines be shortened?

Saving years in clinical development is not about working harder. It requires smart strategies, robust technologies, and early consideration of GMP requirements.

In CAR-T cell research, non-viral gene modification approaches such as transposase systems like Sleeping Beauty are increasingly being used to reduce manufacturing complexity, improve scalability, and accelerate translational workflows. Clinically validated manufacturing protocols utilize electroporation for the delivery of CAR-encoding Minicircle DNA and transposase-encoding mRNA into target cells. Commercial providers, such as PlasmidFactory GmbH, offer Minicircle DNA as an ideal format for non-viral gene delivery, as it is small, supercoiled, and lacks functional bacterial backbone sequences.

One key challenge is the transition from research-grade processes to GMP-compliant manufacturing. As our experts have experienced firsthand: "I would not have expected how much time it takes to validate lab processes for GMP manufacturing. Things that are routine in the laboratory first need to be carefully transferred, scaled up, and validated before they can be implemented in a GMP environment," says Michael Hudecek, who leads the Würzburg branch of Fraunhofer IZI and conducts clinical trials at University Hospital Würzburg. “Through our preclinical research and clinical studies, we have built substantial expertise in non-viral technologies, enabling us to significantly accelerate the development of our programs.”

Lentiviral vectors remain one of the most established tools for ex vivo gene modification, especially for stable transgene expression. Many approved gene-modified cell therapies were developed using lentiviral delivery, and the clinical and manufacturing experience behind this system is substantial.

However, lentiviral workflows have practical upstream constraints: long vector manufacturing and release timelines, high cost burden, viral vector-specific testing, biosafety requirements, payload limitations and regulatory complexity. Therefore, many teams evaluate virus-free engineering routes for next-generation cell therapies.

For stable non-viral CAR-T engineering, the combination of Minicircle DNA and the Sleeping Beauty transposon system is particularly attractive. It enables therapeutic transgene integration, while using a DNA format suited for efficient delivery.

Meanwhile, delivery technologies continue to evolve. For ex vivo workflows, electroporation remains a central route for introducing DNA, RNA or gene-editing components into target cells. In parallel, LNP- and polymer-based delivery platforms are being explored for ex vivo applications and emerging in vivo cell engineering.

Importantly, this is not about replacing one established technology with a single alternative. It is about expanding the engineering toolbox. Each route creates its own development questions: 

• Lentiviral workflows require robust viral vector production, release testing, infectivity assays and appropriate biosafety infrastructure.
• Minicircle/Sleeping Beauty workflows require control over transposase activity, integration pattern, vector copy number and cell viability.
• Genome editing workflows need to balance editing efficiency, off-target activity, repair outcomes and persistence of editing components.
• Nanoparticle-based delivery approaches raise questions around targeting, cargo loading, formulation behavior and expression kinetics.

This changes the role of the entering DNA: For stable non-viral integration, DNA architecture is no longer a background detail. Construct size, bacterial backbone content, topology, purity, supercoiled proportion and quality grade can influence how the workflow performs.

This is where Minicircle DNA becomes relevant. By retaining the functional expression cassette while removing bacterial backbone sequences, Minicircle DNA reduces unnecessary sequence burden. In cell engineering workflows, this can support improved transfection performance, reduced DNA-associated toxicity and a streamlined virus-free manufacturing path.

The key question is not simply “viral or non-viral?” It is: Which delivery system, DNA format and material quality best fit the intended cell product, development timeline, and manufacturing strategy?

Both lentiviral and virus-free routes can generate functional CAR-T cells. The main differences increasingly lies upstream - in time, cost burden, biosafety setup and regulatory complexity.

Transposons are nature's simple and effective gene moving machines. We crafted a transposon-based gene delivery platform, called Sleeping Beauty (SB), that unites the advantages of nonviral genome engineering technologies including scalability, long-term storage of GMP vector components, S1 safety requirements and GMP manufacturing at affordable costs with a fundamental feature no other nonviral system has: integration of a cargo gene into the genome of a target cell.

The SB transposon system offers an advantageous integration profile in comparison to the commonly used lentiviral vector system, resulting in a safer genome-wide footprint:

• https://pubmed.ncbi.nlm.nih.gov/27491640/
• https://pubmed.ncbi.nlm.nih.gov/28101686/

Nucleic acid components of nonviral technologies need to be delivered into primary human cells when developing cell-based therapies. Such delivery has been relying on physical methods such as electroporation and lipid nanoparticle-based technologies and represents a formidable barrier that manifests itself in reduced efficiencies of genetic engineering of the cells and associated cellular toxicities.

One key advancement to overcome the above barriers was to combine SB transposons with PlasmidFactory`s Minicircle technology. We have shown in both hematopoietic stem cells and T cells that by delivering SB transposon vectors as Minicircles the viable fraction of the electroporated cell population can be dramatically increased. Furthermore, a key enabling feature of a Minicircle in the context of SB vectors is that by deleting the bacterial plasmid backbone sequences the two ends of the transposon are situated close to one another in the Minicircle, thereby significantly enhancing a rate-limiting step during transposition. This biochemical step enables delivery of large cargo sequences that are significantly beyond the cargo capacity of viral systems.

Sleeping Beauty has claimed ground in nonviral CAR-T cell engineering and Minicircles are a defining component of the technology. We are offering an integrated service package to develop, characterize and manufacture SB transposon-engineered human T cells for therapy. This covers all aspects of developing, functionally testing and manufacturing a genetically engineered cell product. The offer/package is agnostic with respect to the gene of interest (GOI) to be transferred into the T cell genome. A prominent example for a relevant GOI is a CAR. The package includes transgene design and optimization, cloning of GOI into the transposon vector, stable gene transfer into T cells, functional in vitro testing including GOI expression and biological performance, cytotoxicity and proliferation, functional tests in vivo and genomic safety analysis, including vector integration site analysis and vector copy number analysis.

The service package addresses need of manufacturing cell-based therapeutic products with nonviral technologies that offer superior scalability, enhanced genomic safety and lower manufacturing costs.