Transposons and Transposases: A Dynamic Duo in Genetic Engineering

Welcome to the world of genetic innovation and transformation, where transposases take center stage. In this article, we will explore its significance and the latest developments. Our journey through the mysteries of transposase also explains the interaction between transposase and transposon and why transposons are becoming more and more important on the way to a sophisticated system of modern times.

Content

Understanding Transposase

Transposases and Their Continuous Optimization

What Do the Transposases Actually Move?

The Next Generation of Transposase Technology

Impact of DirectedLuck^® in the Biopharma Field

Understanding Transposase

What Is a Transposase?

A transposase is a fundamental enzyme in genetic biology, playing a pivotal role in facilitating the movement of genetic material within a genome. At its core, a transposase is responsible for catalyzing the transposition of DNA segments, typically called DNA transposons, within an organism's genome. This type of transposon is divided into four sub-classes based on their mobilization mechanism: "cut and paste", "self-synthesizing", "replicative" and "rolling-circle". Today, the "cut and paste" transposase/transposon systems in particular play a central role in the genetic manipulation of cells and thus have far-reaching implications for non-viral gene transfer.

Historical Context and Milestones in Transposase Research

The study of transposases dates back to the mid-20th century when Barbara McClintock, an American geneticist, made groundbreaking discoveries regarding transposons in maize. Through meticulously conducted experiments on a variety of corn with different colors, she was able to demonstrate that these distinct, externally visible traits were attributable to certain regions in the genome being mobile. These mobile regions had the capacity to cut themselves out from one location in the genome and then reinsert themselves at another location. Today, we recognize these regions as "transposons". This was an extraordinary discovery, as it was previously believed that an individual's genome remains unaltered.

However, McClintock's ideas were not readily accepted by many researchers. Her thinking diverged from established paradigms and was dismissed as nonsensical. In 1983, four decades after she proposed her theories, and after they had been substantiated through experimentation, Barbara McClintock received the Nobel Prize in Medicine for her groundbreaking work.

As scientific investigation evolved, researchers unveiled the remarkable diversity of transposases, identifying different types and systems present in various organisms, ranging from bacteria to mammals. This diversity provided the impetus for versatile applications in genetic engineering and biopharmaceuticals.

Over the years, transposase technologies advanced considerably. Scientists continually honed their understanding of transposases and their mechanisms, striving to optimize the targeting of specific genomic regions.

This historical journey has significantly contributed to the enhancement of genetic engineering techniques, allowing for more accurate and effective genetic modifications. Nowadays transposases are even used in gene therapy for the treatment of diseases.

Transposases and Their Continuous Optimization

Transposases have garnered widespread recognition for their pivotal role in genetic engineering and biopharmaceutical applications. They serve as invaluable tools for the introduction, deletion, or rearrangement of genes within an organism's genome.

The various transposases, including for example, PiggyBac-based transposases, Tol2, and Sleeping Beauty, represent powerful tools in genetic engineering and molecular biology. Each of these transposases was discovered and developed to introduce DNA into the genomes of organisms, but they differ in their origins and application.

Strategies of Transposase Optimization

Continual advancements in transposase technologies have focused on improving efficiency, specificity, and safety – especially in the context of gene therapy for patients. Researchers have pursued various strategies to enhance transposase systems and optimize the precision. This once more demonstrates the dedicated efforts of the scientific community to realize the full potential of genetic engineering and modification.

A few notable strategies include:

• Hyperactive mutants: The development of hyperactive mutant transposases that exhibit increased transposition efficiency, leading to more effective gene integration.
• Optimized targeting: Precision engineering of transposases for better recognition of specific target sites within the genome, reducing random insertions and unwanted effects.
• Regulated transposition: The implementation of controlled systems to fine-tune the timing and location of gene insertion, offering greater control over the genetic modification process.
• Enhanced delivery techniques: Innovations in the delivery of transposase components, such as viral vectors or nanoparticle-based approaches, have improved the overall efficiency of transposase-mediated gene transfer.

These strategies reflect a dedicated effort within the scientific community to harness the full potential of genetic engineering and modification, making these technologies safer and more effective.

What Do the Transposases Actually Move?

The Role of Transposons

The latest trends in transposase systems cannot be understood without taking a closer look at the interaction partner of transposases - and that is transposons. Transposase, as already described, is the enzyme responsible for DNA movement within or into an organism's genome. It catalyzes the process of transposition, which involves the precise relocation of DNA segments. Transposons are the mobile segments of DNA that transposases move. In a natural biological context, transposases do not shuffle DNA segments or transposons around at random. Instead, transposons harbor genetic information, such as antibiotic resistance genes, which are amplified or transmitted. Transposases are part of the transposon and keep things moving. So it's all about genomic plasticity and evolution.

In the context of genetic engineering, two factors are of crucial importance when you think of transposons. The first is the direct interaction between the transposase enzyme and the sequence parts of the transposon that are involved in the intermolecular reaction. While this point is mainly the subject of scientific research, many of the transposase/transposon systems currently used in the biotech industry focus only on optimizing the transposase enzyme sequence or other transposon elements. However, a little later we will take up a system that also addresses the transposon parts that are important for the direct interaction with the transposase. But let's first look at another important aspect of why transposons have recently come back into focus. After all, it is really important which functional elements are added to the transposon in addition to the gene of interest.

The Significance of the Transposon’s Cargo

So what is it that a transposon can carry that really matters? In principle, transposons represent our traditional expression vectors and replace them at the same time. The main difference is that the transposase cuts out the vector parts required for bacterial plasmid amplification and integrates only the desired gene and the vector elements that define its expression. This means that transposons make their cargo space available for a wide range of genes and DNA elements that can facilitate optimal expression of the target genes. Prominent examples are:

• Selection marker genes: An efficient selection strategy restricts the resulting recombinant population specifically to cells with the highest expression performance. This reduces the screening effort for producer clones.
• Promoters and polyA signals: A careful selection of promoters and polyA signals not only ensures high and stable target gene expression and efficient termination of transcripts. If desired, it also allows the coordinated expression of different molecules or molecule chains.
• Additional beneficial sequence elements and factors: In addition to promoters, there is a broad variety of other cis-acting DNA sequences, such as enhancers, MAR and UCOE elements, which can increase or stabilize the expression of target genes at the genomic integration position. It is also possible to add genes for metabolic cell engineering or small regulatory RNAs that interact favorably with cellular or recombinant factors.

It is important to understand that it is not the individual effects but the concerted interaction of such transposon elements that matters. In summary, the transposon is a hidden hero in transposase systems, providing the system with the efficiency, and stability needed for optimal performance. While transposase development is an exciting trend, it is essential to recognize the integral role of transposons in these advancements.

The Next Generation of Transposase Technology

Today, there are a number of sequence-optimized transposases with higher activity, and just as there were many good classical expression vectors, there are equally good transposons. But is there anything new in this field that can really make a difference? In fact, there is a novel, unique transposase system developed by ProBioGen.

In addition to the transposon, there is another important player that dictates target gene expression and that is the host genome, or more precisely the chromosomal environment of the integration position of the transposon. No matter whether a transposase is more or less active, it integrates the transposons into chromosomal regions that are generally transcriptionally accessible which is helpful. However, there are also subregions with significantly higher or significantly lower transcriptional activity so it still depends on where your transposon lands. In general, the accessibility and activity of DNA embedded in chromatin is regulated by the epigenetic code. This code is based on post-translational modifications of amino acids of histone proteins and can be highly specific. For example, there are certain modifications that are strongly enriched in the areas with the highest transcriptional activity.

This is the reason why they have named their technology DirectedLuck^®. However, DirectedLuck^® is not based on this cutting-edge transposase alone, but comprises three inventive steps:

Epigenetic targeting:

When the scientists at ProBioGen thought about the possibility of directing their desired target genes to the most optimal chromosomal locations while remaining flexible and inserting multiple copies per genome, they came across epigenetics. The “sweet spots” of a genome are already highlighted by specific histone modifications. As the epigenetic code has to be maintained by the cells, there are also protein complexes that can read, add or remove certain modifications. This also requires binding to the specific histone motif. This is the reason why they fused a chromatin reader domain, which identifies a histone modification that is characteristic of genome sites with the highest transcriptional activity, with a transposase. Indeed, this transposase fusion variant directs transposons precisely to the desired spots and integrates them there. As a result, the cell line carries multiple transgene copies at most preferred loci. As this histone code itself is universal, the DirectedLuck^® transposase works in a broad variety of host cells from different species and tissue origin.

Optimized transposase sequence:

Naturally occurring transposases often show little or no activity and are unsuitable for industrial use in this form. Therefore, specific mutations were introduced into the DirectedLuck^® transposase sequence to increase the stability of the enzyme and the transposition rate. The result is a new hyperactive transposase variant.

Optimized transposon:

Besides the transposase enzyme, the architecture of the transposon plays an important role in impact on excision and integration efficiency. In simple terms, the transposases bind to short inverted repeats (ITR) that flank "cut and paste" class transposons. For the DirectedLuck^® system, an artificial transposon with modified ITRs was designed to further promote the interaction between the Dimerization and DNA-binding domain of the transposase and the transposon ITRs. In addition, these ITR elements avoid promoter and activator activities of commonly used transposon configurations.

The DirectedLuck^® system unfolds its full potential through a powerful transposon with a careful combination of strong promoters and DNA elements that boost and stabilize expression.

Impact of DirectedLuck^® in the Biopharma field

The DirectedLuck^® system precisely guides the desired target genes to integration sites exhibiting the highest and most stable transcriptional activity. This results in significantly higher robustness and elevated titers. Extensive screening of high-producing clones is therefore no longer required, allowing for a more dedicated focus on product quality - especially for complex antibody formats like Bi-specifics.

The approach yields an unparalleled level of consistency among bulk pools, resulting in superior producer clones characterized by exceptional expression stability.

As bulk pools are highly representative of the clone to be selected later on, they can be used to manufacture early material for development work, TOX studies or even DS1 material, reducing overall timelines to the clinic. Increased productivity and reduced timelines have also an impact on the cost of goods.

In summary, DirectedLuck^® stands out as a groundbreaking and profitable system due to its significant time savings, enhanced product quality, and exceptionally low risk of failure. Applications range from cell lines for the production of biologics and viral packaging cell lines to cell and gene therapy.

For more information on DirectedLuck^® visit: https://lp.probiogen.de/transposase

For more information on DirectedLuck^® download our Factsheet.