Molecule design for genetic therapy

Computer-aided nucleic acid design for gene and cell therapy to treat type 2 Gaucher's disease

Nucleic acid molecules which specifically deactivate, correct or encode human gene functions now play a fundamental role at every stage of the value chain for biotechnology products. Key applications for nucleic acids include validating unknown gene functions, genetic vaccination and somatic gene therapy. The Steinbeis Transfer Center for Nucleic Acids Design provides highly specialized services in the field of biotechnology and the development of active pharmaceutical ingredients. The center designs nucleic acid molecules tailored to individual requirements using powerful software developed in-house.

Gaucher’s disease is a hereditary condition, and the commonest of the lysosomal storage diseases – a group of disorders which disrupt lipometabolism. Different subtypes of the disease exist: non-neuropathic (type 1) and neuropathic (types 2 and 3). All types lead to changes in internal organs which are characteristic of Gaucher’s disease. Additionally, the two neuropathic forms cause serious and acute (type 2) or chronic changes (type 3) in the central nervous system.

The only treatment currently available is enzyme replacement therapy with imiglucerase – a chemically modified form of the human GCase enzyme which acts to replace the faulty enzyme. The replacement enzyme is administered intravenously and is absorbed well by the body’s phagocytes. Therapy for one patient costs several hundred thousand euros per year, but its success in combating type 1 Gaucher’s disease is undisputed. The therapy reduces damage to the spleen, liver and bones, and can even bring disease progression to a standstill. However, this therapy is ineffective in combating type 2 Gaucher’s disease, as the replacement enzyme cannot penetrate the blood-brain barrier. As a result, causal treatment of brain damage in patients with type 2 Gaucher’s disease is currently impossible.

To address this issue, the consortium “Innovative gene and cell therapy for Gaucher’s Disease Type 2” was set up in Germany in 2009. Also known by the abbreviation In-
TherGD, the consortium is funded by the German Federal Ministry for Education and Research. To effectively treat type 2 Gaucher’s disease, it must be made possible to introduce a functional form of the GCase enzyme into all bodily organs. The consortium is working towards this goal by developing innovative new gene therapy methods to treat the disease.

Instead of directly administering the replacement enzyme, these methods focus on introducing the enzyme’s DNA to target cells – including in the brain – in a variety of ways. This “genetic blueprint” means the body’s own cells can then produce the healthy enzyme on their own, on a longterm basis.

In the cell nucleus, the DNA “blueprint” is first transcribed into an mRNA (messenger ribonucleic acid) molecule, which exports the code from the cell nucleus into the surrounding cytoplasm. This mRNA then acts as the direct blueprint for protein biosynthesis of the healthy GCase enzyme. However, the poor efficiency of cellular infiltration presents a significant problem. To overcome this hurdle, the consortium is applying optimized viral and non-viral gene transfer methods to help introduce the genetic blueprint for GCase into muscle cells or hematopoietic stem cells, thereby ensuring ongoing production and secretion of GCase within the body. Two different strategies are also being applied to help reach a therapeutic level of GCase in the central nervous system. The first strategy exploits the retrograde transport mechanism of adeno-associated viruses by using them as gene vectors. Carrying the healthy GCase code, the viruses are injected into different muscles, where they are transported into peripheral nerve cells in the central nervous system. Here, the healthly GCase enzyme is separated and can enter the lysosomes of other cells. In the second strategy, hematopoietic stem cells are genetically manipulated using vectors based on transposons (“jumping genes”) and minicircles (small circular DNA sequences) so that they can stably produce a soluble form of GCase which is able to penetrate the blood-brain barrier.

For this therapy to work, the GCase gene – after having been painstakingly introduced into the target cells – must be optimally exploited to ensure it releases the maximum possible amount of the healthy enzyme needed for therapy. The Steinbeis Transfer Center for Nucleic Acids Design in Berlin provides expert support in this area. The center specializes in modeling the molecular structure of mRNA using advanced bioinformatics methods, allowing cells to process the mRNA far more efficiently into the enzyme required for therapy. These methods have been proven experimentally, and some are even patented.

These methods exploit a variety of functional sections of the mRNA. As well as optimizing the section of the gene sequence which contains the code for the enzyme, these methods also focus on modulating the regulatory 5’ and 3’ untranslated regions (5’ and 3’UTR). Using a new algorithm, molecules of functional RNA can be connected in a way which allows their individual structures – and thus their individual gene functions – to be retained. Based on this form of active fusion, structural domains can then be attached to the GCase mRNA to simplify its processing and its export from the cell nucleus into the cytoplasm. They also protect the processed mRNA from attack by decomposition enzymes. As an example, one particularly effective structure is the post-transcriptional regulatory element of the woodchuck Hepatitis B virus.

This RNA processing is also aided by an intron, which can be removing via splicing. This intron is a section of DNA which does not act as a blueprint for the enzyme, but rather modifies the structure of the artificial gene so that it behaves in a similar manner to most natural human genes. It also makes the 5’ UTR of the mRNA more available to interact with ribosomes – complexes of RNA and protein which initiate the production of GCase in the cytoplasm. Although none of these methods actually alter the therapeutically active molecule, they can significantly boost its intracellular concentration. The potential for optimization by modifying the structure of the mRNA is one which has barely been exploited. As such, the expected results when this method is combined with other synergetic methods are highly anticipated. For gene therapy to be successful, the required level of GCase enzymes must be reached in the target cells. This strategy for producing healthy GCase enzymes can also be complemented by inhibiting the defective form of the enzyme in the patient, so that it cannot disrupt the functioning of the intact enzyme needed for therapy. The Steinbeis Transfer Center for Nucleic Acids Design in Berlin is currently developing a specific inhibitor for this purpose.

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