Emerging genetic engineering in medical biotechnology
After the DNA helix was uncovered in 1953 and in 1968 Rogers and Pfuderer demonstrated a proof-of-concept for virus-mediated gene transfer, about two decades ago the first gene therapy trials were performed. In 2003 the sequencing of the human genome was completed, which provided new opportunities for further development of molecular medicine. In 2003 Gendicine was the first gene therapy product approved for clinical use in humans in China. In July 2012, the European Medicines Agency recommended Glybera for approval, which was the first recommendation for a gene therapy in either Europe or the United States. With the increased understanding of molecular medicine, the field is now developing even more specific and efficient therapeutics that repair gene function, which is now producing clinical results.
Commissioned by the Ministry of Infrastructure and Environment (Ministry of I&M) and the GMO office (Part of the Netherlands Institute for Public Health and the Environment, RIVM) Xendo executed a scientific literature evaluation on novel and trending molecular genetic techniques applied in medical biotechnology.The GMO Office is responsible for the processing of license applications with respect to GMO handling on behalf of the Ministry of I&M and it is intended to develop new policies for medicine based on new molecular biotechnologies.
Trending themes within molecular medicine such as genomics-based medicine, epigenetics, nanomedicine, personalized medicine and synthetic biology are all impacted by the development of techniques that facilitate and improve genetic engineering. Four technology areas were identified: genome editing, epigenome editing, gene expression regulation and gene delivery. Within these technology areas, the following technologies were identified: ZNF (Engineered nuclease), TALENs (Engineered nuclease), CRISPR/Cas9 (Engineered nuclease system), siRNA and miRNA and Modified Antisense Oligonucleotides. The table below presents the technology areas and the underlying techniques as well as possible applications.
Genome editing by engineered nucleases (ZFN, TALENs and CRISPR/Cas9) is of great value in research to understand functions of individual genes but also as medicine for genetic disease treatment. Currently, genome editing strategies are developed as therapeutic agents. A critical breakthrough for gene targeting approaches was the discovery that by creating a site-specific DNA double-stranded break (DSB) at the targeted locus it is possible to strongly stimulate genome editing by homologous recombination. Engineered nucleases are not only used to introduce permanent deletions or insertions in the host genome but can be re-designed to control epigenome modification and gene expression. Engineered DNA binding domains of artificial endonucleases can be fused to functional domains of chromatin-modifying enzymes or a transcription activator/repressor. This type chimeric protein is able to control chromatin modification status, or regulate gene expression at the transcriptional level.
Small noncoding RNAs
Micro RNAs (miRNA) and Small Interfering RNAs (siRNA) have been discovered two decades ago and added a new dimension to our understanding of complex RNA-mediated gene regulatory networks. These RNA molecules can exert regulation of gene expression. As such, molecular medicine base on these small RNA molecules can be applied at an additional level, for example, to regulate developmental and physiological processes or to treat a wide range of disorders including cancers and infections.
Therapeutic oligonucleotides (including small noncoding RNAs) that intend to have an effect on gene expression in general need to be able to enter the targeted cells and stay biologically active to be able to reach their DNA or RNA target sequence. As nucleotides composing RNA and DNA are linked to each other by phosphodiester linkages that are easily cleaved by endo- and exonucleases such molecules often are not suitable for the intended medical use. Many types of modifications have been described, and besides backbone modification; sugar modification (Locked Nucleic Acids, Bridged Nucleic Acids), nucleobase modification (Base Analogues), and terminal modification (coupled sugar, lipid, and peptide) have been applied to improve oligonucleotides properties.
In most cases, the described technologies and their future development depend on efficient delivery systems. About 70% of gene therapy clinical trials carried out so far have used modified viruses to deliver genes. Although they have substantially advanced the field of gene therapy, several limitations are associated with viral vectors, including patient safety issues and difficulty of virus production. The development of non-viral vectors is attractive because of advantages such as fewer safety issues and fairly simple manufacturing processes. Many non-viral systems have been developed for delivery of genetic material, including the injection of naked DNA alone or in combination with physical methods such as gene gun, electroporation, hydrodynamic delivery, sonoporation, and magnetofection. These techniques are generally less applicable to systemic gene delivery in humans than in small animals such as mice. Therefore, a range of synthetic delivery vectors has also been developed, including lipids and liposomes, polymers (linear and branched polymers, dendrimers and polysaccharides), polymersomes and inorganic nanoparticles.
The most attractive aspect of the novel therapeutics based on the technologies described is their ability to target virtually any gene(s), which may not be possible with classical small molecules or protein-based drugs. While the efficacy of these novel therapeutics has been successfully demonstrated in vivo, several technical barriers still need to be overcome in order for many clinical applications. The novel therapeutics allow for direct and sustained interference with disease related gene expression and gene regulation, in most cases without the necessity to change the endogenous sequences of the genome itself. The ethical and safety concerns of changing genome sequences are herewith in most cases circumvented and a clear paradigm shift from gene repair and replacement to gene regulation can be observed. Nevertheless, some concern remains related to the transgenerational effects of medical treatments in general and specifically for treatments that strongly affect gene expression. New insights into epigenetic mechanisms revealed a new high-speed evolution system independent of random DNA changes: epigenetic evolution by chromatin modifications, such as acetylation and methylation, in response to environmental changes including medical treatments and even psychological experiences, which are transmitted between generations.
With the recent surge in intensive research investigating new therapeutic mechanisms and combinations of new tools, it can be expected that significant advance will be made for their future role in therapeutics.
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Blog & report by:
Harm Hermsen - Managing Consultant
Paul Joosten - Sr. Consultant
Xiaoxi Zhu - Associate Consultant