Nucleic Acid Therapeutics

Downregulating a disease-causing gene in a sequence-specific manner has a profound impact on mitigating human diseases conditions. Traditionally this is achieved by targeting the mRNA, the fundamental cellular machinery that creates protein using antisense technology or by using interfering RNA (RNAi technology).

RNAi is an endogenous pathway for the post-transcriptional silencing of gene expression that is triggered by double-stranded RNA (dsRNA), including endogenous microRNA (miRNA) and synthetic short interfering RNA (siRNA). By this pathway, RNAi molecules can silence the expression of virtually any gene with high efficiency and specificity, highlighting its potential for treating several diseases. Though researchers around the world have made significant achievements in understanding how these RNA based systems work, there are several challenges to overcome, both at the fundamental level as well as at the application level.

Our research group is focused on these aspects by employing engineering tools that interfere with the intracellular enzymatic processes and biological function.

(a) Chemically modified RNAi drug design

Structural design of chemically modified oligonucleotides with enhanced enzymatic stability and bioactivity.

For designing effective antisense and RNAi drugs, structural and chemical modifications are necessary to improve the therapeutic potential of oligonucleotides drugs. In our laboratory, we use synthetic organic chemistry to create new chemically modified DNA and RNA molecules by employing multistep synthetic organic chemistry to generate designer drugs that are more potent than traditional drugs. Such modifications not only improve the nuclease stability of drugs in human serum, but also improve the drug efficacy by enhancing the biochemical reaction. These chemically modified interfering RNA molecules also modulate thermodynamic asymmetry of the duplexes which facilitate selective recruitment of the desired strand within the RNA induced silencing complex (RISC). Such a chemical design of modified siRNA that can favourably interact with the enzymes within the RISC complex and modulate RNAi machinery is of great significance to develop next-generation RNAi therapeutics. In our laboratory, we are constantly exploring strategies to manipulate the nucleic acid structure to improve the therapeutic index of the drug molecules.

Recent related publications:

1. Barman, J.; Gurav, D.; Oommen, O. P.; Varghese, O. P. 2′-N-Guanidino,4′-C-ethylene bridged thymidine (GENA-T) modified oligonucleotide exhibits triplex formation with excellent enzymatic stability. RSC Advances 2015, 5, 12257-12260. http://dx.doi.org/10.1039/C4RA14721A
    

(b) Structural design of asymmetric RNAi drugs

Structural design of unsymmetrical siRNA and miRNA with tailored thermodynamic asymmetry.

The RNAi technology is primarily based on two different double-stranded RNA molecules namely; short interfering RNA (siRNA) and microRNA (miRNA). We have discovered the first asymmetric siRNA, which is derived from natural nucleotides, which possesses endosomolytic properties and promote carrier-free transfection to human cancer cells and hard-to-transfect primary cells. We achieved a durable and long-lasting silencing effect without triggering an immune response using our modified siRNA. This innovative siRNA and miRNA design will open new avenues to develop effective RNAi therapeutics. We are currently exploring this technology to develop drug molecules for different clinical indications with partners across Europe.

Please visit www.uppsalatherapeutics.com for our commercial activities.

Recent related publications

1. ‘Improved small interfering ribonucleic acid molecules’ O. P. VARGHESE and O. P. Oommen, 2017, US Patent App. 15/127,616.
2. ‘Nucleic acid molecules with enhanced activity’ O. P. VARGHESE and O. P. Oommen, 2018, US Patent App. 15/761,753.
    

(c) Mimicking Nature to deliver plasmid DNA and RNAi molecules for in vivo applications

Novel glycosaminoglycan-based intracellular delivery strategies of plasmid DNA and RNAi drugs

Cellular delivery of nucleic acid drugs has been at the forefront of research for many years. Even though we could delivery molecules inside the cells, it remains trapped within the endolysosomal compartments. With well-known lipid-based delivery, only 1-2% of the cargo molecules reaches the target site. Our ambition is to identify novel methods by mimicking Nature to develop novel delivery strategies. We have recently shown that nanoparticles coated with glycosaminoglycans can control plasmid release from the endosome and improve transfection efficiency. Such delivery carriers are promising owing to their unique tumour targeting ability (e.g. HA targets CD44 receptor). We have also recently developed the first non-cationic non-viral siRNA delivery methods by exploiting naturally occuring Van der Waal’s and H-bonding interactions. These methods will provide next-generation transfection reagent that does not rely on electrostatic interactions between cationic polymers and the phosphate backbone of DNA.

Recent related publication:

1. Paidikondala, M.; Rangasami, V. K.; Nawale, G. N.; Casalini, T.; Perale, G.; Kadekar, S.; Mohanty, G.; Salminen, T.; Oommen, O. P.; Varghese, O. P. An Unexpected Role of Hyaluronic Acid in Trafficking siRNA Across the Cellular Barrier: The First Biomimetic, Anionic, Non-Viral Transfection Method. Angew. Chem. Int. Ed. 2019, 58, 2815-2819. https://onlinelibrary.wiley.com/doi/abs/10.1002/anie.201900099
2. Paidikondala, M.; Kadekar, S.; Varghese, O. P. Innovative Strategy for 3D Transfection of Primary Human Stem Cells with BMP-2 Expressing Plasmid DNA: A Clinically Translatable Strategy for Ex Vivo Gene Therapy. Int. J. Mol. Sci. 2019, 20, 56. http://www.mdpi.com/1422-0067/20/1/56
3. Paidikondala, M.; Nawale, G. N.; Varghese, O. P. Insights into siRNA Transfection in Suspension: Efficient Gene Silencing in Human Mesenchymal Stem Cells Encapsulated in Hyaluronic Acid Hydrogel. Biomacromolecules 2019. https://doi.org/10.1021/acs.biomac.8b01712
4. Yan, H.; Oommen, O. P.; Yu, D.; Hilborn, J.; Qian, H.; Varghese, O. P. Chondroitin Sulfate-Coated DNA-Nanoplexes Enhance Transfection Efficiency by Controlling Plasmid Release from Endosomes: A New Insight into Modulating Nonviral Gene Transfection. Adv. Funct. Mater. 2015, 25, 3907-3915. https://onlinelibrary.wiley.com/doi/abs/10.1002/adfm.201500695