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Gene and cell therapies are emerging technologies with the potential of having an important impact on the future treatment of human diseases. Over the last few years, highly promising results have been obtained in patients with inherited immunodeficiencies, storage diseases, blindness, and hemophilia, just to mention a few.

Five subprojects describe this research:

1. Development of ONs with sequence-specific binding to double-stranded (ds) DNA.

The recently designed locked-nucleic acids (bisLNAs) have the capacity to strand-invade and form highly stable complexes with supercoiled DNA (Fig. 1a; Moreno et al. 2013). Another construct that we have developed, designated Zorro-LNA, has the unique feature of simultaneously hybridizing to both strands of the DNA duplex (Fig. 1b; Ge et al., 2007, Zaghloul et al., 2011). Zorro-LNAs are investigated as possible anti-gene constructs for silencing mutated genes in dominant genetic diseases.

2. Splice-switching ONs as tools to alter the processing of pre-mRNA.

We have recently been able to correct the splicing of a mutated BTK pre-mRNA, thus rescuing the production of functional BTK protein (Fig. 2; Bestas et al. manuscript in preparation). ONs were optimized in terms of sequence and chemical modifications and studied both in primary patient cells and in cells from humanized mice.

3. Nuclear transport of nucleic acids for gene therapy.

Here we have used the trimethylated Cap (m3G-Cap) as a new intranuclear transport enhancer. The m3G-Cap specifically binds to the nuclear transport protein, Snurportin and was used to ferry splice-switching ONs into the nucleus, thereby enhancing their activity (Fig. 3; Moreno et al., 2009).

4. Minicircle technology

In a study addressing the stability issue of plasmid -derived vectors we have recently highlighted the advantages of small mini-circle vectors devoid of bacterial sequences (Stenler et al., 2014).

5. Exosomes, cell-derived vesicles for transport of protein and nucleic acids.

Exosomes are vesicles naturally exported from cells into extracellular fluids. In this project we have established a new purification method to increase the yield and purity of the exosomes (Nordin et al., submitted). The possible role of exosomes in transporting signals in the form of nucleic acids or proteins between cells is currently being studied, as well as how to use exosomes as a means to transfer genetic material.