DNA analogs for the purpose of gene therapy

Abstract: This thesis describes DNA analogs and their potential use for gene therapeutic purposes, primarily by sequence specific strand-invasion of double-stranded DNA. Gene therapy is classically thought of as restoration of a defect gene. Correction of dysfunctional genes is not feasible with today's technology, instead a new therapeutic gene is introduced. Nature's own gene delivery system, the virus, has gained its potency through evolution. Viruses can be modified, to instead of delivering its genome, be exploited as a vector to convey a therapeutic gene. Biotechnologically engineered viruses are expensive to produce, involving extensive monitoring for recombinant competent viruses, and if to become readministrated, the isotype has to be changed in order to prevent a specific immune response. Furthermore, humans have also developed defense mechanisms to prevent pathogens as viruses to fulfill their purpose. Non-viral gene delivery techniques are being developed to avoid viral associated complications. Bioplex (biological complex) is based on attachment of biologically active molecules, called functional entities (FE), on double-stranded DNA. Gene delivery is a complex process where FEs of different sources and origins are anticipated to guide the genetic material through the body, taken up by the correct cell-type and gain entrance to the nuclei. The FEs are anchored to DNA at specific positions by DNA analogs with superior binding affinity. Analogs presented here are peptide nucleic acid (PNA) and locked nucleic acid (LNA). Paper I addresses the assembly of the Bioplex. Instead of having a FE directly conjugated to the anchor, multiple molecules could be added to each anchor by selfassembling of singlestranded oligonucleotides, presenting exponentially growing number of anchoring sites without compromising the structure of the DNA. There are circumstances when it is advantageous to detach the FE from the cargo. Paper II presents a possible strategy by insertion of an amino acid recognition sequence for the endosomal protease, cathepsin L. In this way, the FE is released when the pH drops and the enzyme is activated. The FE could be carbohydrates or peptides of endogenous or viral origin, or synthetic. Furthermore, combinations of FEs are needed for optimal effect. Literature offers many alternatives and to try all of them in combinations is manually undoable, even for small ligand libraries. Paper III utilizes robots for transfection and analyzation. Ligands in combinations of two are screened for combinatorial effect on cellular association of 6 different human cell-lines. Even though only 22 ligands are included, it equals 484 unique combinations, resulting in 10 000 transfections. The field of oligonucleotide mediated gene therapy was founded with the discovery of antisense in the early 1970s. Paper IV is a study aiming to optimize our own oligonucleotide based construct called Zorro LNA. It inhibits transcription by directly blocking the polymerase machinery sterically by stably hybridizing to both strands of the helical DNA. Zorro has two arms of LNA oligonucleotides, each hybridizing to one strand of the DNA strands. They are interconnected by base paring, creating a Zlike structure within the double-stranded DNA.