Development of nonviral gene delivery and of anti-gene reagents

Abstract

Although significant progress has been made in the basic science and applications of various non-viral gene delivery systems, the majority of non-viral approaches are still much less efficient than viral vectors, especially for in vivo gene delivery, Therefore, non-viral gene delivery must be developed in order to increase the efficiency of in vivo delivery. Moreover, a novel anti-gene reagent was developed for sequence-specific gene silencing.

Peptide Nucleic Acid (PNA), a mimic of oligonucleotide with a neutral pseudopeptide backbone consisting of repeating N- (2-amino-ethyl)-glycine units, can invade double-stranded DNA to generate a stable single-strand D-loop. Locked Nucleic Acid (LNA) are synthetic analogs of nucleic acids, which contain a bridging methylene carbon between the 2’ and 4’ positions of the ribose ring. Over the years, a variety of chemically modified small molecules have been developed. Among them, PNA and LNA have shown great promise for a number of applications.

In order to further develop a technology for linking functional entities to non-viral vectors, the Bioplex technology, we investigated how hybridization of PNA to supercoiled plasmids would be affected by the binding of multiple PNA-peptides to DNA strands. Cooperative effects were found at a distance of up to three bases. With a peptide present at the end of one of the PNA, steric hindrance occurred, reducing the increase in binding rate when the distance between the two sites was less than two bases. In addition, we found enhanced kinetics when PNA binding to overlapping sites on opposite DNA strands were added, without the use of further chemically modified bases in the PNAs (I).

We also generated a novel, sequence-specific anti-gene molecule “Zorro LNA”, in which a 14- mer LNA binds to the coding strand, while a 16-mer connected LNA binds to the template strand. Our data suggest that the “Zorro LNA” induced effective and specific strand invasion into DNA duplexes and potent inhibition of gene transcription, also in mammalian cells. This offers a novel type of anti-gene drug, which is easy to synthesize (II). We also found that the Zorro LNA construct efficiently inhibited pol III-dependent transcription in a cellular context, including in vivo in a mouse model. Thus, this new form of gene silencer could potentially serve as a versatile regulator of pol III-dependent transcription, including various forms of shRNAs (III).

Finally, In order to further evaluate the in vivo activity of Zorro LNA and trace its long-term effect in inhibiting gene expression, we inserted two adjacent Zorro LNA-binding sites flanked by loxP sites within the transcribed region of a reporter gene. As previously observed, after Zorro LNA was hybridized to the plasmid, it significantly induced gene silencing in the mammalian cells. Both in transfected cells and in mouse model the silencing effect was lost when Cre-recombinase expressing plasmids were subsequently transfected. This suggests that binding of Zorro LNA is stable over a period of days.