Synthesis and Evaluation of MGB Polyamide-Oligonucleotide Conjugates as Gene Expression Control Compounds.

MGB polyamide-oligonucleotide conjugates ON 1-4 with linked MGB polyamides at the 2-exocyclic amino group of a guanine base using aminoalkyl linkers were synthesized and evaluated in terms of binding affinity for complementary DNA containing the MGB polyamide binding sequence using T m and CD analyses. The MGB polyamides comprised pyrrole polyamides (Py4- and Py3-), which possess binding affinity for A-T base pairs, and imidazole (Im3-) and pyrrole-γ-imidazole (Py3-γ-Im3-) polyamide hairpin motifs, which possess binding affinity for C-G base pairs. It was found that the stability of modified dsDNA was greatly influenced by the linker length. Py4- and Py3-oligonucleotide conjugates (ON 1 (n = 4) and ON 2 (n = 4)) containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA. Although Im3-oligonucleotide conjugate ON 3 (n = 4) containing the 4-aminobutyl linker formed stable dsDNA with complementary DNA, stabilization of dsDNA by the imidazole amide moiety of ON 3 (n = 4) was lower compared with the pyrrole amide moiety of ON 2 (n = 4). The Py3-γ-Im3-oligonucleotide conjugate ON 4 (n = 2), which possesses binding affinity for C-G base pairs via a pyrrole/imidazole combination and contains a 2-aminoethyl linker, showed high binding ability for complementary DNA. Furthermore, the DNA sequence recognition of MGB polyamide-oligonucleotide conjugates was investigated using single-base mismatch DNAs, which possess a mismatch base in the MGB polyamide binding sequence. The Py3-γ-Im3-oligonucleotide conjugate ON 4 (n = 2) showed high sequence recognition ability for complementary DNA.

ICP0 inhibits the decrease of HSV amplicon-mediated transgene expression.

The herpes simplex virus (HSV) amplicon vector produces an initial host response that limits transgene expression. In this study, we hypothesized that restoration of the HSV gene infected cell protein (ICP0) into the amplicon could circumvent this host response and thus overcome silencing of encoded transgenes. To test this, we constructed an amplicon vector that encodes the ICP0 under control of its native promoter (ICP0+ amplicon). Expression of ICP0 was transient and, at a multiplicity of infection (MOI) of 1, did not significantly alter interferon (IFN)-based responses against the vector or cell kinetics/apoptosis of infected cells. Chromatin immunoprecipitation (ChIP) PCR analysis revealed that conventional amplicon DNA became associated with histone deacetylase 1 (HDAC1) immediately after infection, whereas ICP0+ amplicon DNA remained relatively unbound by HDAC1 for at least 72 hours after infection. Mice administered systemic ICP0+ amplicon exhibited significantly greater and more sustained transgene expression in their livers than did those receiving conventional amplicon, likely due to increased transcriptional or post-transcriptional activity rather than increased copy numbers of vector DNA. These findings indicate that restoration of ICP0 expression may be employed within HSV amplicon constructs to decrease transgene silencing in vitro and in vivo.