Peptide nucleic acid (PNA) binding-mediated induction of human  -globin gene expression

Abstract

Peptide nucleic acids (PNAs) can bind to homopurine/homopyrimidine sequences of double-stranded DNA targets in a sequence-specific manner and form [PNA]2/DNA triplexes with single-stranded DNA D-loop structures at the PNA binding sites. These D-loop structures have been found to have a capacity to initiate transcription in vitro. If this strategy can be used to induce transcription of endogenous genes, it may provide a novel approach for gene therapy of many human diseases. Human [beta] globin disorders such as sickle cell anemia and beta-thalassemia are very common genetic diseases that are caused by mutations in the beta-globin gene. When gamma-globin genes are highly expressed in sickle cell patients, the presence of high levels of fetal hemoglobin (HbF, alpha2gamma2) can compensate for the defective beta-globin gene product and such patients have much improved symptoms or are free of disease. However, the gamma-globin genes are developmentally regulated and normally expressed at very low levels (>1%) in adult blood cells. We have investigated the possibility of inducing gamma-globin gene expression with PNAs. Using PNAs designed to bind to the 5' flanking region of the gamma-globin gene, induction of expression of a reporter gene construct was demonstrated both in vitro and in vivo. More importantly, PNA-mediated induction of endogenous gamma-globin gene expression was also demonstrated in K562 human erythroleukemia cells. This result suggests that induction of gamma-globin gene expression with PNAs might provide a new approach for the treatment of sickle cell disease. PNA-induced gene expression strategy also may have implications in gene therapy of other diseases such as genetic diseases, cancer and infectious diseases.

Design of embedded chimeric peptide nucleic acids that efficiently enter and accurately reactivate gene expression in vivo

“…In an earlier study (38), the level of γ-globin expression was increased in K562 cells using triple helix-forming PNA targeted to a homopurine region at the −300 region of γ-globin promoter. However, this study was not a true reactivation test, and neither site-specific binding in vivo nor efficiency of cell/nuclear entry were monitored.…”

Peptide nucleic acid (PNA) binding-mediated gene regulation

“…In comparison, the binding of PNAs to single-stranded targets such as mRNA or single-stranded DNA targets is not affected by the presence of salts. For example, in our previous studies the presence of 150 mM KCl, which is relevant to the physiological K + concentration, significantly inhibited the binding of PNA-1 to the target [35].…”

“…Early studies revealed that the PNA binding-generated D-loops could initiate transcriptions in vitro using T7 RNA polymerase [34]. The work of our laboratory demonstrated that the PNA binding-generated D-loops could initiate transcription in a HeLa nuclear extract in vitro transcription system and induce a GFP reporter gene expression in the CV-1 monkey kidney cells [35]. Most importantly, when PNAs designed to bind to the human γ-globin gene 5' untranslated region (5' UTR) sequence were used to treat the K562 human erythroleukemia cells, transcription of the endogenous γ-globin gene from the PNA binding sites was achieved [35].…”

“…The work of our laboratory demonstrated that the PNA binding-generated D-loops could initiate transcription in a HeLa nuclear extract in vitro transcription system and induce a GFP reporter gene expression in the CV-1 monkey kidney cells [35]. Most importantly, when PNAs designed to bind to the human γ-globin gene 5' untranslated region (5' UTR) sequence were used to treat the K562 human erythroleukemia cells, transcription of the endogenous γ-globin gene from the PNA binding sites was achieved [35]. These results suggest that PNA binding-mediated transcription may provide an innovative strategy to induce expression of specific target genes.…”

Chemical Tools for Targeted Mutagenesis of DNA Based on Triple Helix Formation

“…13,14) The binding characteristics and sequence specificity of TFOs give these oligonucleotides vast potential in gene modification, such as inhibition of gene expression, inhibition of replication, and induction of site-specific mutagenesis. [15][16][17][18][19][20][21][22][23][24][25] However, there are a number of obstacles to TFO activity under physiologic conditions. [26][27][28][29] Recent developments in nucleoside and oligonucleotide analogue chemistry show great promise for solving the problems of TFO bioactivity and target options.…”