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

Abstract

Pharmacological treatments designed to reactivate fetal γ-globin can lead to an effective and successful clinical outcome in patients with hemoglobinopathies. However, new approaches remain highly desired because such treatments are not equally effective for all patients, and toxicity issues remain. We have taken a systematic approach to develop an embedded chimeric peptide nucleic acid (PNA) that effectively enters the cell and the nucleus, binds to its target site at the human fetal γ-globin promoter, and reactivates this transcript in adult transgenic mouse bone marrow and human primary peripheral blood cells. In vitro and in vivo DNAbinding assays in conjunction with live-cell imaging have been used to establish and optimize chimeric PNA design parameters that lead to successful gene activation. Our final molecule contains a specific γ-promoter-binding PNA sequence embedded within two amino acid motifs: one leads to efficient cell/nuclear entry, and the other generates transcriptional reactivation of the target. These embedded PNAs overcome previous limitations and are generally applicable to the design of in vivo transcriptional activation reagents that can be directed to any promoter region of interest and are of direct relevance to clinical applications that would benefit from such a need.γ-globin gene reactivation | primary human CD34+ cells

Functional Interactions between Erythroid Kruppel-like Factor (EKLF/KLF1) and Protein Phosphatase PPM1B/PP2Cβ

“…2a), followed by differentiation of HSCs into erythroid progenitors (29). Consistent with data suggesting that Ppm1b is a negative regulator of Cdk9, whose activity is required for globin expression during both zebrafish and human erythropoiesis (36), we observed an increase in ␤-globin RNA concomitant with a decrease in Ppm1b protein levels (Fig.…”

“…After 7 days of expansion, 1.5 ϫ 10 6 cells per sample were nucleofected with 200 nM siPpm1b (supplemental Table S3) or untargeted siRNA (Qiagen) using the Amaxa Human CD34 ϩ nucleofection kit (program U-008) (28). Erythroid differentiation was induced with Epo (4 units/ml) and SCF (100 ng/ml) (29) in SFEM media 24 h after nucleofection. Cells were collected 72 h after differentiation for analysis by RT-PCR.…”

γ sulphate PNA (PNA S): Highly Selective DNA Binding Molecule Showing Promising Antigene Activity

“…Our hypothesis is consistent with data recently published in the literature [9] and is supported by the results of fluorescence experiments; other assays, as for example the Chromatin Association Assay might be carried out to demonstrate that PNA S bind to DNA in vivo . [52] Data obtained so far underline how critical is the efficiency in the uptake to determine the biological activity of PNA analogues. In conclusion PNA S is the first example reported so far of antigene γ PNA bearing a negative charge.…”

Antisense peptide nucleic acid-functionalized cationic nanocomplex for in vivo mRNA detection

“…Studies that show direct and specific targeting of iNOS mRNA both in vitro and in vivo are limited. Our work and that of others have shown that peptide nucleic acid (PNA) has a number of useful properties that make it an ideal choice for targeting candidate mRNA [13][14][15][16][17][18][19].…”

“…PNA, a synthetic analogue of DNA [20], is an ideal platform for the development of antisense diagnostic agents owing to its high resistance to enzymatic degradation, high affinity for RNA, ability to invade regions of secondary structure and inability to activate RNase H, which would otherwise degrade the target mRNA sequence and lead to signal loss [13,15,16,[21][22][23]. Although PNAs have been used in many biological applications as biosensors and imaging agents [14,18], poor membrane permeability and rapid clearance have limited its in vitro and in vivo applications [22,24].…”