Exploring a peptide nucleic acid-based antisense approach for CD5 targeting in chronic lymphocytic leukemia

Abstract

We herein report an innovative antisense approach based on Peptide Nucleic Acids (PNAs) to down-modulate CD5 expression levels in chronic lymphocytic leukemia (CLL). Using bioinformatics tools, we selected a 12-mer tract of the CD5 mRNA as the molecular target and synthesized the complementary and control PNA strands bearing a serine phosphate dipeptide tail to enhance their water solubility and bioavailability. The specific recognition of the 12-mer DNA strand, corresponding to the target mRNA sequence by the complementary PNA strand, was confirmed by non-denaturing polyacrylamide gel electrophoresis, thermal difference spectroscopy, circular dichroism (CD), and CD melting studies. Cytofluorimetric assays and real-time PCR analysis demonstrated the downregulation of CD5 expression due to incubation with the anti-CD5 PNA at RNA and protein levels in Jurkat cell line and peripheral blood mononuclear cells from B-CLL patients. Interestingly, we also observed that transfection with the anti-CD5 PNA increases apoptotic response induced by fludarabine in B-CLL cells. The herein reported results suggest that PNAs could represent a potential candidate for the development of antisense therapeutic agents in CLL.

Biological relevance of ZNF224 expression in chronic lymphocytic leukemia and its implication IN NF-kB pathway regulation

“…Peripheral Blood Mononuclear Cells (PBMCs) were isolated by a Ficoll-PaqueTM density gradient (Merck, Darmstadt, Germany) from the heparinized peripheral blood of B-CLL of untreated patients in the stationary phase of the disease for in vitro silencing of ZNF224. CLL patients who had ≥80% CD19 + /CD5+ co-espressing cells were included in this study ( Cesaro et al, 2022 ). CLL PBMCs were cultured in RPMI 1640 (Sigma-Aldrich, Milan, Italy) supplemented with 10% human serum and stimulating anti-Human CD3 (10μg/1*10 6 cells) (eBioscience Thermo Fisher, Inc, Waltham, MA) at 37°C in a humidified 5% CO 2 atmosphere ( Romano et al, 2008 ).…”

Unlocking the potential of chemically modified peptide nucleic acids for RNA-based therapeutics

“…Peripheral blood mononuclear cells from B-CLL patients treated with PNA showed a ~60% reduction in CD5 mRNA levels 35 . PNAs have also been used to block miRNA binding sites on the 3'UTR of mRNAs resulting in stabilization and increased protein production.…”

Exploring the DNA2-PNA heterotriplex formation in targeting the Bcl-2 gene promoter: A structural insight by physico-chemical and microsecond-scale MD investigation

“…PNAs are also capable of behaving as G-quadruplex binders [ 2 ] or triplex-forming (TF) agents [ 3 , 4 ] depending on their length, sequence, and the structural features of the DNA or RNA target. These remarkable properties, together with the higher chemical and enzymatic stability and lower toxicity [ 5 ], render PNAs suitable molecular tools in biosensing [ 6 , 7 ] and biomedical applications using the antigene [ 2 , 8 ], antisense [ 9 , 10 ], antimiRNA [ [11] , [12] , [13] , [14] , [15] ], and gene editing [ 4 , 16 ] strategies. Several anticancer approaches based on PNA technology have been reported [ 9 , 10 , 17 ].…”

“…These remarkable properties, together with the higher chemical and enzymatic stability and lower toxicity [ 5 ], render PNAs suitable molecular tools in biosensing [ 6 , 7 ] and biomedical applications using the antigene [ 2 , 8 ], antisense [ 9 , 10 ], antimiRNA [ [11] , [12] , [13] , [14] , [15] ], and gene editing [ 4 , 16 ] strategies. Several anticancer approaches based on PNA technology have been reported [ 9 , 10 , 17 ]. In this context, we studied the ability of PNA oligomers to target the loops of DNA G-quadruplexes (G4s) formed in the promoters of human oncogenes [ 2 , 18 ] aiming at downregulating their expression.…”