Targeted correction of a thalassemia-associated beta-globin mutation induced by pseudo-complementary peptide nucleic acids

TY - JOUR

T1 - Targeted correction of a thalassemia-associated beta-globin mutation induced by pseudo-complementary peptide nucleic acids

AU - Lonkar, Pallavi

AU - Kim, Ki-Hyun

AU - Kuan, Jean Y

AU - Chin, Joanna Y

AU - Rogers, Faye A

AU - Knauert, Melissa P

AU - Kole, Ryszard

AU - Nielsen, Peter E

AU - Glazer, Peter M

PY - 2009

Y1 - 2009

N2 - Beta-thalassemia is a genetic disorder caused by mutations in the beta-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. However, the use of these molecules for gene targeting requires homopurine tracts to facilitate triple helix formation. Alternatively, to achieve binding to mixed-sequence target sites for the induced gene correction, we have used pseudo-complementary PNAs (pcPNAs). Due to steric hindrance, pcPNAs are unable to form pcPNA-pcPNA duplexes but can bind to complementary DNA sequences via double duplex-invasion complexes. We demonstrate here that pcPNAs, when co-transfected with donor DNA fragments, can promote single base pair modification at the start of the second intron of the beta-globin gene. This was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta globin fusion gene. We also demonstrate that pcPNAs are effective in stimulating recombination in human fibroblast cells in a manner dependent on the nucleotide excision repair factor, XPA. These results suggest that pcPNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells without purine sequence restriction.

AB - Beta-thalassemia is a genetic disorder caused by mutations in the beta-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. However, the use of these molecules for gene targeting requires homopurine tracts to facilitate triple helix formation. Alternatively, to achieve binding to mixed-sequence target sites for the induced gene correction, we have used pseudo-complementary PNAs (pcPNAs). Due to steric hindrance, pcPNAs are unable to form pcPNA-pcPNA duplexes but can bind to complementary DNA sequences via double duplex-invasion complexes. We demonstrate here that pcPNAs, when co-transfected with donor DNA fragments, can promote single base pair modification at the start of the second intron of the beta-globin gene. This was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta globin fusion gene. We also demonstrate that pcPNAs are effective in stimulating recombination in human fibroblast cells in a manner dependent on the nucleotide excision repair factor, XPA. These results suggest that pcPNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells without purine sequence restriction.

U2 - 10.1093/nar/gkp217

DO - 10.1093/nar/gkp217

M3 - Journal article

C2 - 19364810

SN - 0305-1048

VL - 37

SP - 3635

EP - 3644

JO - Nucleic Acids Research

JF - Nucleic Acids Research

IS - 11

ER -