Document Detail


Triplex-forming peptide nucleic acids induce heritable elevations in gamma-globin expression in hematopoietic progenitor cells.
MedLine Citation:
PMID:  23337982     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Potentiating homologous recombination using triplex-forming peptide nucleic acids (PNAs) can be used to mediate targeted sequence editing by donor DNAs and thereby induce functional gene expression to supplant non-functional counterparts. Mutations that disrupt the normal function of the β-globin subunit cause hemoglobinopathies such as sickle cell disease and β-thalassemias. However, expression of the functional γ-globin subunit in adults, a benign condition called hereditary persistence of fetal hemoglobin (HPFH), can ameliorate the severity of these disorders, but this expression is normally silenced. Here, we harness triplex-forming PNA-induced donor DNA recombination to create HPFH mutations that increase the expression of γ-globin in adult mammalian cells, including β-yeast artificial chromosome (YAC) bone marrow and hematopoietic progenitor cells (HPCs). Transfection of human cells led to site-specific modification frequencies of 1.63% using triplex-forming PNA γ-194-3K in conjunction with donor DNAs, compared with 0.29% using donor DNAs alone. We also concurrently modified the γ-globin promoter to insert both HPFH-associated point mutations and a hypoxia-responsive element (HRE), conferring increased expression that was also regulated by oxygen tension. This work demonstrates application of oligonucleotide-based gene therapy to induce a quiescent gene promoter in mammalian cells and regulate its expression via an introduced HRE transcription factor binding site for potential therapeutic purposes.
Authors:
Joanna Y Chin; Faisal Reza; Peter M Glazer
Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural     Date:  2013-01-22
Journal Detail:
Title:  Molecular therapy : the journal of the American Society of Gene Therapy     Volume:  21     ISSN:  1525-0024     ISO Abbreviation:  Mol. Ther.     Publication Date:  2013 Mar 
Date Detail:
Created Date:  2013-03-01     Completed Date:  2013-10-22     Revised Date:  2014-03-06    
Medline Journal Info:
Nlm Unique ID:  100890581     Medline TA:  Mol Ther     Country:  United States    
Other Details:
Languages:  eng     Pagination:  580-7     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Adult
Animals
Antigens, CD34 / metabolism
Cell Line
Chromosomes, Artificial, Yeast / metabolism
DNA / chemistry,  genetics*
Fetal Hemoglobin / genetics,  metabolism
Gene Expression Regulation
Genetic Therapy
Hematopoietic Stem Cells / cytology,  metabolism*
Hemoglobinopathies / therapy
Humans
K562 Cells
Mice
Mice, Transgenic
Mutation
Peptide Nucleic Acids / chemistry,  genetics*
Promoter Regions, Genetic
Transfection
gamma-Globins / genetics*,  metabolism
Grant Support
ID/Acronym/Agency:
P30DK072442/DK/NIDDK NIH HHS; R01HL082655/HL/NHLBI NIH HHS; T32DK007556/DK/NIDDK NIH HHS; T32GM07205/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Antigens, CD34; 0/Peptide Nucleic Acids; 0/gamma-Globins; 0/triplex DNA; 9007-49-2/DNA; 9034-63-3/Fetal Hemoglobin
Comments/Corrections

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