Sequence-Specific Correction of Genomic Hypoxanthine-Guanine Phosphoribosyl Transferase Mutations in Lymphoblasts by Small Fragment Homologous Replacement

Abstract

Oligo/polynucleotide-based gene targeting strategies provide new options for achieving sequence-specifi c modifi cation of genomic DNA and have implications for the development of new therapies and transgenic animal models. One such gene modifi cation strategy, small fragment homologous replacement (SFHR), was evaluated qualitatively and quantitatively in human lymphoblasts that contain a single base substitution in the hypoxanthine-guanine phosphoribosyl transferase (HPRT1) gene. Because HPRT1 mutant cells are readily discernable from those expressing the wild type (wt) gene through growth in selective media, it was possible to identify and isolate cells that have been corrected by SFHR. Transfection of HPRT1 mutant cells with polynucleotide small DNA fragments (SDFs) comprising wild type HPRT1 (wtHPRT1) sequences resulted in clones of cells that grew in hypoxanthine-aminopterin-thymidine (HAT) medium. Initial studies quantifying the effi ciency of correction in 3 separate experiments indicate frequencies ranging from 0.1% to 2%. Sequence analysis of DNA and RNA showed correction of the HPRT1 mutation. Random integration was not indicated after transfection of the mutant cells with an SDF comprised of green fl uorescent protein (GFP) sequences that are not found in human genomic DNA. Random integration was also not detected following Southern blot hybridization analysis of an individual corrected cell clone.

An update on targeted gene repair in mammalian cells: methods and mechanisms

“…Studies conducted using mammalian cells indicate no difference in correction efficiency between ss- and ds-SDFs [63,64]. However, a study carried out using E. Coli indicates a higher efficiency using ss-SDFs compared to ds-SDFs [35].…”

Oligo/Polynucleotide-Based Gene Modification: Strategies and Therapeutic Potential

“…Previous studies have demonstrated SFHR-mediated modification of genes associated with sickle cell disease (Goncz et al, 2006) and b-thalassemia (Colosimo et al, 2007) (b-globin), cystic fibrosis (CFTR) (Kunzelmann et al, 1996;Goncz et al, 1998Goncz et al, , 2001Goncz et al, , 2002Sangiuolo et al, 2002Sangiuolo et al, , 2008, Duchene's muscular dystrophy (dystrophin) (Kapsa et al, 2001(Kapsa et al, , 2002Todaro et al, 2007), severe combined immune deficiency (DNA-dependent kinase catalytic subunit) (Zayed et al, 2006), alpha-1 antitrypsin deficiency (alpha-1 antitrypsin) (McNab et al, 2007), spinal muscular atrophy (survival motor neuron-1) (Sangiuolo et al, 2005), and LeschNyhan syndrome (hypoxanthine-guanine phosphoribosyl transferase, HPRT) (Bedayat et al, 2010).…”

“…It will be important to develop assay systems that can be readily used to quantify homologous exchange in the context of particular repair or replication mechanisms. There are a number of gene and cell systems that have already been developed, including the HPRT gene (Doetschman et al, 1987;Hunger-Bertling et al, 1990;Valancius and Smithies, 1991;Kenner et al, 2002;Hendrie et al, 2003;Ohbayashi et al, 2005;Pierce and Jasin, 2005;Hinz et al, 2006;Bedayat et al, 2010), green fluorescent protein (GFP) (Thorpe et al, 2002;Radecke et al, 2004;Olsen et al, 2005a;Tsuchiya et al, 2005c;Vasileva et al, 2006;Sakamoto et al, 2007;Kamiya et al, 2008), neomycin resistance (G418) (Song et al, 1985;Campbell et al, 1989;Manivasakam et al, 2001;Hendrie et al, 2003;Murphy et al, 2007), zeocin resistance , and bgalactocidase (Nickerson and Colledge, 2003). Although the reporter/selectable marker gene systems are often used, the HPRT system represents a gene in its natural genomic context and may more accurately reflect homologous exchange at other genomic loci.…”

“…Although it is possible that a randomly integrated oligo/polynucleotide may result in detrimental insertional mutagenesis, the potential will be minimized because of the lack of regulatory elements directly associated with the therapeutic DNA. In studies evaluating a clonal population of cells modified by HPRT-specific SDFs, random integration was not apparent (Bedayat et al, 2010). However, the potential for random integration needs to be thoroughly evaluated.…”

Nuclease-Mediated Double-Strand Break (DSB) Enhancement of Small Fragment Homologous Recombination (SFHR) Gene Modification in Human-Induced Pluripotent Stem Cells (hiPSCs)

“…SFHR does not rely on drug selection and thereby avoids the introduction of nonnative DNA sequences. SFHR has been previously shown to successfully modify genomic DNA in mouse ESCs [4] (Gruenert and Emamekhoo, unpublished data), immortalized and primary human epithelial cell line [1, 2, 5–7], hematopoietic stem cells [8, 9], peripheral blood monocytes [10], human lymphoblasts [11], mouse T cells [12], and myoblasts [13–15] of a number of target genes, at efficiencies approaching 1–10 %.…”