The ultimate form of gene therapy for inherited diseases is to reverse the phenotype by correcting the genetic mutation at its endogenous location in the chromosome. We have been developing a gene repair strategy that relies on DNA oligonucleotides to enter the cell, hybridize to the mutant sequence and direct single base exchanges in the target gene. During the initial grant period, we created several model systems in yeast and mammalian cells that enabled the elucidation of pathways that control the frequency of gene correction. The data indicate that the gene repair process is controlled by the activation of homologous recombination and rate at which DNA replication takes place. We now propose to transition from model systems to a clinically relevant cell type that is likely to serve as a target in the initial clinical application. Results from several laboratories indicate that liver cells, particularly hepatocytes, are highly responsive to this technique and enable gene correction to take place at robust levels. We shall target a integrated, mutant eGFP gene and the endogenous HPRT gene in clonal isolates of HepG2 and THLE cells, two established hepatocytic cell lines that have been used in the development phase of therapies aimed at liver diseases. Guided by the results of our first grant term, we will focus on the activation of homologous recombination as a means to support enhanced levels of correction in a reproducible and sustainable fashion. The experiments outlined in this grant will address the following questions;1) are random ds breaks required for attaining high levels of gene correction?;2) do lesions at replication forks or stalled forks themselves provide enough stimulus for elevating the levels of gene correction in the absence of DNA damage;3) is the process of gene repair itself mutagenic at non-targeted sites and are cells undergoing gene repair more prone to genome rearrangement?;4) how does the cell respond to the intemalization of the ssODN in terms of DNA damage response pathways. The key to developing this technique in the long term, even for liver disease and cancer, lies in the ability to regulate, predict and reliably attain correction efficiencies that have therapeutic effects. These goals support the choice of liver as a target for clinical applications of gene repair but there are other important reasons for focusing on hepatocvtes: they are the target cell for gene therapy for Alpha-1 Antitrypsin Deficiency. Crigler-Naiiar. OTC. MPSVII. Hemophilia A and B and many lysosomal storage disorders among others. Our work will uncover restrictions or limitations for gene repair in hepatocvtes with the goal of treating hepatic cancer and genetic diseases of the liver.

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National Cancer Institute (NCI)
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Special Emphasis Panel (ZRG1-GGG-J (10))
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Arya, Suresh
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Marshall University
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Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan et al. (2015) Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems. PLoS One 10:e0129308
Kmiec, Eric B (2015) Is the age of genetic surgery finally upon us? Surg Oncol 24:95-9
Strouse, Bryan; Bialk, Pawel; Niamat, Rohina A et al. (2014) Combinatorial gene editing in mammalian cells using ssODNs and TALENs. Sci Rep 4:3791
Rivera-Torres, Natalia; Strouse, Bryan; Bialk, Pawel et al. (2014) The position of DNA cleavage by TALENs and cell synchronization influences the frequency of gene editing directed by single-stranded oligonucleotides. PLoS One 9:e96483
Borjigin, Mandula; Eskridge, Chris; Niamat, Rohina et al. (2013) Electrospun fiber membranes enable proliferation of genetically modified cells. Int J Nanomedicine 8:855-64
Livingston, Paula; Strouse, Bryan; Perry, Haley et al. (2012) Oligonucleotide delivery by nucleofection does not rescue the reduced proliferation phenotype of gene-edited cells. Nucleic Acid Ther 22:405-13
Falgowski, Kerry; Falgowski, Carly; York-Vickers, Cassie et al. (2011) Strand bias influences the mechanism of gene editing directed by single-stranded DNA oligonucleotides. Nucleic Acids Res 39:4783-94
Bonner, Melissa; Kmiec, Eric B (2009) DNA breakage associated with targeted gene alteration directed by DNA oligonucleotides. Mutat Res 669:85-94
Ferrara, Luciana; Engstrom, Julia U; Schwartz, Timothy et al. (2007) Recovery of cell cycle delay following targeted gene repair by oligonucleotides. DNA Repair (Amst) 6:1529-35
Maguire, Katie Kennedy; Kmiec, Eric B (2007) Multiple roles for MSH2 in the repair of a deletion mutation directed by modified single-stranded oligonucleotides. Gene 386:107-14

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