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Generation of CCR5-defective CD34 cells from ZFN-driven stop codon-integrated mesenchymal stem cell clones.

Manotham K, Chattong S, Setpakdee A - J. Biomed. Sci. (2015)

Bottom Line: However, the rarity of this mutation and the safety risks associated with current BMT protocols are the major obstacles to this treatment.We here reported the novel approach on generation of patients own CD34 cells from high fidelity ZFN-mediated HDR MSC clones.We believe that our approach will be beneficial in future HIV treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Molecular and Cell Biology Unit, Lerdsin General Hospital, Bangkok, Thailand. kmanotham@hotmail.com.

ABSTRACT

Backgrounds: Homozygous 32-bp deletion of the chemokine receptor 5 gene (CCR5) is associated with resistance to human immunodeficiency virus (HIV) infection, while heterozygosity delays HIV progression. Bone marrow transplantation (BMT) from a 32/32 donor has been shown to cure an HIV-infected patient. However, the rarity of this mutation and the safety risks associated with current BMT protocols are the major obstacles to this treatment. Zinc finger nuclease (ZFN) targeting is a powerful method for achieving genomic disruption at specific DNA sites of interest.

Results: Taking advantage of the self-renewal and plasticity properties of stem cells, in this study, we successfully generated isogenic and six-cell clones of bone marrow-derived mesenchymal stem cells that carry the stop codon of the CCR5 gene by using a ZFN-mediated homology-directed repair technique. These cells were expandable for more than 5 passages, and thus show potential to serve as an individual's cell factory. When Oct4 was overexpressed, the mutated cells robustly converted to CD34+ progenitor cells.

Conclusion: We here reported the novel approach on generation of patients own CD34 cells from high fidelity ZFN-mediated HDR MSC clones. We believe that our approach will be beneficial in future HIV treatment.

No MeSH data available.


Related in: MedlinePlus

Primers map and donor plasmid generation. Illustration of the CCR5 locus and primer locations. Upper panel: ZFN-targeted DNA sequence (underlined) and the interspace. Lower panel: DNA sequences of the d-plasmid around the ZFN site and the insertion-specific primer (IP); a silent T-A mutation adjacent to the left ZFN-targeted site was intentionally selected for confirmation and marking of the vicinity of integration between the ZFN-binding sites.
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Fig1: Primers map and donor plasmid generation. Illustration of the CCR5 locus and primer locations. Upper panel: ZFN-targeted DNA sequence (underlined) and the interspace. Lower panel: DNA sequences of the d-plasmid around the ZFN site and the insertion-specific primer (IP); a silent T-A mutation adjacent to the left ZFN-targeted site was intentionally selected for confirmation and marking of the vicinity of integration between the ZFN-binding sites.

Mentions: cDNA of human CCR5 (1791 bp), from −733 bp upstream of the left-hand ZFN-binding site to 1038 bp downstream of the right-hand ZFN-binding site, was amplified from genomic DNA of peripheral blood using the primers D1 (5′-GTGGACAGGGAAGCTAGCAG-3′) and D2 (5′-CCATACCTTGGAGGGGAAAT-3′). The polymerase chain reaction (PCR) products were ligated into a TA cloning vector (RBC TA Cloning Vector Kit, RBC Bioscience; Taipei, Taiwan). Next, the ligated vectors were transformed into E. coli competent cells (Solo Pack Gold; Agilent Technologies; Santa Clara, CA, USA) and subjected to sequencing analysis. We designed the universal stop codon “TAGATAGTTAG” and inserted it between two ZFN-binding sites by PCR-induced mutagenesis (Agilent Technologies). The insertion was confirmed by DNA sequencing and the plasmid was designated as d-stop plasmids (Figure 1).Figure 1


Generation of CCR5-defective CD34 cells from ZFN-driven stop codon-integrated mesenchymal stem cell clones.

Manotham K, Chattong S, Setpakdee A - J. Biomed. Sci. (2015)

Primers map and donor plasmid generation. Illustration of the CCR5 locus and primer locations. Upper panel: ZFN-targeted DNA sequence (underlined) and the interspace. Lower panel: DNA sequences of the d-plasmid around the ZFN site and the insertion-specific primer (IP); a silent T-A mutation adjacent to the left ZFN-targeted site was intentionally selected for confirmation and marking of the vicinity of integration between the ZFN-binding sites.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4379539&req=5

Fig1: Primers map and donor plasmid generation. Illustration of the CCR5 locus and primer locations. Upper panel: ZFN-targeted DNA sequence (underlined) and the interspace. Lower panel: DNA sequences of the d-plasmid around the ZFN site and the insertion-specific primer (IP); a silent T-A mutation adjacent to the left ZFN-targeted site was intentionally selected for confirmation and marking of the vicinity of integration between the ZFN-binding sites.
Mentions: cDNA of human CCR5 (1791 bp), from −733 bp upstream of the left-hand ZFN-binding site to 1038 bp downstream of the right-hand ZFN-binding site, was amplified from genomic DNA of peripheral blood using the primers D1 (5′-GTGGACAGGGAAGCTAGCAG-3′) and D2 (5′-CCATACCTTGGAGGGGAAAT-3′). The polymerase chain reaction (PCR) products were ligated into a TA cloning vector (RBC TA Cloning Vector Kit, RBC Bioscience; Taipei, Taiwan). Next, the ligated vectors were transformed into E. coli competent cells (Solo Pack Gold; Agilent Technologies; Santa Clara, CA, USA) and subjected to sequencing analysis. We designed the universal stop codon “TAGATAGTTAG” and inserted it between two ZFN-binding sites by PCR-induced mutagenesis (Agilent Technologies). The insertion was confirmed by DNA sequencing and the plasmid was designated as d-stop plasmids (Figure 1).Figure 1

Bottom Line: However, the rarity of this mutation and the safety risks associated with current BMT protocols are the major obstacles to this treatment.We here reported the novel approach on generation of patients own CD34 cells from high fidelity ZFN-mediated HDR MSC clones.We believe that our approach will be beneficial in future HIV treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Molecular and Cell Biology Unit, Lerdsin General Hospital, Bangkok, Thailand. kmanotham@hotmail.com.

ABSTRACT

Backgrounds: Homozygous 32-bp deletion of the chemokine receptor 5 gene (CCR5) is associated with resistance to human immunodeficiency virus (HIV) infection, while heterozygosity delays HIV progression. Bone marrow transplantation (BMT) from a 32/32 donor has been shown to cure an HIV-infected patient. However, the rarity of this mutation and the safety risks associated with current BMT protocols are the major obstacles to this treatment. Zinc finger nuclease (ZFN) targeting is a powerful method for achieving genomic disruption at specific DNA sites of interest.

Results: Taking advantage of the self-renewal and plasticity properties of stem cells, in this study, we successfully generated isogenic and six-cell clones of bone marrow-derived mesenchymal stem cells that carry the stop codon of the CCR5 gene by using a ZFN-mediated homology-directed repair technique. These cells were expandable for more than 5 passages, and thus show potential to serve as an individual's cell factory. When Oct4 was overexpressed, the mutated cells robustly converted to CD34+ progenitor cells.

Conclusion: We here reported the novel approach on generation of patients own CD34 cells from high fidelity ZFN-mediated HDR MSC clones. We believe that our approach will be beneficial in future HIV treatment.

No MeSH data available.


Related in: MedlinePlus