Gene Therapy by Insertion, Editing, or Ablation.1) Gene insertion – The mutation is depicted by the red spot. A vector (e.g., self-inactivating gammaretroviral or lentiviral vector) is used to introduce the normal cDNA encoding the entire protein into the cell under regulation by an appropriate promoter, and this vector integrates into the host cell genomic DNA. Transcription then proceeds, and the normal protein is produced. Both the mutated gene and the normal gene are present in the cell. 2) Gene editing – The affected cell shows the location of the mutation leading to the disorder with a red spot. A zinc finger nuclease (ZFN) is introduced, and cells are transduced with a vector, in this case, an integrase deficient lentiviral vector (IDLV) containing the normal partial cDNA for the affected gene, resulting in high-fidelity homology directed repair (HDR) and, thereby, correction of the defect. 3) Gene ablation – The CRISPR (clustered, regularly interspaced, palindromic repeats) –associated protein 9 (Cas9) nuclease is introduced in combination with short guide RNAs (gRNAs) that target the nuclease to the gene of interest (represented in green), resulting in cleavage of specific DNA sequence. Certain pairs of gRNAs successfully abrogate gene expression (depicted by red X).

Gene Therapy by Insertion, Editing, or Ablation.1) Gene insertion – The mutation is depicted by the red spot. A vector (e.g., self-inactivating gammaretroviral or lentiviral vector) is used to introduce the normal cDNA encoding the entire protein into the cell under regulation by an appropriate promoter, and this vector integrates into the host cell genomic DNA. Transcription then proceeds, and the normal protein is produced. Both the mutated gene and the normal gene are present in the cell. 2) Gene editing – The affected cell shows the location of the mutation leading to the disorder with a red spot. A zinc finger nuclease (ZFN) is introduced, and cells are transduced with a vector, in this case, an integrase deficient lentiviral vector (IDLV) containing the normal partial cDNA for the affected gene, resulting in high-fidelity homology directed repair (HDR) and, thereby, correction of the defect. 3) Gene ablation – The CRISPR (clustered, regularly interspaced, palindromic repeats) –associated protein 9 (Cas9) nuclease is introduced in combination with short guide RNAs (gRNAs) that target the nuclease to the gene of interest (represented in green), resulting in cleavage of specific DNA sequence. Certain pairs of gRNAs successfully abrogate gene expression (depicted by red X).

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