Development of a Novel Gene Therapy Strategy for SCID-X1 and a Method for Measuring Gene Targeting Outcomes at Endogenous Loci




Kildebeck, Eric James

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Two decades of gene therapy trials for primary immunodeficiencies have seen tremendous clinical success with a significant majority of patients developing functional immune systems. The development of leukemia in some patients has led to the development of precise gene targeting tools to correct genetic deficits without inducing genomic instability. In this thesis I report the development of a novel gene therapy strategy for SCID-X1 and the development of a useful method for measuring gene editing outcomes at endogenous loci in any cell type. TALENs designed to target IL2RG exon 1 are shown to be highly active and stimulate precise integration of IL2RG cDNA under the control of the endogenous IL2RG promoter. Activity levels of IL2Rγ in cells targeted with a codon-optimized cDNA and an artificial intron are also shown to be as high or higher than WT levels, demonstrating the potential for this approach to correct the functional deficit seen in SCID-X1. Furthermore, these TALENs successfully stimulate gene targeting in CD34+ hematopoietic stem and progenitor cells at frequencies 10-fold higher than the highest levels previously reported, while displaying less toxicity than ZFNs already in use in clinical trials. The high activity and low toxicity of these TALENs in combination with the potential for gene targeting at exon 1 to correct more than 98% of SCID-X1-causing mutations make this a promising strategy for gene therapy, which could one day form the basis for a safe and effective cure for SCID-X1.

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