The goal of Project 2 is correction of the sickle cell disease (SCD) and B-thalassemia (B-thal) mutations, using patient derived somafic cells and their induced pluripotent stem (iPS) cell derivatives. These cells will ultimately be converted to hematopoietic stem cells (HSCs) to reconstitute patient hematopoietic systems. As an alternative to conventional cDNA-based genetic therapies, IPS cells or their somafic precursors, will be modified by a sequence-specific gene targeting strategy, small fragment homologous replacement (SFHR), triplex forming oligonucleotide (TFO)-mediated homologous exchange and by classical homologous recombination (HR). These studies will be carried out in the presence and absence of zinc finger nucleases (ZFNs) and meganucleases and meganucleases (MNs) potent stimulators of recombination. Previous studies from our lab have shown that oligo/polynucleotide small DNA fragments (SDFs) carrying the A>T sickle mutation (B[S]-DFs), when microinjected into wild-type human hematopoietic stem cells (HSCs), will convert the endogenous wild-type globin (B[A]-globin) into the sickle cell disease B[S]-globin at frequencies of at least 7%. This project will test several hypotheses in 2 Specific Aims.
Aim 1 will test the hypotheses that SDF-modified and TFO-modified human SCD and B-thal somafic cells and their IPS cell derivatives can be converted to clonal isoaltes IPS cells that are viable, karyotypically stable, and can be differentiated into engraftable hematopoietic precursors.
Aim 2 : will test the hyopthesis that classical HR is a reliable alterative method for correcting specific genomic mutations in the B-globin globin gene. Somatic and IPS cells homozygous for the B[S]-globin or defined B-thal mutations, will be genetically corrected by SDF, TFO, and classical HR mediated homologous exchange, to generate clonal populations of B-globin heterozygote IPS cells. If patient somafic cells are used for correction, they will be converted to IPS cells when a clonal population has been isolated. The cells will be transfected with SDFs by electroporation or microinjection and in the presence and absence of ZFNs or sequence-specific MNs.
Each Aim will evaluate the corrected cells in terms of their karyotypic stability, genetic integrity, the ability to generate or maintain IPS cell phenotype, and the ability to differentiate in vitro into HSC. Teratoma formation will be evaluated by Core C and gene expression patterns after correction will also be evaluated in through Core B ad the UCSF Array Core. While initial studies will be carried out using retrovirally reprogrammed IPS cells, as new patient-specific IPS cell lines become available through Project 1 and Core B, they will be corrected and evaluated. Finally, corrected IPS cells generated in Project 2 will be evaluated in Project 3 for their ability to differentiate into hematopoietic precursors that will engraft into NOD-SCID mice for hematopoietic reconstitution.

Public Health Relevance

This project will develop protocols for correcting disease-causing mutations in sickle cell disease (SCD) and B-thalassemia (B-thal) patient-specific somafic and induced pluripotent stem (IPS) cells. Autologous, corrected iPS cells that can be differentiated into hematopoietic stem cells (HSC) will be a critical component of a comprehensive cell and gene therapy for these diseases. The impact of developing these protocols to generate autologous, SCD and B-thal corrected HSC from corrected patient IPS cells will be significant since these hemoglobinopathies are among the most prevalent inherited diseases worldwide.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Program Projects (P01)
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Special Emphasis Panel (ZDK1-GRB-6)
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University of California San Francisco
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