Sickle cell disease (SCD) is a genetic disorder that affects millions of people worldwide, mainly of African descent. A single point mutation (S) in the -globin gene allows deoxygenated hemoglobin to polymerize, which leads to deformation of red blood cells, hemolysis, vaso-occlusion, and multiple other complications, including early death. Allogeneic stem cell transplantation can be curative, but carries a risk of mortality, and finding blood matched donors is difficult. Recent advances in gene-editing and generating autologous inducible pluripotent stem cells (iPS) holds promise for replenishing a patient's blood with healthy cells, while avoiding tissue rejection. Previous studies have demonstrated that forced-expression of hemoglobin genes by lentiviruses, or correcting the S mutation by homologous recombination can reduce symptoms. However, these methods are inefficient and carry a risk of inducing oncogenic mutations. Moreover, the resultant erythroid progenitor cells have impaired -globin production. The newly engineered enzyme FokI-dCas9 has been shown to cleave a target site without inducing harmful mutations. We hypothesize that FokI-dCas9 will provide superior gene-editing of the S mutation in autologous iPS cells for use in transplantation. To investigate this hypothesis, this proposal aims to 1) target the HBB gene with FokI-dCas9, 2) generate integration-free iPS cells, 3) correct the S mutation using FokI-dCas9 without inducing off-target mutations, 4) determine the phenotype of erythroid progenitors derived from gene-corrected iPS cells.
Aim 1 will optimize the delivery of Foki-dCas9 for correcting the S mutation in SCD-iPS cells. First, we will target the HBB gene in human (HEK293) and mouse (MEF) somatic cells. The cells will be transfected with a PiggyBac vector carrying -globin donor sequences, and plasmids expressing FokI- dCas9 and specific sgRNA for inducing nicks. The -globin donor DNA will be site-directed for integration into the TTAA sit of HBB intron 2 by PiggyBac, and verified by Sanger sequencing. Next, we will correct the S mutation in a human SCD-iPS cell line. The rate of off-target mutations will be determined by whole-exome deep sequencing.
In Aim 2, iPS cells will be generated from sickle mice by temporary expression of Yamanaka factors using non-integrating Sendai viruses. The mouse iPS cells will be injected into immunodeficient NSG mice to determine pluripotentcy. Next, the S mutation in patient and mouse iPS cells will be corrected to include either the wild-type sequence or the wild-type sequence with the point mutations that have been shown to prevent sickling. Finally, we will use an in vivo differentiation approach which has been shown to generate erythroid progenitor cells that express mature -globin protein. Progenitor cells from bone marrow and teratoma will be sorted and characterized for SCD by hematocrit, morphology, and hemoglobin production. Thus, the generation of gene-corrected, fully functional progenitor cells will be a crucial step for the treatment of SCD. In summary, the goal of this proposal is to generate clinical-grade iPS cells by efficiently correcting the S mutation with FokI- dCas9 and PiggyBac technology.
Sickle cell disease is a severe form of anemia caused by a single point mutation in the -globin gene. Previous studies have corrected this mutation by random gene insertion, or using inefficient gene-editing systems which may induce cancer. To further these studies, this proposal aims to correct the mutation using the highly specific nuclease FokI-dCas9 in clinical-grade iPS cells for the treatment of sickle cell disease.
