Genetic correction of human beta-thalassemic induced pluripotent stem cells

Summary

Principal Investigator: EIRINI PAPAPETROU
Abstract: PROJECT SUMMARY/ABSTRACT Candidate: I obtained my M.D. and Ph.D. in Greece. For the past four years I have been a postdoctoral fellow in the laboratory of Dr Michel Sadelain at Memorial Sloan-Kettering Cancer Center in New York, where I worked on the engineering of lineage- and developmental stage-specific expression of lentivirally-encoded transgenes in the hematopoietic system by exploiting microRNA-mediated gene regulation and on the generation and genetic modification of patient-specific induced pluripotent stem cells (iPSCs). My long-term goal is to develop safer genetic engineering approaches for the treatment of blood disorders. Obtaining an NIH Pathway to Independence Award (K99/R00) will allow me to gain additional training in the mentored phase of the award with activities such as seminars, courses, scientific conferences, development of mentoring skills and training in research techniques such as bioinformatics analyses of the human genome and hematopoietic differentiation of human iPCSs. With additional training, I will be able to pursue an independent research position in a highly ranked academic research institution and focus my career in translational stem cell research. Environment: Memorial Sloan-Kettering Cancer Center (MSKCC) is a center of biomedical research bringing together scientists and physicians working together towards translation of basic science to preclinical and clinical research. This environment strongly encourages interdisciplinary and collaborative investigative projects and offers many training and educational opportunities. Additionally, the Tri-Institutional Collaboration Network is a joint initiative comprising MSKCC, The Rockefeller University, and Weill Cornell Medical College and supports broader networking opportunities as well as sharing of core facility resources across institutions. Dr Sadelain's laboratory is part of the Center for Cell Engineering (CCE), which brings together researchers from areas that encompass stem cell biology, genetic engineering, autologous cell delivery and transgene regulation. Research: For the promise of induced pluripotent stem cells (iPSCs) in regenerative medicine to be realized, strategies for their precise and safe genetic modification and for purging of residual differentiation-resistant cells are needed. The objective of this K99/R00 application is to develop and evaluate a gene addition strategy for autologous cell therapy of a common inherited blood disorder, beta halassemia major, using patient-specific iPSCs. The approach uses genetic engineering of iPSCs, integrating disease correction with protection against undifferentiated cells, with the aim to circumvent risks of oncogenesis posed by both random integration and persistence of undifferentiated pluripotent stem cells. The specific aims are: (1) To generate beta halassemia iPSCs (thal-iPSCs) harboring a lentivirally-encoded ss-globin transgene integrated at "safe harbor" genomic sites. Transgene-free thal-iPSCs will be transduced with a lentiviral vector encoding ss-globin and an exchangeable Neo-eGFP selection cassette. Single vector copy integrants will be screened according to silencing-resistant transgene expression and vector chromosomal position and "safe harbor" integration sites will be selected, based on proximity to endogenous genes, especially cancer-related genes. (2) To engineer a "suicide gene" strategy for purging of differentiation-resistant thal-iPSCs. An Herpes Simplex Virus-thymidine kinase (HSV-tk) "suicide gene" with regulated expression by tissue-specific promoters/enhancers and/or miR- NAs to selectively eliminate undifferentiated thal-iPSCs but not their differentiated progeny will be engineered and incorporated in pre-selected thal-iPSC clones through recombinase-mediated cassette exchange (RMCE). (3) To characterize the therapeutic and safety features conferred by a ss-globin and an HSV-tk transgene integrated at "safe harbor" sites in thal-iPSCs. The tissue specificity and levels of expression of the ss-globin and HSV-tk transgenes integrated at "safe harbor" sites, as well as the expression of neighboring genes, will be determined in undifferentiated thal-iPSC clones and their erythroid progeny. Purging of undifferentiated tumor- initiating cells after administration of ganciclovir will be assessed in vitro and in teratoma formation assays. This study proposes a definition and framework for the prospective identification of "safe harbor" sites for transgene integration in the human genome, using bioinformatics analyses and gene expression profiling of iPSCs and their differentiated progeny. This project also harnesses novel mechanisms of post-transcriptional regulation to engineer robust control of transgene expression by exploiting distinct microRNA expression patterns during development. The "suicide gene" strategy for purging of undifferentiated cells can be broadly applicable to all pluripotent stem cell-based therapies in regenerative medicine. In the new era of human pluripotent stem cell technology, this proof-of-principle study can provide a new paradigm of integrated iPS-based cell and gene therapy, generally applicable to genetic disorders and advance this new field towards translation to the clinic.
Funding Period: 2012-12-01 - 2015-11-30
more information: NIH RePORT

Research Grants

  1. STEM CELL GENE THERAPY FOR HEMOGLOBINOPATHIES
    George Stamatoyannopoulos; Fiscal Year: 2013
  2. Immune Cells in Atherosclerosis and Vascular Disease
    Catherine C Hedrick; Fiscal Year: 2013
  3. CENTER FOR GASTROINTESTINAL BIOLOGY AND DISEASE
    Robert S Sandler; Fiscal Year: 2013
  4. Center for Molecular Imaging Research at MGH/HMS
    Ralph Weissleder; Fiscal Year: 2013
  5. University of Maryland Greenebaum Cancer Center Support Grant
    Kevin J Cullen; Fiscal Year: 2013
  6. Neural Mechanisms of Itch
    ROBERT H LA MOTTE; Fiscal Year: 2013
  7. Directing Tumor-specific T cells to Tumors
    Pawel Kalinski; Fiscal Year: 2013
  8. CURE: DIGESTIVE DISEASES RESEARCH CENTER
    JUAN ENRIQUE ROZENGURT; Fiscal Year: 2013
  9. DIABETES AND ENDOCRINOLOGY RESEARCH CENTER
    Domenico Accili; Fiscal Year: 2013
  10. DNA Methylation Control Hematopoietic Stem Cell Lineage Differentiation
    GRANT ANTHONY CHALLEN; Fiscal Year: 2013

Detail Information

Research Grants30

  1. STEM CELL GENE THERAPY FOR HEMOGLOBINOPATHIES
    George Stamatoyannopoulos; Fiscal Year: 2013
    ..The focus of this Program Project, Gene Therapy, can provide a new paradigm for the treatment of these hemoglobinopathies as well as for other blood diseases. (End of Abstract) ..
  2. Immune Cells in Atherosclerosis and Vascular Disease
    Catherine C Hedrick; Fiscal Year: 2013
    ..Each project will utilize the Human Core to study immune cells from human subjects to establish functional links between candidate genes of interest and immune cell function in atherogenesis. ..
  3. CENTER FOR GASTROINTESTINAL BIOLOGY AND DISEASE
    Robert S Sandler; Fiscal Year: 2013
    ..Through all of its activities, the Center improves communication, promotes collaboration, develops careers and generally enriches the intellectual climate for digestive disease research. ..
  4. Center for Molecular Imaging Research at MGH/HMS
    Ralph Weissleder; Fiscal Year: 2013
    ....
  5. University of Maryland Greenebaum Cancer Center Support Grant
    Kevin J Cullen; Fiscal Year: 2013
    ..Reflecting our remarkable and continued growth, UMGCC seeks to renew its CCSG to enhance and expand its efforts in high-quality and clinically relevant cancer research. ..
  6. Neural Mechanisms of Itch
    ROBERT H LA MOTTE; Fiscal Year: 2013
    ..abstract_text> ..
  7. Directing Tumor-specific T cells to Tumors
    Pawel Kalinski; Fiscal Year: 2013
    ..abstract_text> ..
  8. CURE: DIGESTIVE DISEASES RESEARCH CENTER
    JUAN ENRIQUE ROZENGURT; Fiscal Year: 2013
    ....
  9. DIABETES AND ENDOCRINOLOGY RESEARCH CENTER
    Domenico Accili; Fiscal Year: 2013
    ....
  10. DNA Methylation Control Hematopoietic Stem Cell Lineage Differentiation
    GRANT ANTHONY CHALLEN; Fiscal Year: 2013
    ..I believe the basic research outlined in this project can contribute to the fundamental understanding of epigenetic regulation of stem cell function and has direct relevance to progression of human disease states. ..