Fanconi anemia (FA) is a complex genetic disorder characterized by a progressive bone marrow aplasia, genomic instability, and the acquisition of both myeloid malignancies and squamous cell carcinoma. Previous work by our group identified for the first time, major limitations in the use of murine oncoretroviruses to correct FA hematopoietic stem cells (HSCs). These disease-specific challenges include: the increased predisposition of FA cells to undergo apoptosis and clonal evolution to MDS/AML in non-corrected cells following prolonged ex vivo culture. Consequently, in recent work, we developed foamyviral (FV) and lentiviral (LV) self-inactivating vectors and demonstrated that these constructs can correct the repopulating ability of murine FA HSCs in a short 10-14 hour transduction period without pre-stimulation. We also generated separate plasmids for the FV gag, pol, and env genes and adapted the codons of the three FV genes for improved expression in human cells. This has allowed us to dramatically increase the number of infectious recombinant particles (titers) and reduce the potential for syncytial formation and subsequent toxicity especially in human CD34+ cells. Finally, modifications of the FV envelope protein enabled us to utilize the same envelope for both FV as well as LV vectors. Work proposed in this application will build on these observations. Specifically, we will generate the constructs that will provide optimized expression in human FANCC-/- stem and progenitor cells. We will test the expression of these constructs in vitro and in vivo in a series of studies in FANCC-/- cell lines, primary murine Fancc-/- progenitor and stem cells and human FANCC-/- hematopoietic progenitors and SCID- repopulating cells (SRC) in NOD/LtSz-scidIL2R gammanull (NSG) mice. We will also test the transduction efficiency of a novel FV envelope on both normal human and FANCC-/- progenitor and primary human SRCs. Finally, a major limitation in testing non-genotoxic engraftment protocols in FA has been the lack of a murine model that accurately recapitulates the spontaneous progression of bone marrow failure observed in patients. We have now developed a murine model with these characteristics and we will use it to test engraftment protocols.

Public Health Relevance

Achievement of these goals will permit the translation of this basic work to the development of clinical protocols for effective viral mediated gene correction of Fanconi anemia. On a broader basis, these studies should provide insight into the biological behavior of hematopoietic stem cells and the ability to manipulate them ex vivo prior to transplantation and engraftment.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
Therapeutic Approaches to Genetic Diseases (TAG)
Program Officer
Forry, Suzanne L
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Indiana University-Purdue University at Indianapolis
Schools of Medicine
United States
Zip Code
Hanenberg, Helmut; Roellecke, Katharina; Wiek, Constanze (2017) Stem Cell Genetic Therapy for Fanconi Anemia - A New Hope. Curr Gene Ther 16:309-320
Roellecke, K; Virts, E L; Einholz, R et al. (2016) Optimized human CYP4B1 in combination with the alkylator prodrug 4-ipomeanol serves as a novel suicide gene system for adoptive T-cell therapies. Gene Ther 23:615-26
Barthelemy, Joanna; Hanenberg, Helmut; Leffak, Michael (2016) FANCJ is essential to maintain microsatellite structure genome-wide during replication stress. Nucleic Acids Res 44:6803-16
Sumpter Jr, Rhea; Sirasanagandla, Shyam; Fernández, Álvaro F et al. (2016) Fanconi Anemia Proteins Function in Mitophagy and Immunity. Cell 165:867-81
Park, Jung-Young; Virts, Elizabeth L; Jankowska, Anna et al. (2016) Complementation of hypersensitivity to DNA interstrand crosslinking agents demonstrates that XRCC2 is a Fanconi anaemia gene. J Med Genet 53:672-680
Virts, Elizabeth L; Jankowska, Anna; Mackay, Craig et al. (2015) AluY-mediated germline deletion, duplication and somatic stem cell reversion in UBE2T defines a new subtype of Fanconi anemia. Hum Mol Genet 24:5093-108
Raghunandan, Maya; Chaudhury, Indrajit; Kelich, Stephanie L et al. (2015) FANCD2, FANCJ and BRCA2 cooperate to promote replication fork recovery independently of the Fanconi Anemia core complex. Cell Cycle 14:342-53
Mantere, T; Haanpää, M; Hanenberg, H et al. (2015) Finnish Fanconi anemia mutations and hereditary predisposition to breast and prostate cancer. Clin Genet 88:68-73
Schmidt, Eva M; Wiek, Constanze; Parkinson, Oliver T et al. (2015) Characterization of an Additional Splice Acceptor Site Introduced into CYP4B1 in Hominoidae during Evolution. PLoS One 10:e0137110
Wiek, Constanze; Schmidt, Eva M; Roellecke, Katharina et al. (2015) Identification of amino acid determinants in CYP4B1 for optimal catalytic processing of 4-ipomeanol. Biochem J 465:103-14

Showing the most recent 10 out of 31 publications