The long-term goal of the Project is to explore fetal gene transfer for sickle cell disease (SCD) in a fetal rhesus monkey model. Gene therapy strategies that target hematopoietic stem cells (HSC) have been proposed as a long-term treatment for hemoglobinopathies such as SCD. However, traditional ex vivo gene therapy approaches for transducing CD34+ HSC in humans and large animal models have proven disappointing due to low gene transfer efficiencies, poor expression of the introduced gene, and technical difficulties associated with the transplant procedures. In this Project we are proposing direct in vivo gene transfer into pre-immune fetal rhesus monkeys because this approach eliminates the many problems and limitations associated with removal of HSC, in vitro transduction, and subsequent re-introduction in vivo, and avoids significant immune responses by administering the transgene while the fetus is immunologically immature. Initial studies will compare viral vector systems in the transgene while the fetus is immunologically immature. Initial studies will compare viral vector systems in order to determine the best method for transduction of fetal HSC in utero (Specific Aim 1). The vector systems we will explore include Moloney murine leukemia virus (MLV), HIV-1-derived lentivirus, and adeno-associated the vectors to early gestation rhesus fetuses either by the intrahepatic or intraportal routes in order to determine the most efficient method for targeting fetal HSC in utero. Fetal samples will be collected in utero )blood, liver) then at birth and monthly thereafter (blood, marrow) until 1 year of age for assessments of transduction and transgene expression (flow cytometry, PCR, hematopoietic progenitor assays). Once we have determined the best vector system and approach for the efficient marking of fetal HSC, we will introduce the human beta-gamma globin gene into the HSC of preimmune fetal rhesus monkeys (Specific Aim 2). We will assess MND (which enhances expression in HSC) or the beta-globin LCR (which has been shown to increase the frequency of globin-expressing erythroid cells) to determine which is most efficient in achieving stable gene expression. Fetal samples will be collected in utero transduction and gene expression. Finally, to test whether human HSC with the erythropoietin receptor (EpoR) insert have a proliferative advantage, we will use our human: rhesus xenograft model to perform a competitive repopulation study by administering a 50:50 mixture of HSC expressing the EpoR (marked with EGFP) and HSC without the inset (marked with EYFP) (Specific Aim 3). These studies will allow us to assess whether the addition of EpoR to the HSC provides a method for expanding targeted populations of genetically altered cells in vivo, which could facilitate gene therapy techniques. Thus, we propose to explore methods for safely transferring healthy genes into fetal monkeys that can be persistently expressed in hematopoietic cells as a model for the human fetus diagnosed with hemoglobinopathies such as SCD.
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