Primate in Utero Gene Transfer Comes of Age (2001)

Perhaps the Holy Grail of gene therapy is the transfer of therapeutic genes to prenatal infants. With the ever-expanding database of human genetic disease information, it is now possible to conceive of prenatal genetic diagnosis tightly coupled with prenatal gene therapy. Until recently, the only model systems for testing prenatal gene delivery have been in nonprimates. Because of anatomical and developmental differences between primates and nonprimates, there has been a need to develop and test a primate model for prenatal gene transfer. If treatment of human fetuses with therapeutic genes is to become a reality, there first needs to be substantive testing of gene delivery and its consequences in nonhuman primates. The publication by Tarantal et al. in this issue of Molecular Therapy addresses this very issue (1Tarantal et al.Rhesus monkey model for fetal gene transfer: Studies with retroviral-based vector systems.Mol. Ther. 2001; 3Abstract Full Text Full Text PDF Scopus (56) Google Scholar). Their study demonstrates effective in utero gene transfer into rhesus monkey fetuses. Experiments conducted as part of this study are of special significance to the gene therapy community as a whole since they address several important problems. These include in vivo gene transfer efficiency of retroviral versus lentiviral vectors, potential for crossing of the placental barrier by the infused vectors, potential transfer of genes into the germ lines of the fetuses, and the duration of transgene expression. Tarantal et al. utilized ultrasound guidance to deliver viral vector supernatants either intraperitoneally or intra-hepatically to rhesus monkey fetuses in late trimester (55 ± 5 days). They compared the frequency of gene marking [enhanced green fluorescent protein (EGFP)] when the transgene was delivered by an amphotropic retroviral vector, VSV-G envelope pseudotyped retroviral vector or VSV-G envelope pseudotyped lentiviral vector. Not surprisingly, the lentiviral vector provided the most efficient transduction of hematopoietic progenitor cells at all concentrations of infectious particles used. The lentiviral transduction efficiency was two to three times greater than the most effective retroviral transduction. It is noteworthy that the amphotropic retroviral vector was about twice as efficient as the VSV-G pseudotyped version of this same vector. The gene transfer had no adverse affects on fetal growth and development, and postnatal testing of the treated animals revealed no impairment of growth or developmental patterns. The level of gene marking in the peripheral blood mononuclear cells of animals treated with the lentiviral vectors averaged 2% from birth up to final necropsy at 6 months. A very encouraging result from these studies is the high frequency of gene marking in hematopoietic progenitor cells transduced with the HIV-based vector. Analyses using polymerase chain reaction demonstrated an overall average of 25% gene marking in bone marrow-derived colony-forming cells. Interestingly, this transduction efficiency was not dependent on vector titers since the same percentage of marking was observed whether the titer was 3.1 × 106 or 1.5 × 108 infectious particles. Since a 48-fold difference in vector titer had no enhancing effect on transduction efficiency, the upper limit of gene transfer for the HIV-based vector may have been reached in this study. It remains to be determined if this is a function of the VSV-G envelope or some host factor. Since the transgene had no therapeutic benefit for the animal, there was no clonal selection of transduced cells. It will be of interest to see if the percentage of transduced cells expands under conditions where a selective advantage is imposed (2Abonour R. et al.Efficient retrovirus-mediated transfer of the multidrug resistance 1 gene into autologous human long-term repopulating hematopoietic stem cells.Nat. Med. 2000; 6: 652-658Crossref PubMed Scopus (244) Google Scholar). A primary concern for in utero gene transfer is the safety of the mother. The investigators in this study looked for transmission of the infused vectors across the placental barrier. No vector sequences were observed in any of the animals studied. Of more concern was the consistent detection of vector sequences in the gonads of the treated fetuses, although the authors add that these specimens were not exhaustively perfused before analyses, and therefore the proviral DNA detected could have been derived from infiltrating hematopoietic cells. Gene silencing was observed in all cell and tissue samples taken at birth and 3 months postnatally, regardless of the vector system. The results from cell and tissue assays suggested a significant diminution of gene expression from prenatal to postnatal samplings out to 10 months postgestation. All of the vectors incorporated the CMV-IE promoter to drive expression of the EGFP transgene. This is a strong promoter, but has a notorious reputation for susceptibility to epigenetic silencing. Murine long terminal repeats are also well-known targets for epigenetic silencing, but since the HIV- and murine-based vectors were equally silenced, either the CMV promoter element or the transgene itself could have provided the silencing centers. Clearly, this is a major obstacle for all of gene therapy, and it must be overcome. Exploitation of chromatin insulator elements has been proposed as a possible solution for the silencing problem in hematopoietic stem cell gene therapy (3Emery D.W. Stamatoyannopoulos G. Stem cell gene therapy for the beta-chain hemoglobinopathies.Problems and progress. Ann. N.Y. Acad. Sci. 1999; 872 (discussion 107-08.): 94-107Crossref PubMed Scopus (26) Google Scholar, 4Neff T. Shotkoski F. Stamatoyannopoulos G. Stem cell gene therapy, position effects and chromatin insulators.Stem Cells. 1997; 15: 265-271Crossref PubMed Scopus (32) Google Scholar) and perhaps should be tested in future rounds of in utero gene therapy in rhesus monkeys. The demonstration of successful and efficient in utero gene transfer in the rhesus monkey model using retroviral and lentiviral vector supernatants now opens up many possible experimental applications. Certainly this system is amenable to testing anti-SIV gene therapy strategies which could be translated to anti-HIV applications in humans. The relatively high transduction efficiencies obtained with both retroviral and lentiviral vectors make this approach very attractive for treating diseases such as adenosine deaminase deficiency which does not require 100% of the defective cells to be corrected (5Mullen C.A. Snitzer K. Culver K.W. Morgan R.A. Anderson W.F. Blaese R.M. Molecular analysis of T lymphocyte-directed gene therapy for adenosine deaminase deficiency: Long-term expression in vivo of genes introduced with a retroviral vector.Hum. Gene Ther. 1996; 7: 1123-1129Crossref PubMed Scopus (42) Google Scholar, 6Cornetta K. Wieder R. Anderson W.F. Gene transfer into primates and prospects for gene therapy in humans.Prog. Nucleic Acid Res. Mol. Biol. 1989; 36: 311-322Crossref Scopus (21) Google Scholar). Of importance equal to the exciting data derived from this study are results that serve to emphasize the many challenges that lie ahead prior to application of in utero gene therapy in humans. Among these are development of modified vectors and methods of gene transfer that add to the safety of both the fetus and the mother. In particular, it must be clearly established that in utero gene therapy does not lead to germ cell transduction. For some hematopoietic diseases, it may also be necessary to increase the transduction efficiency of progenitor cells to positively impact on the disease. Strategies using insulators or locus control regions that prevent or minimize epigenetic silencing of transgenes should be tested in the rhesus in utero model. Other important considerations such as the use of cell- or tissue-targeted vectors (7Kasahara N. Dozy A.M. Kan Y.W. Tissue-specific targeting of retroviral vectors through ligand-receptor interactions.Science. 1994; 266: 1373-1376Crossref PubMed Scopus (389) Google Scholar) can now be rigorously examined for transduction efficiency and tropism. In summary, this study paves the way for more widespread use of in utero gene transfer to test new vectors and therapeutic genes. In this regard, it would be of great use to the gene therapy community if one or more NIH-subsidized rhesus monkey in utero gene transfer centers could be established. These would foster the rapid development of in utero gene therapy approaches for humans.