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Lentiviral vectors are widely used in basic research and clinical applications for gene transfer and long-term expression; however, safety issues have not yet been completely resolved. In this study, we characterized hepatocarcinomas that developed in mice 1 year after in utero administration of a feline-derived lentiviral vector. Mapped viral integration sites differed among tumors and did not coincide with the regions of chromosomal aberrations. Furthermore, gene expression profiling revealed that no known cancer-associated genes were deregulated in the vicinity of viral integrations. Nevertheless, five of the six tumors exhibited highly significant upregulation of E2F target genes, of which a majority are associated with oncogenesis, DNA damage response, and chromosomal instability. We further show in vivo and in vitro that E2F activation occurs early on following transduction of both fetal mice and cultured human hepatocytes. On the basis of the similarities in E2F target gene expression patterns among tumors and the lack of evidence implicating insertional mutagenesis, we propose that transduction of fetal mice with a feline lentiviral vector induces E2F-mediated major cellular processes that drive hepatocytes toward uncontrolled proliferation culminating in tumorigenesis.

Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis.

Over the past few years, considerable progress in prenatal diagnosis and surgery combined with improvements in vector design vindicate a reappraisal of the feasibility of in utero gene therapy for serious monogenetic diseases. As adult gene therapy gathers pace, several apparent obstacles to its application as a treatment may be overcome by pre- or early postnatal treatment. This review will examine the concepts and practice of prenatal vector administration. We aim to highlight the advantages of early therapeutic intervention focusing on diseases that could benefit greatly from a prenatal gene therapy approach. We will pay special attention to the strategies and vectors that are most likely to be used for this application and will speculate on their expected developments for the near future.

Gene therapy for Duchenne muscular dystrophy has so far not been successful because of the difficulty in achieving efficient and permanent gene transfer to the large number of affected muscles and the development of immune reactions against vector and transgenic protein. In addition, the prenatal onset of disease complicates postnatal gene therapy. We have therefore proposed a fetal approach to overcome these barriers. We have applied beta-galactosidase expressing equine infectious anaemia virus (EIAV) lentiviruses pseudotyped with VSV-G by single or combined injection via different routes to the MF1 mouse fetus on day 15 of gestation and describe substantial gene delivery to the musculature. Highly efficient gene transfer to skeletal muscles, including the diaphragm and intercostal muscles, as well as to cardiac myocytes was observed and gene expression persisted for at least 15 months after administration of this integrating vector. These findings support the concept of in utero gene delivery for therapeutic and long-term prevention/correction of muscular dystrophies and pave the way for a future application in the clinic.

Current concerns over insertional mutagenesis by retroviral vectors mitigate investigations into alternative, potentially persistent gene therapy vector systems not dependent on genomic integration, such as Sendai virus vectors (SeVV). Prenatal gene therapy requires efficient gene delivery to several tissues, which may not be achievable by somatic gene transfer to the adult. Initially, to test the potential and tropism of the SeVV for gene delivery to fetal tissues, first-generation (replication- and propagation-competent) recombinant SeVV, expressing beta-galactosidase was introduced into late gestation immunocompetent mice via the amniotic and peritoneal cavities and the yolk sac vessels. At 2 days, this resulted in very high levels of expression particularly in the airway epithelium, mesothelium and vascular endothelium, respectively. However, as expected, substantial vector toxicity was observed. The efficiency of gene transfer and the level of gene expression were then examined using a second-generation SeVV. The second generation was developed to be still capable of cytoplasmic RNA replication and therefore high-level gene expression, but incapable of vector spread due to lack of the gene for viral F-protein. Vector was introduced into the fetal amniotic and peritoneal cavities, intravascularly, intramuscularly and intraspinally; at 2 days, expression was observed in the airway epithelia, peritoneal mesothelia, unidentified cells in the gut wall, locally at the site of muscle injection and in the dorsal root ganglia, respectively. Mortality was dramatically diminished compared with the first-generation vector.

The fundamental hypotheses behind fetal gene therapy are that it may be possible (1) to achieve immune tolerance of transgene product and, perhaps, vector; (2) to target cells and tissues that are inaccessible in adult life; (3) to transduce a high percentage of rapidly proliferating cells, and in particular stem cells, with relatively low absolute virus doses leading to clonal transgene amplification by integrating vectors; and (4) to prevent early disease manifestation of genetic diseases. This study provides evidence vindicating the first hypothesis; namely, that intravascular prenatal administration of an adenoviral vector carrying the human factor IX (hFIX) transgene can induce immune tolerance of the transgenic protein. Following repeated hFIX protein injection into adult mice, after prenatal vector injection, we found persistence of blood hFIX and absence of hFIX antibodies in 5 of 9 mice. Furthermore, there was substantial hFIX expression after each of 2 reinjections of vector without detection of hFIX antibodies. In contrast, all adult mice that had not been treated prenatally showed a rapid loss of the injected hFIX and the development of high hFIX antibody levels, both clear manifestations of a strong immune reaction.

Objective: To access the fetal airways percutaneously using ultrasound-guided injection of the fetal trachea in sheep.Methods: Adenoviral gene therapy vectors and transduction-enhancing agents were delivered to the trachea via a needle inserted through the thorax or the neck of late-gestation (0.9 term, n = 3) or mid-gestation (0.5-0.8 term, n = 18) fetal sheep using ultrasound guidance.Results: Injection of the trachea in the fetal thorax was successful in 16 out of 18 fetuses and achieved at the first attempt in 9 fetuses within 12 min [mean 7 min and 31 s +/- (SD) 3 min and 4 s]. Survival was 100%. Injecting the trachea in the neck was less successful.Conclusions: The fetal trachea of the sheep can be safely accessed by percutaneous ultrasound-guided injection to deliver vectors directly to the fetal airways for gene therapy. It may also enable tracheal occlusion for the antenatal treatment of congenital diaphragmatic hernia without the need for endoscopy or open surgery. [ABSTRACT FROM AUTHOR]

Investigates the possibility of gene targeting to the mouse fetus with two different viral vector systems and at different times of gestation. Aim of developing fetal gene therapy for cystic fibrosis; Limitations of low retroviral titre for in vivo fetal gene transfer; Highest levels of transgene expression.

Features research on fetal. Twin-twin transfusion syndrome; Cardiomyocyte contraction in the presence of bile acid taurcholate; Effect of elective preterm delivery on postnatal bowel function in fetuses with gastroschisis.