The fetal mouse is a sensitive genotoxicity model that exposes lentiviral-associated mutagenesis resulting in liver oncogenesis.

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.

Murine leukemia following irradiation conditioning for transplantation of lentivirally-modified hematopoietic stem cells.

Emerging reports are conclusively demonstrating the mutagenic risks involved in using retroviral vectors for gene therapy. Animal studies, as well as cases from a human clinical trial, have proven the potential of insertional leukemogenesis caused by a retroviral vector. Here, we report the observation of six T-lymphoblastic leukemia cases arising during the course of a gene therapy study for hemophilia B after transplantation of ex vivo transduced hematopoietic stem cells (HSCs) by a lentivirus vector. Three of these animals comprised secondary recipients of the same donor and LAM-PCR was performed to identify the vector integration loci. We located integrations in repeat elements of known genes, including a candidate brain-tumor locus, but none of these clones could be tracked in the leukemic blasts. Although transduced clones with an intact proviral cassette were detected in the spleen of the leukemic animals, they comprised a very small proportion, not correlating to the levels of leukemic blasts. After propagation of the latter in NOD/SCID mice, we could no longer detect the proviral cassette suggesting that the leukemic blasts were untransduced. We did, however, detect increased levels of reverse transcriptase activity in the leukemic blasts which may suggest activation of endogenous retroviruses. This study demonstrates that tumors arising in these type of gene therapy protocols are not necessarily due to vector insertional mutagenesis and highlights the importance of careful functional studies to delineate the nature of tumorigenesis.

Targeting the respiratory muscles of fetal sheep for prenatal gene therapy for Duchenne muscular dystrophy.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is a lethal degenerative muscular disease. Fetal gene therapy may correct the primary genetic defect. Our aim was to achieve expression of a reporter gene in the respiratory muscles of early gestation fetal sheep. STUDY DESIGN: An adenovirus vector containing the beta-galactosidase reporter gene (AdRSVbetagal) was injected into the thoracic musculature (n = 3) and pleural cavity (n = 6) of fetal sheep (61-67 days' gestation) under ultrasound guidance. Tissues were harvested after 48 hours and site and intensity of beta-galactosidase expression were assessed. RESULTS: Limited transgene expression observed after a single injection was improved by multiple injections, but remained localized. Ultrasound-guided creation of a hydrothorax led to an increase in the intensity of beta-galactosidase expression (ELISA). X-gal staining and immunohistochemistry showed that vector spread was confined to the innermost intercostal musculature. CONCLUSION: Ultrasound-guided injection can deliver gene therapy vectors to the fetal pleural cavity and achieve transduction of the respiratory muscles.

Comparison of HIV- and EIAV-based vectors on their efficiency in transducing murine and human hematopoietic repopulating cells.

The use of lentiviral vectors for gene transfer into hematopoietic stem cells has raised considerable interest as these vectors can permanently integrate their genome into quiescent cells. Vectors based on alternative lentiviruses would theoretically be safer than HIV-1-based vectors and could also be used in HIV-positive patients, minimizing the risk of generating replication-competent virus. Here we report the use of third-generation equine infectious anemia virus (EIAV)- and HIV-1-based vectors with minimal viral sequences and absence of accessory proteins. We have compared their efficiency in transducing mouse and human hematopoietic stem cells both in vitro and in vivo to that of a previously documented second-generation HIV-1 vector. The third-generation EIAV- and HIV-based vectors gave comparable levels of transduction and transgene expression in both mouse and human NOD/SCID repopulating cells but were less efficient than the second-generation HIV-1 vector in human HSCs. For the EIAV vector this is possibly a reflection of the lower protein expression levels achieved in human cells, as vector copy number analysis revealed that this vector exhibited a trend to integrate equally efficiently compared to the third-generation HIV-1 vector in both mouse and human HSCs. Interestingly, the presence or absence of Tat in viral preparations did not influence the transduction efficiency of HIV-1 vectors in human HSCs.

Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice.

Gene therapy by use of integrating vectors carrying therapeutic transgene sequences offers the potential for a permanent cure of genetic diseases by stable vector insertion into the patients' chromosomes. However, three cases of T cell lymphoproliferative disease have been identified almost 3 years after retrovirus gene therapy for X-linked severe combined immune deficiency. In two of these cases vector insertion into the LMO2 locus was implicated in leukemogenesis, demonstrating that a more profound understanding is required of the genetic and molecular effects imposed on the host by vector integration or transgene expression. In vivo models to test for retro- and lentiviral vector safety prior to clinical application are therefore needed. Here we present a high incidence of lentiviral vector-associated tumorigenesis following in utero and neonatal gene transfer in mice. This system may provide a highly sensitive model to investigate integrating vector safety prior to clinical application.

Permanent phenotypic correction of hemophilia B in immunocompetent mice by prenatal gene therapy.

Hemophilia B, also known as Christmas disease, arises from mutations in the factor IX (F9) gene. Its treatment in humans, by recombinant protein substitution, is expensive, thus limiting its application to intermittent treatment in bleeding episodes and prophylaxis during surgery; development of inhibitory antibodies is an associated hazard. This study demonstrates permanent therapeutic correction of his disease without development of immune reactions by introduction of an HIV-based lentiviral vector encoding the human factor IX protein into the fetal circulation of immunocompetent hemophiliac and normal outbred mice. Plasma factor IX antigen remained at around 9%, 13%, and 16% of normal in the 3 hemophilia B mice, respectively, until the last measurement at 14 months. Substantial improvement in blood coagulability as measured by coagulation assay was seen in all 3 mice and they rapidly stopped bleeding after venipuncture. No humoral or cellular immunity against the protein, elevation of serum liver enzymes, or vector spread to the germline or maternal circulation were detected.

Ultrasound-guided percutaneous delivery of adenoviral vectors encoding the beta-galactosidase and human factor IX genes to early gestation fetal sheep in utero.

In utero gene therapy may provide treatment of genetic diseases before significant organ damage, allow permanent genetic correction by reaching stem cell populations, and provide immune tolerance against the therapeutic transgenes and vectors. We have used percutaneous ultrasound-guided injection as a minimally invasive fetal procedure. First-generation adenoviruses encoding the nuclear localizing beta-galactosidase reporter gene or the human factor IX (hFIX) gene, or colloidal carbon were delivered via the umbilical vein (UV, n = 4), heart (intracardiac [IC], n = 2), liver parenchyma (intrahepatic [HE], n = 11), peritoneal cavity (intraperitoneal [IP], n = 14), skeletal musculature ([intramuscular [IM], n = 11), or the amniotic cavity (intraamniotic [IA], n = 14) to early-gestation fetal sheep (0.3 gestation = day 33-61). Postmortem analysis was performed at 2, 9, or 28 days after injection. Although fetal survival was between 77% and 91% for IP, HE, IA, and IM routes, no fetuses survived UV or IC procedures. The hFIX levels reaching 1900 and 401 ng/ml (IP), 30 ng/ml (HE), 66.5 and 39 ng/ml (IA), and 83 and 65.5 ng/ml (IM), respectively, were determined 2 days after injection and decreased at birth to 16.5 ng/ml (IP), 7 ng/ml (HE), 4.5 ng/ml (IA), and 4 and 0 ng/ml (IM). Polymerase chain reaction (PCR) and immunohistochemistry showed broadest hFIX transgene spread and highest localised beta-galactosidase expression, respectively, after IP administration. Antibodies were observed against vector but not against hFIX.

In utero gene transfer of human factor IX to fetal mice can induce postnatal tolerance of the exogenous clotting factor.

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.