M Themis

Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice

Inefficient gene transfer, inaccessibility of stem cell compartments, transient gene expression, and adverse immune and inflammatory reactions to vector and transgenic protein are major barriers to successful in vivo application of gene therapy for most genetic diseases. Prenatal gene therapy with integrating vectors may overcome these problems and prevent early irreparable organ damage. To this end, high-dose attenuated VSV-G pseudotyped equine infectious anaemia virus (EIAV) encoding β-galactosidase under the CMV promoter was injected into the fetal circulation of immuno-competent MF1 mice. We saw prolonged, extensive gene expression in the liver, heart, brain and muscle, and to a lesser extent in the kidney and lung of postnatal mice. Progressive clustered hepatocyte staining suggests clonal expansion of cells stably transduced. We thus provide proof of principle for efficient gene delivery and persistent transgene expression after prenatal application of the EIAV vector and its potential for permanent correction of genetic diseases.

Relative proximity of chromosome territories influences chromosome exchange partners in radiation-induced chromosome rearrangements in primary human bronchial epithelial cells

It is well established that chromosomes exist in discrete territories (CTs) in interphase and are positioned in a cell-type specific probabilistic manner. The relative localisation of individual CTs within cell nuclei remains poorly understood, yet many cancers are associated with specific chromosome rearrangements and there is good evidence that relative territorial position influences their frequency of exchange. To examine this further, we characterised the complexity of radiation-induced chromosome exchanges in normal human bronchial epithelial (NHBE) cells by M-FISH analysis of PCC spreads and correlated the exchanges induced with their preferred interphase position, as determined by 1/2-colour 2D-FISH analysis, at the time of irradiation. We found that the frequency and complexity of aberrations induced were reduced in ellipsoid NHBE cells in comparison to previous observations in spherical cells, consistent with aberration complexity being dependent upon the number and proximity of damaged CTs, i.e. lesion proximity. To ask if particular chromosome neighbourhoods could be identified we analysed all radiation-induced pair-wise exchanges using SCHIP (statistics for chromosome interphase positioning) and found that exchanges between chromosomes (1;13), (9;17), (9;18), (12;18) and (16;21) all occurred more often than expected assuming randomness. All of these pairs were also found to be either sharing similar preferred positions in interphase and/or sharing neighbouring territory boundaries. We also analysed a human small cell lung cancer cell line, DMS53, by M-FISH observing the genome to be highly rearranged, yet possessing rearrangements also involving chromosomes (1;13) and (9;17). Our findings show evidence for the occurrence of non-random exchanges that may reflect the territorial organisation of chromosomes in interphase at time of damage and highlight the importance of cellular geometry for the induction of aberrations of varying complexity after exposure to both low and high-LET radiation.

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.

Transduction of fetal mice with a feline lentiviral vector induces liver tumors which exhibit an E2F activation signature.

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.

Reduced chromosome aberration complexity in normal human bronchial epithelial cells exposed to low-LET γ-rays and high-LET α-particles.

Abstract Purpose: Cells of the lung are at risk from exposure to low and moderate doses of ionizing radiation from a range of environmental and medical sources. To help assess human health risks from such exposures, a better understanding of the frequency and types of chromosome aberration initially-induced in human lung cell types is required to link initial DNA damage and rearrangements with transmission potential and, to assess how this varies with radiation quality. Materials and methods: We exposed normal human bronchial lung epithelial (NHBE) cells in vitro to 0.5 and 1 Gy low-linear energy transfer (LET) γ-rays and a low fluence of high-LET α-particles and assayed for chromosome aberrations in premature chromosome condensation (PCC) spreads by 24-color multiplex-fluorescence in situ hybridization (M-FISH). Results: Both simple and complex aberrations were induced in a LET and dose-dependent manner; however, the frequency and complexity observed were reduced in comparison to that previously reported in spherical cell types after exposure to comparable doses or fluence of radiation. Approximately 1–2% of all exposed cells were categorized as being capable of transmitting radiation-induced chromosomal damage to future NHBE cell generations, irrespective of dose. Conclusion: One possible mechanistic explanation for this reduced complexity is the differing geometric organization of chromosome territories within ellipsoid nuclei compared to spherical nuclei. This study highlights the need to better understand the role of nuclear organization in the formation of exchange aberrations and, the influence three-dimensional (3D) tissue architecture may have on this in vivo.

Erratum: "Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice" (Molecular Therapy (2005) vol. 12 (763-771) 10.1016/j.ymthe.2005.07.358)

The authors regret that in Table 2 on page 768, one of the insertion sites of the SMART 2 provirus vector identified using LAM-PCR as present on chromosome 5 positioned 32374 bp upstream of Cyp3a11 was incorrectly assigned to Mouse (tumour) 2 T1. This insertion site should be assigned to an independent mouse not listed in Table 2. This animal had only a single provirus insertion found by Southern and LAM-PCR analyses and should be labeled as mouse 7.

Corrigendum to “Oncogenesis Following Delivery of a Nonprimate Lentiviral Gene Therapy Vector to Fetal and Neonatal Mice”

The authors regret that in Table 2 on page 768, one of the insertion sites of the SMART 2 provirus vector identified using LAM-PCR as present on chromosome 5 positioned 32374 bp upstream of Cyp3a11 was incorrectly assigned to Mouse (tumour) 2 T1. This insertion site should be assigned to an independent mouse not listed in Table 2. This animal had only a single provirus insertion found by Southern and LAM-PCR analyses and should be labeled as mouse 7.

827. Oncogenesis Following Delivery of Lentiviral Vectors 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 due to the ability of these vectors to integrate in a stable manner into the patients’ chromosomes. Since three cases of T-cell leukaemia have been identified after retrovirus gene therapy for X-linked severe combined immune deficiency as being associated with the integrating vector used for gene therapy the need for animal models to test for vector safety has become of paramount importance. Our previous work has shown that a high frequency of hepatocellular carcinomas has occurred following in utero and neonatal injection with certain lentivirus vectors. It has been hypothesized that the woodchuck post regulatory element (WPRE) carried by the vectors used in this study could be implicated in the tumour development process. Our recent study using novel vectors with mutations in the WPRE shows that mice treated with these vectors still develop liver tumours. In this report we discuss these findings and preliminary data to support an alternative cause for tumorigenesis. We also discuss the fetal and neonatal system as a novel and sensitive in vivo model to test the effects and safety of integrating vectors under consideration for clinical applications.