Maxine V. Holder

Highly efficient EIAV-mediated in utero gene transfer and expression in the major muscle groups affected by Duchenne muscular dystrophy

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 β-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.

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.

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.