M Nivsarkar

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.

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.

Gene therapy progress and prospects: fetal gene therapy--first proofs of concept--some adverse effects.

Somatic gene delivery in utero is a novel approach to gene therapy for genetic disease based on the hypothesis that prenatal intervention may avoid the development of severe manifestations of early-onset disease, allow targeting of otherwise inaccessible tissues including expanding stem cell populations, induce tolerance against the therapeutic transgenic protein and thereby provide permanent somatic gene correction. This approach is particularly relevant in relation to prenatal screening programmes for severe genetic diseases as it could offer prevention as a third option to families faced with the prenatal diagnosis of a genetically affected child. Most investigations towards in utero gene therapy have been performed on mice and sheep fetuses as model animals for human disease and for the application of clinically relevant intervention techniques such as vector delivery by minimally invasive ultrasound guidance. Other animals such as dogs may serve as particular disease models and primates have to be considered in immediate preparation for clinical trials. Proof of principle for the hypothesis of fetal gene therapy has been provided during the last 2 years in mouse models for Crigler Najjar Disease, Lebers congenital amaurosis, Pompes disease and haemophilia B showing long-term postnatal therapeutic effects and tolerance of the transgenic protein after in utero gene delivery. However, recently we have also observed a high incidence of liver tumours after in utero application of an early form of third-generation equine infectious anaemia virus vectors with SIN configuration. These findings highlight the need for more investigations into the safety and the ethical aspects of in utero gene therapy as well as for science-based public information on risks and benefits of this preventive gene therapy approach before application in humans can be contemplated.

The hopes and fears of in utero gene therapy for genetic disease--a review.

Somatic gene delivery in utero is a novel approach to gene therapy for genetic disease. It is based on the concept that application of gene therapy vectors to the fetus in utero may prevent the development of early disease related tissue damage, may allow targeting of otherwise inaccessible organs, tissues and still expanding stem cell populations and may also provide postnatal tolerance against the therapeutic transgenic protein. This review outlines the hypothesis and scientific background of in utero gene therapy and addresses some of the frequently expressed concerns raised by this still experimental, potentially preventive gene therapy approach. We describe and discuss the choice of vectors, of animal models and routes of administration to the fetus. We address potential risk factors of prenatal gene therapy such as vector toxicity, inadvertent germ line modification, developmental aberration and oncogenesis as well as specific risks of this procedure for the fetus and mother and discuss their ethical implications.

Evidence for Contribution of CD4+CD25+ Regulatory T Cells in Maintaining Immune Tolerance to Human Factor IX following Perinatal Adenovirus Vector Delivery

Following fetal or neonatal gene transfer in mice and other species immune tolerance of the transgenic protein is frequently observed; however the underlying mechanisms remain largely undefined. In this study fetal and neonatal BALB/c mice received adenovirus vector to deliver human factor IX (hFIX) cDNA. The long-term tolerance of hFIX was robust in the face of immune challenge with hFIX protein and adjuvant but was eliminated by simultaneous administration of anti-CD25+ antibody. Naive irradiated BALB/c mice which had received lymphocytes from donors immunised with hFIX developed anti-hFIX antibodies upon immune challenge. Cotransplantation with CD4+CD25+ cells isolated from neonatally tolerized donors decreased the antibody response. In contrast, cotransplantation with CD4+CD25− cells isolated from the same donors increased the antibody response. These data provide evidence that immune tolerance following perinatal gene transfer is maintained by a CD4+CD25+ regulatory population.

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.

238. Stem Cell Gene Delivery to Immunocompetent Mice Using a Non-Myeloablative Regimen Results in Long-Term Transgene Expression and Stable Mixed Chimerism

Stem cell gene delivery offers a potential cure for blood related disorders such as haemophilia B and degenerative diseases. We have shown permanent phenotypic correction of a murine model of haemophilia B for over a year by lentiviral delivery of the human factor IX gene to haematopoietic stem cells using a myeloablative conditioning regimen. In addition we were able to show full haematopoietic chimerism and robust tolerance, even after challenge with factor IX.

Towards fetal gene therapy for cystic fibrosis: ultrasound guided delivery of recombinant adenoviral vectors to the fetal sheep trachea results in efficient marker gene expression in the airway epithelia

Fetal gene therapy for cystic fibrosis may have advantages over adult treatment. We aimed (1) to develop a percutaneous ultrasound guided technique to inject adenoviral vectors into the fetal sheep trachea and (2) to improve transgene expression with transduction enhancing agents. Adenoviral vectors containing the β-galactosidase gene (2.0×1011–8.3×1011 particles/kg) and transduction enhancing agents were delivered to the trachea via a needle inserted through the thorax of late (n = 3) or mid gestation (n = 15) fetal sheep using ultrasound guidance. Tissues were analysed 48 hours after injection (n = 17) or 12 hours after birth (n = 1) for transgene expression. Transthoracic injection of the trachea was successful in 16 fetuses with 100% survival. Expression of β-galactosidase, as measured by ELISA, was low after delivery of adenoviral vector alone, but increased 10-fold when the vector was complexed with the polycation DEAE dextran. Pretreatment of the fetal airways with sodium caprate, which opens tight junctions to reach the basolateral surface of lung epithelia, resulted in a 90-fold increase in expression. A synergistic effect of the two agents resulted in widespread staining of the trachea, main bronchi and all airways, confirmed by immunohistochemistry for β-galactosidase. Instillation of perflubron following sodium caprate and vector/DEAE dextran complex injection increased transduction of the peripheral small airways at the expense of tracheal and large airways transduction.