Systemic delivery of scAAV9 in fetal macaques facilitates neuronal transduction of the central and peripheral nervous systems.

Correction of perinatally lethal neurogenetic diseases requires efficient transduction of several cell types within the relatively inaccessible CNS. Intravenous AAV9 delivery in mouse has achieved development stage-specific transduction of neuronal cell types, with superior neuron-targeting efficiency demonstrated in prenatal compared with postnatal recipients. Because of the clinical relevance of the non-human primate (NHP) model, we investigated the ability of AAV9 to transduce the NHP CNS following intrauterine gene therapy (IUGT). We injected two macaque fetuses at 0.9 G with 1 × 10(13) vg scAAV9-CMV-eGFP through the intrahepatic continuation of the umbilical vein. Robust green fluorescent protein (GFP) expression was observed for up to 14 weeks in the majority of neurons (including nestin-positive cells), motor neurons and oligodendrocytes throughout the CNS, with a significantly lower rate of transduction in astrocytes. Photoreceptors and neuronal cell bodies in the plexiform and ganglionic retinal layers were also transduced. In the peripheral nervous system (PNS), widespread transduction of neurons was observed. Tissues harvested at 14 weeks showed substantially lower vector copy number and GFP levels, although the percentage of GFP-expressing cells remained stable. Thus, AAV9-IUGT in late gestation efficiently transduces both the CNS and PNS with neuronal predilection, of translational relevance to hereditary disorders characterized by perinatal onset of neuropathology.

In utero administration of Ad5 and AAV pseudotypes to the fetal brain leads to efficient, widespread and long-term gene expression.

The efficient delivery of genetic material to the developing fetal brain represents a powerful research tool and a means to supply therapy in a number of neonatal lethal neurological disorders. In this study, we have delivered vectors based upon adenovirus serotype 5 (Ad5) and adeno-associated virus (AAV) pseudotypes 2/5, 2/8 and 2/9 expressing green fluorescent protein to the E16 fetal mouse brain. One month post injection, widespread caudal to rostral transduction of neural cells was observed. In discrete areas of the brain these vectors produced differential transduction patterns. AAV2/8 and 2/9 produced the most extensive gene delivery and had similar transduction profiles. All AAV pseudotypes preferentially transduced neurons whereas Ad5 transduced both neurons and glial cells. None of the vectors elicited any significant microglia-mediated immune response when compared with control uninjected mice. Whole-body imaging and immunohistological evaluation of brains 9 months post injection revealed long-term expression using these non-integrating vectors. These data will be useful in targeting genetic material to discrete or widespread areas of the fetal brain with the purpose of devising therapies for early neonatal lethal neurodegenerative disease and for studying brain development.

Efficient gene delivery to the adult and fetal CNS using pseudotyped non-integrating lentiviral vectors.

Non-integrating lentiviral vectors show considerable promise for gene therapy applications as they persist as long-term episomes in non-dividing cells and diminish risks of insertional mutagenesis. In this study, non-integrating lentiviral vectors were evaluated for their use in the adult and fetal central nervous system of rodents. Vectors differentially pseudotyped with vesicular stomatitis virus, rabies and baculoviral envelope proteins allowed targeting of varied cell populations. Efficient gene delivery to discrete areas of the brain and spinal cord was observed following stereotactic administration. Furthermore, after direct in utero administration (E14), sustained and strong expression was observed 4 months into adulthood. Quantification of transduction and viral copy number was comparable when using non-integrating lentivirus and conventional integrating vector. These data support the use of non-integrating lentiviral vectors as an effective alternative to their integrating counterparts in gene therapy applications, and highlight their potential for treatment of inherited and acquired neurological disorders.

Local delivery of VEGF adenovirus to the uterine artery increases vasorelaxation and uterine blood flow in the pregnant sheep.

Impaired materno-placental perfusion causes two important obstetric complications, fetal growth restriction and preeclampsia. This study investigated whether adenoviral vector-mediated overexpression of vascular endothelial growth factor (VEGF) in the uterine arteries (UtAs) increases uterine artery blood flow (UBF). First-generation adenovirus vectors (5 x 10(11) particles) containing the VEGF gene (Ad.VEGF-A or -D) or the beta-galactosidase reporter gene (Ad.lacZ) were injected into the UtAs of pregnant sheep (n=6) at 88-102 days of gestation (term=145 days). UBF was measured using Doppler sonography before, and 4-7 days after injection. Mean UBF increased significantly from 233+/-156 (s.d.) ml min(-1) to 753+/-415 ml min(-1) following Ad.VEGF-A injection (P=0.005, n=5); Ad.lacZ infection had no significant effect. Organ bath experiments on uterine arterial sections 4-7 days after injection showed that, compared with Ad.lacZ vessels, Ad.VEGF-A-transduced vessels had a reduced contractile response to phenylephrine (E max 148+/-10.9 vs E max 228.2+/-27.5, P<0.05) but increased relaxation with bradykinin (pD2 (-log EC50) values 9.11+/-0.01 vs 8.65+/-0.11, P<0.05). Injection of Ad.VEGF-A into the UtAs increases UBF by enhancing vasodilatation. This may provide the basis for therapy in pregnancies complicated by uteroplacental insufficiency.

Lentiviral transduction of the murine lung provides efficient pseudotype and developmental stage-dependent cell-specific transgene expression.

Gene transfer for cystic fibrosis (CF) airway disease has been hampered by the lung's innate refractivity to pathogen infection. We hypothesized that early intervention with an integrating gene transfer vector capable of transducing the lung via the lumen may be a successful therapeutic approach. An HIV-based lentiviral vector pseudotyped with the baculovirus gp64 envelope was applied to the fetal, neonatal or adult airways. Fetal intra-amniotic administration resulted in transduction of approximately 14% of airway epithelial cells, including both ciliated and non-ciliated epithelia of the upper, mid and lower airways; there was negligible alveolar or nasal transduction. Following neonatal intra-nasal administration we observed significant transduction of the airway epithelium (approximately 11%), although mainly in the distal lung, and substantial alveolar transduction. This expression was still detectable at 1 year after application. In the adult, the majority of transduction was restricted to the alveoli. In contrast, vesicular stomatitis virus glycoprotein pseudotyped virus transduced only alveoli after adult and neonatal application and no transduction was observed after fetal administration. Repeat administration did not increase transduction levels of the conducting airway epithelia. These data demonstrate that application at early developmental stages in conjunction with an appropriately pseudotyped virus provides efficient, high-level transgene expression in the murine lung. This may provide a modality for treatment for lung disease in CF.

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, Leber's congenital amaurosis, Pompe's 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.

Factors influencing adenovirus-mediated airway transduction in fetal mice.

Intra-amniotic injection of adenovirus allows transduction of the fetal airways following natural fetal breathing movements. This administration method is promising for use in gene therapy for cystic fibrosis and other diseases for which the main target for exogenous gene expression is the lung. Here we have investigated factors that may affect the efficacy of gene transfer to the murine fetal lung. We examined marker compound distribution and transgene expression (from a first-generation adenoviral vector) at different stages of development. This demonstrated that fetal breathing movements at 15-16 days of gestation are of sufficient intensity to carry marker/vector into the fetal lungs. These movements can be significantly stimulated by the combination of intra-amniotic theophylline administration and postoperative exposure of the dam to elevated CO(2) levels. However, the most important factor for efficient and consistent pulmonary transgene delivery is the dose of adenoviral vector used, as both the degree of transduction and the percentage of lungs transduced increases with escalating viral dose.

Fetal and neonatal gene therapy: benefits and pitfalls.

The current approaches to gene therapy of monogenetic diseases into mature organisms are confronted with several problems including the following: (1) the underlying genetic defect may have already caused irreversible pathological changes; (2) the level of sufficient protein expression to ameliorate or prevent the disease requires prohibitively large amounts of gene delivery vector; (3) adult tissues may be poorly infected by conventional vector systems dependent upon cellular proliferation for optimal infection, for example, oncoretrovirus vectors; (4) immune responses, either pre-existing or developing following vector delivery, may rapidly eliminate transgenic protein expression and prevent future effective intervention. Early gene transfer, in the neonatal or even fetal period, may overcome some or all of these obstacles. The mammalian fetus enjoys a uniquely protected environment in the womb, bathed in a biochemically and physically supportive fluid devoid of myriad extra-uterine pathogens. Strong physical and chemical barriers to infection might, perhaps, impede the frenetic cell division. The physical support and the biochemical support provided by the fetal-maternal placental interface may, therefore, minimize the onset of genetic diseases manifest early in life. The fetal organism must prepare itself for birth, but lacking a mature adaptive immune system may depend upon more primordial immune defences. It is the nature of these defences, and the vulnerabilities they protect, that are poorly understood in the context of gene therapy and might provide useful information for approaches to gene therapy in the young, as well as perhaps the mature organism.

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 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.

Reduced toxicity of F-deficient Sendai virus vector in the mouse fetus.

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.

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 beta-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.

Optimization of gene transfer into primitive human hematopoietic cells of granulocyte-colony stimulating factor-mobilized peripheral blood using low-dose cytokines and comparison of a gibbon ape leukemia virus versus an RD114-pseudotyped retroviral vector.

Primitive human hematopoietic cells in granulocyte-colony stimulating factor (G-CSF)-mobilized peripheral blood (MPB) are more difficult to transduce compared to cells from umbilical cord blood. Based on the hypothesis that MPB cells may require different stimulation for efficient retroviral infection, we compared several culture conditions known to induce cycling of primitive hematopoietic cells. MPB-derived CD34(+) cells were stimulated in the presence or absence of the murine fetal liver cell line AFT024 in trans-wells with G-CSF, stem cell factor (SCF), and thrombopoietin (TPO) (G/S/T; 100 ng/ml) or Flt3-L, SCF, interleukin (IL)-7, and TPO (F/S/7/T; 10-20 ng/ml), and transduced using a GaLV-pseudotyped retroviral vector expressing the enhanced green fluorescence protein (eGFP). Compared to cultures without stroma, the presence of AFT024 increased the number of transduced colony-forming cells (CFC) by 3.5-fold (with G/S/T), long-term culture-initiating cells (LTC-IC) by 4.6-fold (with F/S/7/T), and nonobese diabetic/severe immunodeficiency disease (NOD/SCID)-repopulating cells (SRC) by 6.8-fold (with F/S/7/T). Similar numbers of long-term culture-initiating cells (LTC-IC) and SRC could be transduced using AFT024-conditioned medium (AFT-CM) or a defined medium that had been supplemented with factors identified in AFT-CM. Finally, using our best condition based on transduction with the gibbon ape leukemia virus (GaLV)-pseudotyped vector, we demonstrate a 33-fold higher level of gene transfer (p < 0.001) in SRC using an RD114-pseudotyped vector. In summary, using an optimized protocol with low doses of cytokines, and transduction with an RD114 compared to a GaLV-pseudotyped retroviral vector, the overall number of transduced cells in NOD/SCID mice could be improved 144-fold, with a gene-transfer efficiency in SRC of 16.3% (13.3-19.9; n = 6).

Detection of ventilatory threshold using near infrared spectroscopy in men and women.

The onset of anaerobic (lactate) metabolism during incremental exercise, which may be a result of an imbalance between tissue oxygen supply and demand, has been associated with the gas exchange ventilatory threshold (VT). This study was designed to examine whether near infrared spectroscopy (NIRS) could be used to detect the VT in healthy subjects. Twenty-one men and 19 women completed incremental cycle ergometry during which NIRS measurements were obtained from the right vastus lateralis and gas exchange measurements were monitored simultaneously using a metabolic cart. The VT was identified from the metabolic data by the V-slope method and from NIRS data as the intensity at which tissue absorbency crossed the resting baseline value observed immediately prior to the initiation of exercise. Pearson correlations for the relative oxygen uptake and power output observed for the two methods of detecting VT were 0.90 and 0.88, respectively, in men and 0.89 and 0.86, respectively, in women (P < 0.01). No significant differences were observed between the two methods of detecting VT for any of the physiological responses (P > 0.05). No significant (P > 0.05) gender differences were observed in muscle oxygenation values at the VT, 32% in men and 38% in women. These results validate the use of NIRS as an alternate noninvasive method for detecting VT during cycle exercise in healthy subjects.

Falls from moving motor vehicles in New Zealand.

Injuries due to falls from moving motor vehicles have received relatively little attention from the research community. Injury events of this type in New Zealand were examined using national injury mortality and hospitalisation data from the New Zealand National Health Statistics Centre (NHSC). Also used were data obtained from the New Zealand Post Motor Registration Centre and from coroner's investigation reports held by the Department of Justice. Fifty-six fatal falls from moving motor vehicles occurred during the period 1977-1986 (0.18 per 100,000 population per year). The average age of fatalities was 23. The total potential years of life lost due to these fatalities was 2,696, or an average of 48 years per person. Thirty-nine persons (70%) fell while riding on the exterior of a vehicle. None of the 56 fatalities was using a belt restraint when he/she fell. Four hundred and twenty-three admissions to hospital occurred during 1986 and 1987 (6.5 per 100,000 persons per year). The average age of those hospitalised was 18. Incidence rates were highest in the 0-4, 15-19, and 20-24 year age groups. In the case of both deaths and hospitalisations, the incidence rate for males was approximately double the rate for females. In addition, the rate of falls (per unit registered motor vehicle) from trucks was significantly higher than the rate of falls from cars. Means of preventing falls from motor vehicles are discussed.

Injuries due to falls from horses.

This study describes the epidemiology of injuries due to falls from horses in New Zealand. There were 54 fatalities from 1977 to 1986 (0.17 per 100,000 persons per year). There were 773 hospitalisations in 1987 (23.7 per 100,000 persons per year). Head injuries were predominant among both fatal and nonfatal injuries. The incidence of nonfatal head injury in the 10 to 19 age group was significantly higher than the incidence in all older groups (P = 0.003). Young people, particularly females, were the segment of the population most affected by the problem of falls from horses. Reference to data on horse-riding participation rates, however, did not indicate that young people were overrepresented in the series studied. Reference to the same data showed that the rate of hospitalisation due to falls from horses is comparable to the rate for injuries from playing rugby. The magnitude and severity of the problem indicates that there is a need need for helmet use, safe-riding practices, and further research.

Genetic relatedness of the Kemerovo serogroup viruses: I. RNA-RNA blot hybridization and gene reassortment in vitro of the Kemerovo serocomplex.

The dsRNA profiles of the Czechoslovakian and Siberian serotypes of the Kemerovo serocomplex viruses examined were similar in agarose, while their dsRNA profiles were distinct in polyacrylamide gel. Blot hybridization studies of the Kemerovo serocomplex viruses demonstrated that the genes were highly conserved among the members within each type, but not between types. Gene reassortment in vitro was demonstrated among selected pairs of the Kemerovo serocomplex viruses by intra- and inter-typic crosses. The majority of the reassortant progeny from inter-typic crosses were single gene replacements, whereas the majority of the reassortant progeny from intra-typic crosses were multiple gene replacements suggesting that certain gene combinations were restrictive under conditions of the experiment.

Arthropod studies with rabies-related Mokola virus.

A cell culture-adapted variant of the rabies-related Mokola virus was demonstrated to replicate in inoculated Aedes aegypti mosquitoes. Replication was slow compared to many arboviruses in their vectors. Maximum titers were not obtained until after approximately 6 weeks of extrinsic incubation. Mokola virus underwent nine mosquito-mosquito passages at approximately monthly intervals and was thus maintained in insects for 340 days before terminating the study. Virus antigen was detected by immunofluorescence in a variety of mosquito tissues and organs, including salivary glands, but primarily in nervous tissue. Irrefutable virus transmission by bite could not be demonstrated because of equivocal results. Transovarial passage of virus was observed in the mosquito. Viremia in baby mice was demonstrable. Ornithodoros moubata nymphal ticks were exposed to viremic mice but failed to become infected.