Optogenetic Tractography for anatomo-functional characterization of cortico-subcortical neural circuits in non-human primates.

Dissecting neural circuitry in non-human primates (NHP) is crucial to identify potential neuromodulation anatomical targets for the treatment of pharmacoresistant neuropsychiatric diseases by electrical neuromodulation. How targets of deep brain stimulation (DBS) and cortical targets of transcranial magnetic stimulation (TMS) compare and might complement one another is an important question. Combining optogenetics and tractography may enable anatomo-functional characterization of large brain cortico-subcortical neural pathways. For the proof-of-concept this approach was used in the NHP brain to characterize the motor cortico-subthalamic pathway (m_CSP) which might be involved in DBS action mechanism in Parkinson's disease (PD). Rabies-G-pseudotyped and Rabies-G-VSVg-pseudotyped EIAV lentiviral vectors encoding the opsin ChR2 gene were stereotaxically injected into the subthalamic nucleus (STN) and were retrogradely transported to the layer of the motor cortex projecting to STN. A precise anatomical mapping of this pathway was then performed using histology-guided high angular resolution MRI tractography guiding accurately cortical photostimulation of m_CSP origins. Photoexcitation of m_CSP axon terminals or m_CSP cortical origins modified the spikes distribution for photosensitive STN neurons firing rate in non-equivalent ways. Optogenetic tractography might help design preclinical neuromodulation studies in NHP models of neuropsychiatric disease choosing the most appropriate target for the tested hypothesis.

Enhancing titres of therapeutic viral vectors using the transgene repression in vector production (TRiP) system.

A key challenge in the field of therapeutic viral vector/vaccine manufacturing is maximizing production. For most vector platforms, the 'benchmark' vector titres are achieved with inert reporter genes. However, expression of therapeutic transgenes can often adversely affect vector titres due to biological effects on cell metabolism and/or on the vector virion itself. Here, we exemplify the novel 'Transgene Repression In vector Production' (TRiP) system for the production of both RNA- and DNA-based viral vectors. The TRiP system utilizes a translational block of one or more transgenes by employing the bacterial tryptophan RNA-binding attenuation protein (TRAP), which binds its target RNA sequence close to the transgene initiation codon. We report enhancement of titres of lentiviral vectors expressing Cyclo-oxygenase-2 by 600-fold, and adenoviral vectors expressing the pro-apoptotic gene Bax by >150,000-fold. The TRiP system is transgene-independent and will be a particularly useful platform in the clinical development of viral vectors expressing problematic transgenes.

Development of inducible EIAV-based lentiviral vector packaging and producer cell lines.

Large-scale production of gene therapeutics comprising equine infectious anaemia virus (EIAV) -based lentiviral vectors (LVs) would benefit from the development of producer cell lines enabling the generation of larger quantities of vector than achievable by transient systems. Such cell lines would contain three vector components (Gag/Pol, VSV-G envelope and genome expression constructs). As the vesicular stomatitis virus (VSV-G) envelope protein is cytotoxic, its expression must be regulated. It is also desirable to regulate Gag/Pol expression to minimise metabolic burden on the cell. The Tet repressor (TetR) system was selected to regulate expression of VSV-G and Gag/Pol, necessitating the introduction of a fourth construct, encoding TetR, into the cell line. We have generated an inducible packaging cell line that shows tight control of the packaging components, and high-titre vector production on transient transfection of the EIAV genome. The cell line is stable for at least 7 weeks in the absence of selective pressure. To verify that this packaging cell line can support the generation of producer cell lines it was transfected stably with an EIAV genome cassette encoding ProSavin; a gene therapeutic for Parkinson's disease. Producer cell lines were generated, which on induction, yielded ProSavin with titres comparable to the transient system.

Analysis of factor VIII mediated suppression of lentiviral vector titres.

Effective gene therapy for haemophilia A necessitates a vector system that is not subject to a pre-existing immune response, has adequate coding capacity, gives long-term expression and preferably can target non-dividing cells. Vector systems based on lentiviruses such as equine infectious anaemia virus (EIAV) fulfil these criteria for the delivery of factor VIII (FVIII). We have found that B domain-deleted (BDD) FVIII protein inhibits functional viral particle production when co-expressed with the EIAV vector system. Although particle numbers (as measured by reverse transcriptase activity) are near normal, RNA genome levels are reduced and measurement of integrated copies revealed the virus is severely defective in its ability to transduce target cells. This is due to the absence of sufficient vesicular stomatitis virus glycoprotein (VSV-G) envelope on viral particles derived from cells expressing FVIII. By using an internal tissue-specific promoter, that has low activity in the producer cells, to drive expression of FVIII we have overcome this inhibitory effect allowing us to generate titres approaching those obtained with vector genomes encoding reporter genes. Furthermore, we report that codon optimization of the full-length FVIII gene increased vector titres approximately 10-fold in addition to substantially improving expression per integrated vector copy.

Oviduct-specific expression of two therapeutic proteins in transgenic hens.

Recent advances in avian transgenesis have led to the possibility of utilizing the laying hen as a production platform for the large-scale synthesis of pharmaceutical proteins. Ovalbumin constitutes more than half of the protein in the white of a laid egg, and expression of the ovalbumin gene is restricted to the tubular gland cells of the oviduct. Here we describe the use of lentiviral vectors to deliver transgene constructs comprising regulatory sequences from the ovalbumin gene designed to direct synthesis of associated therapeutic proteins to the oviduct. We report the generation of transgenic hens that synthesize functional recombinant pharmaceutical protein in a tightly regulated tissue-specific manner, without any evidence of transgene silencing after germ-line transmission.

In vivo evaluation of an EIAV vector for the systemic genetic delivery of therapeutic antibodies.

Lentiviral-based vectors hold great promise as gene delivery vehicles for the treatment of a wide variety of diseases. We have previously reported the development of a nonprimate lentiviral vector system based on the equine infectious anaemia virus (EIAV), which is able to efficiently transduce dividing and nondividing cells both in vitro and in vivo. Here, we report on the application of EIAV vectors for the systemic delivery of an antibody fusion protein designed for the treatment of cancer. The therapeutic potential of a single chain antibody against the tumour-associated antigen, 5T4, fused to immune enhancer moieties has been demonstrated in vitro and here we evaluate the genetic delivery of a 5T4 scFv fused to B7.1 (scFvB7) using an EIAV vector. The kinetics and concentration of protein produced following both intravenous (i.v.) and intramuscular (i.m.) administration was determined in immune competent adult mice. In addition, the immune response to the EIAV vector and the transgene were determined. Here, we show that a single injection of EIAV expressing scFv-B7 can give rise to concentrations of protein in the range of 1-5 microg/ml that persist in the sera for more than 50 days. After a second injection, concentrations of scFv-B7.1 rose as high as 20 microg/ml and levels greater than 2 microg/ml were present in the sera of all mice injected i.v. after 210 days despite the detection of antibodies against both the transgene and viral envelope for the duration of this study. These results demonstrate the potential of EIAV as a gene therapy vector for long-term production of therapeutic recombinant proteins.

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.

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.

Gene transduction efficiency in cells of different species by HIV and EIAV vectors.

The ability of human immunodeficiency virus (HIV)- and equine infectious anaemia virus (EIAV)-based vectors to transduce cell lines from a range of species was compared. Both vectors carried the vesicular stomatitis virus G (VSV-G) envelope protein and encoded an enhanced green fluorescent protein (eGFP) gene driven by a human cytomegalovirus (CMV) early promoter. Immunostaining for viral core proteins and VSV-G was used to demonstrate that the HIV and EIAV vector preparations contained similar numbers of virus particles. Various cell lines were transduced with these vectors and the transduction efficiency was estimated by measuring eGFP expression. Efficient transduction by both vectors was observed in human, hamster, pig, horse, cat and dog cell lines, although EIAV vector was about 10-fold less efficient in human, hamster and pig cells normalised to the total number of viral particles. This could be partly explained by the lower RNA genome levels per particle for EIAV as measured by real-time RT-PCR. Rodent cells appeared to be transduced inefficiently with both vectors, but when the CMV promoter was substituted with the EF1alpha promoter in the HIV vectors, the expression level increased leading to an increase in the measurable level of transduction.

Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery.

In this report it is demonstrated for the first time that rabies-G envelope of the rabies virus is sufficient to confer retrograde axonal transport to a heterologous virus/vector. After delivery of rabies-G pseudotyped equine infectious anaemia virus (EIAV) based vectors encoding a marker gene to the rat striatum, neurons in regions distal from but projecting to the injection site, such as the dopaminergic neurons of the substantia nigra pars compacta, become transduced. This retrograde transport to appropriate distal neurons was also demonstrated after delivery to substantia nigra, hippocampus and spinal cord and did not occur when vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped vectors were delivered to these sites. In addition, peripheral administration of rabies-G pseudotyped vectors to the rat gastrocnemius muscle leads to gene transfer in motoneurons of lumbar spinal cord. In contrast the same vector pseudotyped with VSV-G transduced muscle cells surrounding the injection site, but did not result in expression in any cells in the spinal cord. Long-term expression was observed after gene transfer in the nervous system and a minimal immune response which, together with the possibility of non-invasive administration, greatly extends the utility of lentiviral vectors for gene therapy of human neurological disease.

Growth factor enhanced retroviral gene transfer to the adult central nervous system.

The use of viral vectors for gene delivery into mammalian cells provides a new approach in the treatment of many human diseases. The first viral vector approved for human clinical trials was murine leukemia virus (MLV), which remains the most commonly used vector in clinical trials to date. However, the application of MLV vectors is limited since MLV requires cells to be actively dividing in order for transduction and therefore gene delivery to occur. This limitation precludes the use of MLV for delivering genes to the adult CNS, where very little cell division is occurring. However, we speculated that this inherent limitation of ML V may be overcome by utilizing the known mitogenic effect of growth factors on cells of the CNS. Specifically, an in vivo application of growth factor to the adult brain, if able to induce cell division, could enhance MLV-based gene transfer to the adult brain. We now show that an exogenous application of basic fibroblast growth factor induces cell division in vivo. Under these conditions, where cells of the adult brain are stimulated to divide, MLV-based gene transfer is significantly enhanced. This novel approach precludes any vector modifications and provides a simple and effective way of delivering genes to cells of the adult brain utilizing MLV-based retroviral vectors.

A Rev-independent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits a codon-optimized HIV-1 gag-pol gene.

The human immunodeficiency virus (HIV) genome is AU rich, and this imparts a codon bias that is quite different from the one used by human genes. The codon usage is particularly marked for the gag, pol, and env genes. Interestingly, the expression of these genes is dependent on the presence of the Rev/Rev-responsive element (RRE) regulatory system, even in contexts other than the HIV genome. The Rev dependency has been explained in part by the presence of RNA instability sequences residing in these coding regions. The requirement for Rev also places a limitation on the development of HIV-based vectors, because of the requirement to provide an accessory factor. We have now synthesized a complete codon-optimized HIV-1 gag-pol gene. We show that expression levels are high and that expression is Rev independent. This effect is due to an increase in the amount of gag-pol mRNA. Provision of the RRE in cis did not lower protein or RNA levels or stimulate a Rev response. Furthermore we have used this synthetic gag-pol gene to produce HIV vectors that now lack all of the accessory proteins. These vectors should now be safer than murine leukemia virus-based vectors.