RetinoStat(®) is an equine infectious anemia virus-based lentiviral gene therapy vector that expresses the angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the wet form of age-related macular degeneration. We initiated 6-month safety and biodistribution studies in two species; rhesus macaques and Dutch belted rabbits. After subretinal administration of RetinoStat the level of human endostatin and angiostatin proteins in the vitreous of treated rabbit eyes peaked at ∼1 month after dosing and remained elevated for the duration of the study. Regular ocular examinations revealed a mild to moderate transient ocular inflammation that resolved within 1 month of dosing in both species. There were no significant long-term changes in the electroretinograms or intraocular pressure measurements in either rabbits or macaques postdosing compared with the baseline reading in RetinoStat-treated eyes. Histological evaluation did not reveal any structural changes in the eye although there was an infiltration of mononuclear cells in the vitreous, retina, and choroid. No antibodies to any of the RetinoStat vector components or the transgenes could be detected in the serum from either species, and biodistribution analysis demonstrated that the RetinoStat vector was maintained within the ocular compartment. In summary, these studies found RetinoStat to be well tolerated, localized, and capable of persistent expression after subretinal delivery.

ProSavin is an equine infectious anemia virus vector-based gene therapy for Parkinson's disease for which inducible HEK293T-based producer cell lines (PCLs) have been developed. These cell lines demonstrate stringent tetracycline-regulated expression of the packaging components and yield titers comparable to the established transient production system. A prerequisite for the use of PCL-derived lentiviral vectors (LVs) in clinical applications is the thorough characterization of both the LV and respective PCL with regard to identity and genetic stability. We describe the detailed characterization of two ProSavin PCLs (PS5.8 and PS46.2) and resultant ProSavin vector. The two cell lines demonstrate stable production of vector over a time period sufficient to allow generation of master and working cell banks, and subsequent large-scale vector production. ProSavin generated from the PCLs performs comparably in vivo to that produced by the standard transient transfection process with respect to transduction efficiency and immunogenicity. The development of ProSavin PCLs, and the detailed characterization described here, will aid the advancement of ProSavin for clinical application.

Top of pageAbstract Lentiviral vectors hold great promise for the treatment of chronic diseases. Vectors based on the equine infectious anemia virus EIAV have shown long-term stable correction in models for Parkinson's disease, spinal muscular atrophy, spinal injury, motor neuron disease and diabetes insipidus. They have also been used to create transgenic chickens and pigs where gene expression is stable over several generations. A notable feature in all of these models is the long term expression and the long term safety (see references below). We were concerned to learn of a study where a very early generation EIAV vector (SMART2 with the pONY3.1 packaging system) had been injected in utero in mice and the resulting pups had developed liver tumours (http://www.advisorybodies.doh.gov.uk/genetics/gtac/lentiviruses-1104.pdf). We compared the properties and vector genome structure with other vectors, in particular HIV vectors, which had not had any side effects or tumours reported. In general, transduction efficiencies reported in mice with HIV vectors are lower than with EIAV, possibly due to the known host cell restriction. One hypothesis was therefore that the EIAV vectors had a higher number of integrations per cell providing an increased opportunity for insertional mutagenesis. There is nothing unusual about the integration properties of EIAV vectors. The findings to date show a preference for active regions of the chromosome but no particular bias to a location or to positions relative to a transcription unit. Furthermore some tumours had only one or two integrations. At present we believe that a role for insertional mutagenesis alone seems unlikely. The SMART series of EIAV vectors contain an open reading frame in the WPRE that could in theory express a truncated form of the oncogenic X protein from the woodchuck hepatitis virus. This could potentially act as a weak oncogene (Kingsman et al 2005), particularly in the rapidly proliferating fetal tissues. Certain vector integrations could contribute to the promotion of the tumour and so it remains to be seen whether this truncated protein is either necessary and/or sufficient to cause tumours in this model. We will present data on the integration characteristics of EIAV in cell lines and in transduced livers and liver tumours from the in utero studies. Our data lead us to favour the hypothesis that the X-like protein acts as an oncogene in this model. Clinical EIAV vectors do not express this protein and lack all extraneous viral sequences. Further studies are required to test all of these hypotheses in order to inform the further development of these vectors for in utero gene therapy.

The use of lentiviral vectors for gene transfer into hematopoietic stem cells has raised considerable interest as these vectors can permanently integrate their genome into quiescent cells. Vectors based on alternative lentiviruses would theoretically be safer than HIV-1-based vectors and could also be used in HIV-positive patients, minimizing the risk of generating replication-competent virus. Here we report the use of third-generation equine infectious anemia virus (EIAV)- and HIV-1-based vectors with minimal viral sequences and absence of accessory proteins. We have compared their efficiency in transducing mouse and human hematopoietic stem cells both in vitro and in vivo to that of a previously documented second-generation HIV-1 vector. The third-generation EIAV- and HIV-based vectors gave comparable levels of transduction and transgene expression in both mouse and human NOD/SCID repopulating cells but were less efficient than the second-generation HIV-1 vector in human HSCs. For the EIAV vector this is possibly a reflection of the lower protein expression levels achieved in human cells, as vector copy number analysis revealed that this vector exhibited a trend to integrate equally efficiently compared to the third-generation HIV-1 vector in both mouse and human HSCs. Interestingly, the presence or absence of Tat in viral preparations did not influence the transduction efficiency of HIV-1 vectors in human HSCs.

Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination.

The ability of lentiviral-based vectors to integrate into non-dividing cells such as neuronal cells has been exploited to correct a number of disease models. Using vectors based on the equine infectious anaemia virus (EIAV) we have previously reported the correction of an animal model of Parkinson's disease (Azzouz et al. J Neurosci. 2002 Dec 1;22(23):10302–12). To use such vectors in the clinic each manufactured lot must be tested and demonstrated to be free of replication competent lentivirus (RCL). We have designed the vectors in such a way to limit the possibility of homologous recombination taking place; the vector genome contains only 824 nt of EIAV sequence with no coding regions; all the accessory genes have been removed entirely from the system; the gag/pol sequence has been codon optimised and a heterologous envelope is used (VSV-G). However, there is still the formal possibility of some form of recombination leading to the generation of an RCL. We have therefore developed an RCL assay based on the property common to all members of the Retroviradae family, namely the presence of reverse transcriptase (RT). RT activity can be detected by a sensitive assay: Product Enhanced Reverse Transcriptase (PERT). The test article is passaged on human cells to amplify any replicating entity and then a PERT assay is carried out on the supernatant. A typical set of assay results will be presented.