Equine Infectious Anemia Virus Vectors Research Articles

The status and progress in gene therapy study of Stargardt disease

Stargardt disease (STGD) is an inherited disorder of retinal pigment epithelium. Three genes have been found to be implicated in STGD including Abca4 (adenosine triphosphate-binding cassette, sub-family A, member 4), Elovl4 (elongation of very long chain fatty acids protein 4) and Prom1 (prominin-1). Target genes can be delivered to the retina by various methods such as lentivirus (LV) vectors, adeno-associated virus (AAV) vectors and non-viral nano-particles. The Abca4-/-, Elovl4-/- and Prom1-/- mice model are used to study the pathogenesis mechanism and treatment of STGD. Retinal function improved significantly upon gene therapy in these models. Based on these works using animal model, phase Ⅰ/Ⅱa clinical trial of Abca4-associated STGD gene therapy are underway. As a LV vector, equine infectious anemia virus (EIAV) is used to carry the Abca4 gene. These studies will evaluate three dose levels of the EIAV vector for safety, tolerability and biological activity. Moreover, some preclinical attempts to deliver Abca4 via AAV have been made using a modified AAV vectors because of the large size of the ABCA4 cDNA. The good responses in animal models render STGD a very attractive object for human gene therapy after the successful of the phase Ⅰ/Ⅱ clinical trials of Leber's congenital amaurosis. Key words: Macular Degenerationcongenital/congenital; Gene therapy; Disease models, animal; Review

Suppression of Neovascularization of Donor Corneas by Transduction with Equine Infectious Anemia Virus-Based Lentiviral Vectors Expressing Endostatin and Angiostatin

Corneal transplantation is the oldest and one of the most successful transplant procedures with a success rate in many studies in excess of 90%. The high success rate is mainly attributable to the relatively immune-privileged status of the eye and the fact that the cornea is largely avascular. However, the success rate in patients with failed grafts is much lower such that regrafting is frequently the top indication for corneal transplantation in many centers. Neovascularization is the most important risk factor for rejection, as it allows access of the immune system to the donor tissue, compromising immune privilege of the graft/eye. We have developed a process to modify donor corneal tissue to prevent rejection by a single exposure to a gene therapy vector before surgery (EncorStat(®)). The vector used is based on clinically relevant equine infectious anemia virus (EIAV)-derived lentiviral platform and contains genes for two potently angiostatic genes, endostatin and angiostatin. We show that incubation of rabbit, primate, and human corneal tissue with the EIAV vector mediates strong, stable expression in the corneal endothelium. We have optimized this process to maximize transduction and, once this is complete, maximize the removal of free vector before transplant. Rabbit corneas treated with two different antiangiogenic expression vectors (EIAV-EndoAngio and to a lesser extent EIAV-Endo:k5) significantly suppressed neovascularization in a rabbit model of corneal rejection. As a result, corneal opacity, edema, and inflammatory infiltrates were reduced in these corneas. This study demonstrates that angiogenesis is a suitable target to prevent corneal rejection, and provides the first proof-of-concept data for the development of EncorStat, an ex vivo gene therapy treatment to prevent corneal rejection.

Transduction of Photoreceptors With Equine Infectious Anemia Virus Lentiviral Vectors: Safety and Biodistribution of StarGen for Stargardt Disease

StarGen is an equine infectious anemia virus (EIAV)-based lentiviral vector that expresses the photoreceptor-specific adenosine triphosphate (ATP)-binding cassette transporter (ABCA4) protein that is mutated in Stargardt disease (STGD1), a juvenile macular dystrophy. EIAV vectors are able to efficiently transduce rod and cone photoreceptors in addition to retinal pigment epithelium in the adult macaque and rabbit retina following subretinal delivery. The safety and biodistribution of StarGen following subretinal delivery in macaques and rabbits was assessed. Regular ophthalmic examinations, IOP measurements, ERG responses, and histopathology were carried out in both species to compare control and vector-treated eyes. Tissue and fluid samples were obtained to evaluate the persistence, biodistribution, and shedding of the vector following subretinal delivery. Ophthalmic examinations revealed a slightly higher level of inflammation in StarGen compared with control treated eyes in both species. However, inflammation was transient and no overt toxicity was observed in StarGen treated eyes and there were no abnormal clinical findings. There was no StarGen-associated rise in IOP or abnormal ERG response in either rabbits or macaques. Histopathologic examination of the eyes did not reveal any detrimental changes resulting from subretinal administration of StarGen. Although antibodies to StarGen vector components were detected in rabbit but not macaque serum, this immunologic response did not result in any long-term toxicity. Biodistribution analysis demonstrated that the StarGen vector was restricted to the ocular compartment. In summary, these studies demonstrate StarGen to be well tolerated and localized following subretinal administration.

Assessment of Integration-defective HIV-1 and EIAV Vectors In Vitro and In Vivo

The interest in integrase-defective lentiviral vectors (IDLVs) stems from their potential advantage of large cloning capacity and broad cell tropism while avoiding the possibility of insertional mutagenesis. Here, we directly compared the transducing potential of IDLVs based on the equine infectious anemia virus (EIAV) to the more commonly described HIV-1 IDLVs. IDLVs were constructed by introducing equivalent single/triple mutations into the integrase catalytic triad. We show that both the single and the triple mutant HIV-1 IDLVs transduce the PC12 cells, but not the C2C12 cells, with similar efficiency to their parental HIV-1 vector. In contrast, the single and triple EIAV IDLVs did not efficiently transduce either differentiated cell line. Moreover, this HIV-1 IDLV-mediated expression was independent of any residual integration activity because reporter expression was lost when cell cycling was restored. Four weeks following stereotactic administration into adult rat brains, only the single HIV-1 IDLV mutant displayed a comparable transduction profile to the parental HIV-1 vector. In contrast, neither EIAV IDLV mutants showed significant reporter gene expression. This work indicates that the transducing potential of IDLVs appears to depend not only on the choice of integrase mutation and type of target cell, but also on the nature of the lentiviral vector.

Equine Infectious Anemia Viral Vector-Mediated Codelivery of Endostatin and Angiostatin Driven by Retinal Pigmented Epithelium-Specific VMD2 Promoter Inhibits Choroidal Neovascularization

Equine infectious anemia virus (EIAV) is a nonprimate lentivirus that does not cause human disease. Subretinal injection into mice of a recombinant EIAV lentiviral vector in which lacZ is driven by a CMV promoter (EIAV CMV LacZ) resulted in rapid and strong expression of LacZ in retinal pigmented epithelial (RPE) cells and some other cells including ganglion cells, resulting in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside within the optic nerve. Substitution of the RPE-specific promoter from the vitelliform macular dystrophy (VMD2) gene for the CMV promoter resulted in prolonged (at least 1 year) expression of LacZ that was restricted to RPE cells, albeit reduced 6- to 10-fold compared with the CMV promoter. Similarly, the amount of FLAG-tagged endostatin detected in eyes injected with the EIAV VMD2 Endo(FLAG) vector was similar to that seen in eyes injected with a vector that expressed both endostatin and angiostatin [EIAV VMD2 Endo(FLAG)/Angio]; expression was approximately 6-fold lower than with identical vectors in which the CMV promoter drove expression. Compared with murine eyes treated with a control EIAV vector, subretinal injection of EIAV vectors expressing murine endostatin alone or in combination with angiostatin driven by either the CMV or VMD2 promoter caused significant suppression of choroidal neovascularization (NV) at laser-induced rupture sites in Bruch's membrane. These data support proceeding toward clinical studies with EIAV-based gene therapy for choroidal NV, using the VMD2 promoter to selectively drive expression of a combination of endostatin and angiostatin in RPE cells.

Correction of the disease phenotype in the mouse model of Stargardt disease by lentiviral gene therapy

Autosomal recessive Stargardt disease (STGD1) is a macular dystrophy caused by mutations in the ABCA4 (ABCR) gene. The disease phenotype that is most recognized in STGD1 patients, and also in the Abca4-/- mouse (a disease model), is lipofuscin accumulation in retinal pigment epithelium. Here, we tested whether delivery of the normal (wt) human ABCA4 gene to the subretinal space of the Abca4 -/- mice via lentiviral vectors would correct the disease phenotype; that is, reduce accumulation of the lipofuscin pigment A2E. Equine infectious anemia virus (EIAV)-derived lentiviral vectors were constructed expressing either the human ABCA4 gene or the LacZ reporter gene under the control of the constitutive (CMV) or photoreceptor-specific (Rho) promoters. Abca4-/- mice were injected subretinally with 1 microl ( approximately 5.0 x 10(5) TU) of each EIAV vector in one eye at postnatal days 4 and 5. An injection of saline, an EIAV-null vector, or an uninjected contralateral eye served as a control. Mice were killed at various times after injection to determine photoreceptor (PR) transduction efficiency and A2E concentrations. EIAV-LacZ vectors transduced from 5 to 20% of the PRs in the injected area in mice. Most importantly, a single subretinal injection of EIAV-CMV-ABCA4 to Abca4-/- mouse eyes substantially reduced disease-associated A2E accumulation compared to untreated and mock-treated control eyes. Treated eyes of Abca4-/- mice accumulated 8-12 pmol per eye (s.d.=2.7) of A2E 1 year after treatment, amounts comparable to wt controls, whereas mock-treated or untreated eyes had 3-5 times more A2E (27-39 pmol per eye, s.d.=1.5; P=0.001-0.005). Although extrapolation to humans requires caution, the high transduction efficiency of both rod and cone photoreceptors and the statistically significant reduction of A2E accumulation in the mouse model of STGD1 suggest that lentiviral gene therapy is a potentially efficient tool for treating ABCA4-associated diseases.

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.

Development of photoreceptor‐specific promoters and their utility to investigate EIAV lentiviral vector mediated gene transfer to photoreceptors

We wanted to investigate the ability of recombinant equine infectious anemia virus (EIAV) vectors to transduce photoreceptor cells by developing a series of photoreceptor-specific promoters that drive strong gene expression in photoreceptor cells. Promoter fragments derived from the rhodopsin (RHO), the beta phosphodiesterase (PDE) and the retinitis pigmentosa (RP1) genes were cloned in combination with an enhancer element, derived from the interphotoreceptor retinoid-binding protein gene (IRBP), into luciferase reporter plasmids. An in vitro transient reporter assay was carried out in the human Y-79 retinoblastoma cell line. The optimal promoters from this screen were then cloned into the recombinant EIAV vector for evaluation in vivo following subretinal delivery into mice. All promoters maintained a photoreceptor-specific expression profile in vitro and the gene expression was further enhanced in combination with the IRBP enhancer. The use of IRBP-combined RHO or PDE promoters showed modest but exclusive expression in photoreceptors following subretinal delivery to mice. By contrast an EIAV vector containing the cytomegalovirus (CMV) promoter drove reporter gene expression in both photoreceptors and retinal pigment epithelium. It may be possible to use recombinant EIAV vectors containing photoreceptor-specific promoters to drive therapeutic gene expression to treat a range of retinal degenerative diseases where the photoreceptor cell is the primary disease target.

The integration profile of EIAV-based vectors

Lentiviral vectors based on equine infectious anemia virus (EIAV) stably integrate into dividing and nondividing cells such as neurons, conferring long-term expression of their transgene. The integration profile of an EIAV vector was analyzed in dividing HEK293T cells, alongside an HIV-1 vector as a control, and compared to a random dataset generated in silico. A multivariate regression model was generated and the influence of the following parameters on integration site selection determined: (a) within/not within a gene, (b) GC content within 20 kb, (c) within 10 kb of a CpG island, (d) gene density within a 2-Mb window, and (e) chromosome number. The majority of the EIAV integration sites (68%; n = 458) and HIV-1 integration sites (72%; n = 162) were within a gene, and both vectors favored AT-rich regions. Sites within genes were examined using a second model to determine the influence of the gene-specific parameters, gene region, and transcriptional activity. Both EIAV and HIV-1 vectors preferentially integrated within active genes. Unlike the gammaretrovirus MLV, EIAV and HIV-1 vectors do not integrate preferentially into the promoter region or the 5′ end of the transcription unit.

Stable and efficient intraocular gene transfer using pseudotyped EIAV lentiviral vectors

We have developed minimal non-primate lentiviral vectors based on the equine infectious anaemia virus (EIAV). We evaluated the in vivo expression profiles of these vectors delivered regionally to ocular tissues to define their potential utility in ocular gene therapy. EIAV vectors pseudotyped with VSV-G or rabies-G envelope proteins were delivered subretinally, intravitreally or into the anterior chambers (intracameral administration) in mice. Reporter gene (eGFP) expression was analysed using in vivo retinal imaging or histological examination of eyes and brains at intervals between 3 days and 16 months. We investigated the effects of vector titre, pseudotype, genome configuration, site of intraocular administration, intentional retinal trauma and the degree of retinal maturation on the spatial and temporal expression profiles of these vectors. Subretinal vector delivery resulted in efficient and stable transduction of retinal pigment epithelial (RPE) cells and variable transduction of photoreceptors up to 16 months post-injection. Retinal trauma facilitated the local transduction of neurosensory retinal cells. Intracameral administration of VSV-G- but not rabies-G-pseudotyped vectors produced stable eGFP expression in corneal endothelial cells and trabecular meshwork. The cellular tropism and expression kinetics of optimised EIAV vectors after intraocular administration make them attractive vehicles for delivering therapeutic genes in the management of inherited and acquired retinal and anterior segment disorders.

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.

Comparison of HIV- and EIAV-Based Vectors on Their Efficiency in Transducing Murine and Human Hematopoietic Repopulating Cells

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.

481. Dynamics of Transduction of Human Cells by EIAV Vectors – The Importance of Cell Entry

Recent reports have described some of the underlying components and mechanisms that contribute to the species barrier to retro/lentiviruses, and vectors derived from them. At least part of this blockade is thought to be due to restriction factors, which act to subvert incoming virions down non-replicative pathways. At high multiplicities of infection (MOI) such factors can be titrated away, allowing the cell to be transduced. Elsewhere it has been demonstrated that, similarly to HIV and MLV, Equine Infectious Anaemia virus (EIAV) vectors may also be restricted in a variety of cell types, including human cells. If true, this may have implications regarding EIAV vectors' utility for human gene therapy applications. We have developed highly engineered completely minimal EIAV vectors, and have demonstrated the vectors' potential in a number of pre-clinical models of human disease, including recent studies in models of Parkinson's disease and amyotrophic lateral sclerosis (ALS). We are currently developing the vector system and an associated bank of safety tests, as a prerequisite for treating Parkinson's disease in patients.

828. Optimizing the Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element (WPRE) for Safety and Function in Lentiviral Vectors

We have been developing a gene transfer system based on the non-primate lentivirus equine infectious anemia virus (EIAV) for human gene transfer. Previously, we reported that recombinant EIAV vectors pseudotyped with the G-glycoprotein of the vesicular stomatitis virus (VSV) are able to efficiently transduce muscle, hematopoietic, and neuronal cells. The vector has been optimized for vector titers and trans-gene expression in transduced cells by incorporating various cis-acting elements (EIAV Rev responsive element (RRE), Mason Pfizer monkey transport element (CTE), post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE)) into the vector. The optimal vector configuration resulted in placement of the RRE upstream of the internal promoter and the WPRE into the 3-prime untranslated region downstream of the reporter gene. Although this configuration of vector yields the highest viral titers and transgene protein expression in target cells, safety concerns have been raised regarding the potential oncogenic activity of the truncated WHV X protein encoded in this element (Kingsman et al, Gene Therapy 12, 3, 2005). The WPRE used in our vectors is a 900 bp tripartite element described by Donello et al (J Virol, 78, 5085, 1998). The gamma and the alpha sub-elements provide the majority of the post-transcriptional effects on localization of viral RNA with some contribution from the beta sub-element which also encodes the WHV X protein, the function of which is still not clearly understood. To test whether the X protein is being expressed from our constructs, we fused a luciferase reporter gene in frame with the X protein. Luciferase activity was observed in transduced cells, which clearly suggests that the truncated X protein is being expressed. We are determining the levels of expression in primary human hepatocytes and in transduced mouse liver. We have constructed a minimal WPRE sequence of 513 bp that retains its complete cis-acting function. This element contains a 32 amino acid portion of protein X. We are currently making mutations in the protein X promoter which is a 21 nucleotide domain immediately upstream of the ATG codon of protein X and mutating the start codon and X ORF to prevent any aberrant X protein expression in target cells.

648. Lentiviral Vector Expressing Retinoic Acid Receptor â2 Promotes Regeneration in a Rat Spinal Cord Injury Model

Top of pageAbstract After spinal cord injury, axons in the central nervous system (CNS) fail to regenerate resulting in paralysis. The factors that contribute to this regenerative failure include the inihibitory environment at the injury site as well as the intrinsic growth potential of injured axons. Previously we have demonstrated that retinoic acid receptor |[beta]|2 (RAR|[beta]|2), a transcription factor activated by retinoic acid, can induce neurite outgrowth in adult spinal cord explants and promote functional regeneration in a rat model of cervical rhizotomy. Here we investigate the utility of a minimal equine infectious anaemia virus (EIAV)-based lentiviral vector expressing RAR|[beta]|2 in stimulating axonal outgrowth in a rat spinal cord injury model. EIAV-RAR|[beta]|2 was injected into the sensorimotor cortex prior to lesioning of dorsal columns in the cervical cord. At 4 weeks after lesion, RAR|[beta]|2-treated animals demonstrated regeneration of descending corticospinal axons up to, and beyond the lesion site, compared to control animals that received an EIAV vector expressing |[beta]|-galactosidase. These anatomical observations correlated to an improvement in task performance in locomotor and proprioceptive behavioural tests as well as an increase in post-synaptic activity below the lesion after electrical stimulation of corticospinal neurons. This suggests that lentiviral-mediated delivery of RAR|[beta]|2 promoted functional regeneration of CNS axons in the injured spinal cord and may thus be exploited as a novel approach to therapeutic applications in the treatment of spinal cord injuries.

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.

Lentiviral-Mediated Delivery of Bcl-2 or GDNF Protects against Excitotoxicity in the Rat Hippocampus

Nutrient deprivation during ischemia leads to severe insult to neurons causing widespread excitotoxic damage in specific brain regions such as the hippocampus. One possible strategy for preventing neurodegeneration is to express therapeutic proteins in the brain to protect against excitotoxicity. We investigated the utility of equine infectious anemia virus (EIAV)-based vectors as genetic tools for delivery of therapeutic proteins in an in vivo excitotoxicity model. The efficacy of these vectors at preventing cellular loss in target brain areas following excitotoxic insult was also assessed. EIAV vectors generated to overexpress the human antiapoptotic Bcl-2 or growth factor glial-derived neurotrophic factor (GDNF) genes protected against glutamate-induced toxicity in cultured hippocampal neurons. In an in vivo excitotoxicity model, adult Wistar rats received a unilateral dose of the glutamate receptor agonist N-methyl-D-aspartate to the hippocampus that induced a large lesion in the CA1 region. Neuronal loss could not be protected by prior transduction of a control vector expressing beta-galactosidase. In contrast, EIAV-mediated expression of Bcl-2 and GDNF significantly reduced lesion size thus protecting the hippocampus from excitotoxic damage. These results demonstrate that EIAV vectors can be effectively used to deliver putative neuroprotective genes to target brain areas and prevent cellular loss in the event of a neurological insult. Therefore these lentiviral vectors provide potential therapeutic tools for use in cases of acute neurotrauma such as cerebral ischemia.

Optimization of equine infectious anemia derived vectors for hematopoietic cell lineage gene transfer

Gene transfer into hematopoietic cells may allow correction of a variety of hematopoietic and metabolic disorders. Optimized HIV-1 based lentiviral vectors have been developed for improved gene transfer and transgene expression into hematopoietic cells. However, the use of HIV-1 based vectors for human gene therapy may be limited due to ethical and biosafety issues. We report that vectors based on the non-primate equine infectious anemia virus (EIAV) transduce a variety of human hematopoietic cell lines and primary blood cells. To investigate optimization of gene expression in hematopoietic cells, we compared a variety of post-transcriptional elements and promoters in the context of EIAV vectors. We observed cell specific increase in the number of transgene expressing cells with the different post-transcriptional elements, whereas the use of elongation factor alpha 1 (EFalpha1) promoter resulted in significant increases in both the number of transgene expressing cells and the level of transgene protein in all cell types tested. We then demonstrate increased transduction of hematopoietic cells using a second-generation EIAV vector containing a self-inactivating EIAV LTR and the EIAV central polypurine tract (cppt). These data suggest that optimized EIAV vectors may be a suitable alternative to HIV-1 vectors for use in hematopoietic gene therapy.

737. Development of a Feline Immunodeficiency Virus-Based Lentiviral Vector for Genetic Modification of CNS Neurons

Background: Insulin-like growth factor (IGF)-I has various effects on proliferation, differentiation, and survival of neurons in the central nervous system. Neurodegenerative disease processes occurring in amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA) and diabetic neuropathy ultimately result in motor neuron (MN) death. Equine infectious anemia virus (EIAV)-based gene transfer vectors have been reported to sustain long-term gene expression in the nervous system. Pseudotyping of EIAV vectors with the rabies-G envelope protein (RabG.EIAV) has rendered EIAV vectors capable of retrograde axonal transport and access to the central nervous system after peripheral injection. This provides an attractive remote nervous system-targeting approach.

636. Reversal of Hypothalamic Diabetes Insipidus Using EIAV-Based Regulatable Lentiviral Vectors Expressing Arginine Vasopressin

Top of pageAbstract Lentiviral vectors, such as the Equine Infectious Anaemia Virus (EIAV)-based vectors, have the potential to be used in the clinic for a number of gene therapy applications. Efficient gene transfer using EIAV vectors and long-term expression has been demonstrated in vivo in the central nervous system. Hypothalamic diabetes insipidus (DI) is a disorder that affects water balance in humans. The cause of DI is the lack of the water retaining hormone arginine vasopressin (AVP) produced by magnocellular neurons of the supraoptic nuclei (SON) of the hypothalamus. AVP is secreted from the pituitary gland into the blood stream and acts on the kidney causing water reabsorption. In the AVP-deficient Brattleboro rat, as a result of a single mutation, AVP is not produced thus causing copious water intake and secretion of dilute urine. This genetic animal model allows the non-invasive monitoring of the efficacy of gene therapy strategies to treat DI (1). In this study, self-inactivating (SIN) minimal EIAV vector genomes with enhanced transduction efficiency (SMART vectors) have been used to express rat AVP. Vector particles were bilaterally injected into the (SON) in the hypothalamus of the Brattleboro rat model of DI. The EIAV-AVP treated animals show a dramatic reduction of daily water intake and urine volume output compared to control animals (injected with EIAV-GFP), reaching levels very similar to the ones observed in the Long Evans (wild type) rats. AVP is also detected in the urine of EIAV-AVP treated animals. These observations indicate that the hormone is now produced in the SON and released in the blood stream where it is correcting water balance. We observed reversal of the phenotype for one year, which is longer and more effective than that obtained with any previous gene therapy interventions. We have also developed tetracycline regulatable (single-vector) lentiviral systems and data showing that AVP expression can be increased following the addition of doxycycline will also be presented.