Vector Stocks Research Articles

Enhanced nigrostriatal neuron-specific, long-term expression by using neural-specific promoters in combination with targeted gene transfer by modified helper virus-free HSV-1 vector particles.

BackgroundDirect gene transfer into neurons has potential for developing gene therapy treatments for specific neurological conditions, and for elucidating neuronal physiology. Due to the complex cellular composition of specific brain areas, neuronal type-specific recombinant gene expression is required for many potential applications of neuronal gene transfer. One approach is to target gene transfer to a specific type of neuron. We developed modified Herpes Simplex Virus (HSV-1) particles that contain chimeric glycoprotein C (gC) – glial cell line-derived neurotrophic factor (GDNF) or brain-derived neurotrophic factor (BDNF) proteins. HSV-1 vector particles containing either gC – GDNF or gC – BDNF target gene transfer to nigrostriatal neurons, which contain specific receptors for GDNF or BDNF. A second approach to achieve neuronal type-specific expression is to use a cell type-specific promoter, and we have used the tyrosine hydroxylase (TH) promoter to restrict expression to catecholaminergic neurons or a modified neurofilament heavy gene promoter to restrict expression to neurons, and both of these promoters support long-term expression from HSV-1 vectors. To both improve nigrostriatal-neuron specific expression, and to establish that targeted gene transfer can be followed by long-term expression, we performed targeted gene transfer with vectors that support long-term, neuronal-specific expression.ResultsHelper virus-free HSV-1 vector packaging was performed using either gC – GDNF or gC – BDNF and vectors that contain either the TH promoter or the modified neurofilament heavy gene promoter. Vector stocks were injected into the midbrain proximal to the substantia nigra, and the rats were sacrificed at either 4 days or 1 month after gene transfer. Immunofluorescent costaining was performed to detect both recombinant gene products and nigrostriatal neurons. The combination of targeted gene transfer with neuronal-specific promoters improved nigrostriatal neuron-specific expression (83 to 93%) compared to either approach alone, and supported long-term (1 month) expression at levels similar to those observed using untargeted gene transfer.ConclusionTargeted gene transfer can be used in combination with neuronal-specific promoters to achieve a high level of nigrostriatal neuron-specific expression. Targeted gene transfer can be followed by long-term expression. Nigrostriatal neuron-specific expression may be useful for specific gene therapy approaches to Parkinson's disease or for genetic analyses of nigrostriatal neuron physiology.

Substitution of the Rev-response element in an HIV-1-based gene delivery system with that of SIVmac239 allows efficient delivery of Rev M10 into T-lymphocytes

BackgroundHuman immunodeficiency virus type 1 (HIV-1)-based gene delivery systems are popular due to their superior efficiency of transduction of primary cells. However, these systems cannot be readily used for delivery of anti-HIV-1 genes that target constituents of the packaging system itself due to inimical effects on vector titer. Here we describe HIV-1-based packaging systems containing the Rev-response element (RRE), of simian immunodeficiency virus (SIV) in place of the HIV-1 RRE. The SIV RRE-containing packaging systems were used to deliver the anti-Rev gene, Rev M10, into HIV-1 susceptible target cells.ResultsAn HIV-1 based packaging system was created using either a 272- or 1045-nucleotide long RRE derived from the molecular clone SIVmac239. The 1045-nucleotide SIV RRE-containing HIV-1 packaging system provided titers comparable to that of the HIV-1 RRE-based one. Moreover, despite the use of HIV-1 Rev for production of vector stocks, this packaging system was found to be relatively refractory to the inhibitory effects of Rev M10. Correspondingly, the SIV RRE-based packaging system provided 34- to 130-fold higher titers than the HIV-1 RRE one when used for packaging a gene transfer vector encoding Rev-M10. Jurkat T-cells, gene modified with Rev M10 encoding HIV-1 vectors, upon challenge with replication defective HIV-1 in single-round infection experiments, showed diminished production of virus particles.ConclusionA simple modification of an HIV-1 gene delivery system, namely, replacement of HIV-1 RRE with that of SIV, allowed efficient delivery of Rev M10 transgene into T-cell lines for intracellular immunization against HIV-1 replication.

Hexamethylene bisacetamide leads to reduced helper virus-free HSV-1 amplicon expression titers via suppression of ICP0.

The herpes simplex virus (HSV)-derived amplicon vector has evolved into a promising gene transfer platform for widespread DNA delivery in gene replacement strategies and vaccine development given its ease of molecular manipulation, large transgene capacity, and transduction efficiencies of numerous cell types in vivo. The recent development of helper virus-free packaging methodologies bodes well for this vector system in its eventual implementation as a clinically viable therapeutic modality. For realization of clinical application, efforts have been made to enhance yields and quality of helper-free amplicon stocks. Hexamethylene bisacetamide (HMBA), a hybrid polar compound that exhibits stimulatory activity of HSV-1 immediate-early gene expression, has been employed as a standard reagent in helper virus-free packaging given its purported mode of action on virus gene expression kinetics. Unexpectedly, we have found that HMBA exhibits no titer-enhancing activity; in contrast, the compound enhances the proportion of amplicon virions that are non-expressive. Omission of HMBA during vector packaging led to a marked reduction in the ratios of vector genome-transducing to transgene-expressing virions. This effect was neither packaging-cell-specific nor amplicon-promoter-dependent. Analysis of resultant vector stocks indicated amplicon genome replication/concatenation was unaffected, but the level of particle-associated ICP0 was reduced in stocks packaged in the presence of HMBA. Inclusion of a co-transfected, ICP0-expressing plasmid into the packaging process led to significant rescue of amplicon expression titers, indicating that regulation of ICP0 concentrations is critical for maintenance of the amplicon genome expressive state.

Multifactorial Optimization of Gammaretroviral Gene Transfer into Human T Lymphocytes for Clinical Application

The ability to genetically modify human T cells to target tumor antigens through retroviral gene transfer constitutes a potentially powerful approach to cancer immunotherapy. However, low transduction efficiencies may hamper the efficacy of such therapeutic strategies in the clinical setting. Most commonly, gammaretroviral gene transfer into T cells is conducted through spinoculation, that is, centrifugation of retroviral particles and T cells on RetroNectin-coated non-tissue culture vessels. Here we present data investigating the impact of temperature, speed, and frequency of spinoculation on T cell transduction efficiencies. We found that all three variables independently impacted gene transfer, with increasing temperature, speed, and frequency of spinoculation all enhancing the transduction of T cells. These improved conditions were additive, with the greatest proportion of transduced T cells being generated at the highest tested temperature and speed, after daily spinoculation for 2 to 3 days. Under these conditions, enhanced gene transfer was observed in T cells derived from healthy donors, using research-grade vector stocks. Whereas both RetroNectin and spinoculation were critical to optimal gene transduction, preloading of gammaretroviral particles before spinoculation did not enhance gene transfer. Significantly, application of these enhanced transduction conditions to T cells derived from previously treated patients with chronic lymphocytic leukemia allowed for adequate gene transfer under both small-scale and large-scale clinically applicable conditions using either preclinical or current Good Manufacturing Practice-grade gammaretroviral vector stocks.

Herpes simplex virus vector-mediated delivery of glial cell line-derived neurotrophic factor rescues erectile dysfunction following cavernous nerve injury

Erectile dysfunction (ED) is frequently associated with injury to the cavernous nerve sustained during pelvic surgery. Functional recovery from cavernous nerve injury is generally incomplete and occurs over an extended time frame. We employed a therapeutic gene transfer approach with herpes simplex virus (HSV) vector expressing glial cell line-derived neurotrophic factor (GDNF). Rat cavernous nerve was injured bilaterally using a clamp and dry ice. For HSV-treated groups, 20 microl of purified vector stock was administered directly to and around the damaged nerve. Delivery of an HSV vector expressing both green fluorescent protein (GFP) and lacZ (HSV-LacZ) was used as a control. Intracavernous pressure along with systemic arterial pressure (ICP/AP) was measured 2 and 4 weeks after the nerve injury. Fluorogold (FG) was injected into the penile crus 7 days before killing to assess nerve survival. Approximately 60% of major pelvic ganglion (MPG) cells were GFP positive after viral administration. At 4 weeks after nerve injury, rats treated with HSV-GDNF exhibited significant recovery of ICP/AP compared with control vector or untreated groups. The HSV-GDNF group also yielded more FG-positive MPG cells than the control vector group. HSV vector-mediated delivery of GDNF presents a viable approach for the treatment of ED following cavernous nerve injury.

Simian immunodeficiency virus vector pseudotypes differ in transduction efficiency and target cell specificity in brain

Lentiviral vectors have proven to be promising tools for transduction of brain cells in vivo and in vitro. In this study, we have examined the central nervous system (CNS) transduction efficiencies and patterns of a self-inactivating simian immunodeficiency virus (SIVmac)-derived lentiviral vector pseudotyped with glycoproteins from the vesicular stomatitis virus (VSV-G), the amphotropic murine leukemia virus (MLV4070Aenv), the lymphocytic choriomeningitis virus (LCMV-GP), the Ross River virus (RRV-GP) and the rabies virus (RV-G). All glycoproteins were efficiently incorporated into SIV virions, allowing efficient transduction of neuronal cell lines as well as of primary dissociated mouse brain cell cultures. After injection of highly concentrated vector stocks into the striatum of adult mice, quantitative analyses revealed high transduction efficiency with VSV-G pseudotypes, while LCMV-GP and RV-G pseudotypes exhibited moderate transduction efficiencies. MLV4070Aenv and RRV-GP pseudotypes, however, showed only weak levels of transduction after stereotactic injection into the brain. Regarding cell tropism in vivo, VSV-G-pseudotyped SIV vectors transduced neuronal as well as glial cells, whereas all other pseudotypes preferentially transduced neuroglial cells. In addition, we analyzed the influence of the central polypurine tract (cPPT) in context of the VSV-G-pseudotyped SIV transfer vector for infection of brain cells. Deletion of the cPPT sequence from the transfer vector decreased the in vivo transduction efficiency by fourfold, and, more importantly, this modification changed the transduction pattern, since these vectors were no longer able to infect neuronal cells in vivo. Vector injection into the brain did elicit a humoral immune response in the injected hemisphere; however, no gross signs of inflammation could be detected. Analysis of the biodistribution of the vector revealed that, besides the injected brain region, no vector-specific sequences could be detected in any of the organs evaluated. These data indicate SIV vectors as efficient gene delivery vehicles for the treatment of neurodegenerative diseases.

CGI-58 facilitates lipolysis on lipid droplets but is not involved in the vesiculation of lipid droplets caused by hormonal stimulation

A lipid droplet (LD)-associated protein, perilipin, is a critical regulator of lipolysis in adipocytes. We previously showed that Comparative Gene Identification-58 (CGI-58), a product of the causal gene of Chanarin-Dorfman syndrome, interacts with perilipin on LDs. In this study, we investigated the function of CGI-58 using RNA interference. Notably, CGI-58 knockdown caused an abnormal accumulation of LDs in both 3T3-L1 preadipocytes and Hepa1 hepatoma cells. CGI-58 knockdown did not influence the differentiation of 3T3-L1 adipocytes but reduced the activity of both basal and cAMP-dependent protein kinase-stimulated lipolysis. In vitro studies showed that CGI-58 itself does not have lipase/esterase activity, but it enhanced the activity of adipose triglyceride lipase. Upon lipolytic stimulation, endogenous CGI-58 was rapidly dispersed from LDs into the cytosol along with small particulate structures. This shift in localization depends on the phosphorylation of perilipin, because phosphorylated perilipin lost the ability to bind CGI-58. During lipolytic activation, LDs in adipocytes vesiculate into micro-LDs. Using coherent anti-Stokes Raman scattering microscopy, we pursued the formation of micro-LDs in single cells, which seemed to occur in cytoplasmic regions distant from the large central LDs. CGI-58 is not required for this process. Thus, CGI-58 facilitates lipolysis in cooperation with perilipin and other factors, including lipases.

310. A Rapid, Accurate and Precise Assay System To Analyze the Quality and the Quantity of Clinical Grade HSV Vector Stocks

310. A Rapid, Accurate and Precise Assay System To Analyze the Quality and the Quantity of Clinical Grade HSV Vector Stocks

Production of lentiviral vectors by large‐scale transient transfection of suspension cultures and affinity chromatography purification

The use of lentiviral vectors as gene delivery vehicles has become increasingly popular in recent years. The growing interest in these vectors has created a strong demand for large volumes of vector stocks, which entails the need for scaleable vector manufacturing procedures. In this work, we present a simple and robust process for the production of lentiviral vectors using scaleable production and purification methodologies. Lentivirus particles were produced by transient transfection of serum-free suspension-growing 293 EBNA-1 cells with four plasmids encoding the vector components using linear polyethylenimine (PEI) as transfection reagent. This process was successfully scaled-up from shake flasks to a 3-L bioreactor from which 10(10) IVP were recovered. In addition, an affinity chromatography protocol designed for purification of bioactive oncoretroviral vectors has been adapted in this work for the purification of VSV-G pseudotyped lentiviral vectors. Using heparin affinity chromatography, lentiviral particles were concentrated and purified directly from the clarified supernatants. During this step, a recovery of 53% of infective lentiviral particles was achieved while removing 94% of the impurities contained in the supernatant.

Gene delivery by lentivirus vectors

The capacity to efficiently transduce nondividing cells, shuttle large genetic payloads, and maintain stable long-term transgene expression are attributes that have brought lentiviral vectors to the forefront of gene delivery vehicles for research and therapeutic applications in a clinical setting. Our discussion initiates with advances in lentiviral vector development and how these sophisticated lentiviral vectors reflect improvements in safety, regarding the prevention of replication competent lentiviruses (RCLs), vector mobilization, and insertional mutagenesis. Additionally, we describe conventional molecular regulatory systems to manage gene expression levels in a spatial and temporal fashion in the context of a lentiviral vector. State of the art technology for lentiviral vector production by transient transfection and packaging cell lines are explicitly presented with current practices used for concentration, purification, titering, and determining the safety of a vector stock. We summarize lentiviral vector applications that have received a great deal of attention in recent years including the generation of transgenic animals and the stable delivery of RNA interference molecules. Concluding remarks address some of the successes in preclinical animals, and the recent transition of lentiviral vectors to human clinical trials as therapy for a variety of infectious and genetic diseases.

Scaleable purification process for gene therapy retroviral vectors

Retroviral vectors (RVs) constitute one of the preferred gene therapy tools against inherited and acquired diseases. Development of scaleable downstream processes allowing purification under mild conditions and yielding viral preparations with high titer, potency and purity is critical for the success of clinical trials and subsequent clinical use of this technology. A purification process for murine leukaemia virus (MLV)-derived vector supernatants was developed based on membrane separation and anion-exchange chromatography (AEXc). Initial clarification of the vector stocks was performed using 0.45 microm membranes followed by concentration with 500 kDa molecular weight cut-off (MWCO) membranes; further purification was performed by AEXc using a tentacle matrix bearing DEAE functional ligands. Finally, concentration/diafiltration was performed by 500 kDa MWCO membranes. To validate final product quality the process was scaled up 16-fold. Optimization of microfiltration membrane pore size and ultrafiltration transmembrane pressure allowed the recovery of nearly 100% infectious particles. Further purification of the RVs by AEXc resulted in high removal of protein contaminants while maintaining high recoveries of infectious vectors (77+/-11%). Up-scaling of the process resulted in high titer vector preparations, 3.2x10(8) infectious particles (IP)/ml (85-fold concentration), with an overall recovery reaching 26%. The process yielded vectors with transduction efficiencies higher than the starting material and more than 99% pure, relative to protein contamination. The combination of membrane separation and AEXc processes results in a feasible and scaleable purification strategy for MLV-derived vectors, allowing the removal of inhibitory contaminants thus yielding pure vectors with increased transduction efficiencies.

Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter

Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons. A PAG promoter was partially characterized using a cultured kidney cell line. The three vesicular glutamate transporters (VGLUTs) are expressed in distinct populations of neurons, and VGLUT1 is the predominant VGLUT in the neocortex, hippocampus, and cerebellar cortex. Glutamic acid decarboxylase (GAD) produces GABA; the two molecular forms of the enzyme, GAD65 and GAD67, are expressed in distinct, but largely overlapping, groups of neurons, and GAD67 is the predominant form in the neocortex. In transgenic mice, an ∼ 9 kb fragment of the GAD67 promoter supports expression in most classes of GABAergic neurons. Here, we constructed plasmid (amplicon) Herpes Simplex Virus (HSV-1) vectors that placed the Lac Z gene under the regulation of putative PAG, VGLUT1, or GAD67 promoters. Helper virus-free vector stocks were delivered into postrhinal cortex, and the rats were sacrificed 4 days or 2 months later. The PAG or VGLUT1 promoters supported ∼ 90% glutamatergic neuron-specific expression. The GAD67 promoter supported ∼ 90% GABAergic neuron-specific expression. Long-term expression was observed using each promoter. Principles for obtaining long-term expression from HSV-1 vectors, based on these and other results, are discussed. Long-term glutamatergic or GABAergic neuron-specific expression may benefit specific experiments on learning or specific gene therapy approaches. Of note, promoter analyses might identify regulatory elements that determine a glutamatergic or GABAergic neuron.

Development of AAV serotype-specific ELISAs using novel monoclonal antibodies

Adeno-associated viruses (AAV) have been developed and evaluated as recombinant vectors for gene therapy. More recently, due to the advantages they offer for gene transfer, several AAV serotypes have gained increasing interest. However, monoclonal antibodies for the characterization and quantitation of vectors derived from different serotypes are at present not available. Serotype-specific monoclonal antibodies (mAbs) against the capsids of the serotypes 1/6, 4 and 5 are described. These antibodies, designated as ADK1a and b, ADK4 or ADK5a and b, reacted specifically with the indicated serotype capsids in cell lysates. ADK 1a and b cross-reacted with its highly related AAV6 serotype, but not with the other serotypes tested. The new antibodies recognized exclusively assembled capsids and neither free nor denatured capsid proteins as shown by fractionation experiments. In immunofluorescence experiments, the mAbs stained only distinct intranuclear foci in cells expressing the capsid protein. The development of capture ELISAs for quantitation of AAV1 and 6, AAV4 or AAV5 capsids illustrates that these novel monoclonal antibodies provide valuable tools for characterization of vector stocks.

Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors

Retroviral vectors with self-inactivating (SIN) long-terminal repeats not only increase the autonomy of the internal promoter but may also reduce the risk of insertional upregulation of neighboring alleles. However, gammaretroviral as opposed to lentiviral packaging systems produce suboptimal SIN vector titers, a major limitation for their clinical use. Northern blot data revealed that low SIN titers were associated with abundant transcription of internal rather than full-length transcripts in transfected packaging cells. When using the promoter of Rous sarcoma virus or a tetracycline-inducible promoter to generate full-length transcripts, we obtained a strong enhancement in titer (up to 4 x 10(7) transducing units per ml of unconcentrated supernatant). Dual fluorescence vectors and Northern blots revealed that promoter competition is a rate-limiting step of SIN vector production. SIN vector stocks pseudotyped with RD114 envelope protein had high transduction efficiency in human and non-human primate cells. This study introduces a new generation of efficient gammaretroviral SIN vectors as a platform for further optimizations of retroviral vector performance.

Quantification of High-Capacity Helper-Dependent Adenoviral Vector Genomes In Vitro and In Vivo, Using Quantitative TaqMan Real-Time Polymerase Chain Reaction

First-generation adenoviral (Ad) and high-capacity adenoviral (HC-Ad) vectors are efficient delivery vehicles for transferring therapeutic transgenes in vivo into tissues/organs. The initial successes reported with adenoviral vectors in preclinical trials have been limited by immune-related adverse side effects. This has been, in part, attributed to the use of poorly characterized preparations of adenoviral vectors and also to the untoward immune adverse side effects elicited when high doses of these vectors were used. HC-Ads have several advantages over Ads, including the lack of viral coding sequences, which after infection and uncoating, makes them invisible to the host's immune system. Another advantage is their large cloning capacity (up to approximately 35 kb). However, accurate characterization of HC-Ad vectors, and of contaminating replication-competent adenovirus (RCA) or helper virus, is necessary before these preparations can be used safely in clinical trials. Consequently, the development of accurate, simple, and reproducible methods to standardize and validate adenoviral preparations for the presence of contaminant genomes is required. By using a molecular method that allows accurate, reproducible, and simultaneous determination of HC-Ad, contaminating helper virus, and RCA genome copy numbers based on real-time quantitative PCR, we demonstrate accurate detection of these three genomic entities, within CsCl-purified vector stocks, total DNA isolated from cells transduced in vitro, and from brain tissue infected in vivo. This approach will allow accurate assessment of the levels and biodistribution of HC-Ad and improve the safety and efficacy of clinical trials.

Removal of Empty Capsids from Type 1 Adeno-Associated Virus Vector Stocks by Anion-Exchange Chromatography Potentiates Transgene Expression

Production of recombinant adeno-associated virus (rAAV) results in substantial quantities of empty capsids or virus-like particles (VLPs), virus protein shells without the vector genome. The contaminating VLPs would interfere with transduction by competing for cell-surface receptors and, when administered in vivo, contribute to antigen load, which may elicit a stronger immune response. Density-gradient ultracentrifugation provides a means to separate VLPs from rAAV particles, but is not feasible for large-scale preparations of vectors. Since the compositions of the VLP and vector differ by the single-stranded DNA genome, we hypothesized that the isoelectric point of the vector may differ from that of the VLP. In an attempt to separate type 1 rAAV particles from VLPs by ion-exchange chromatography, we tested a number of buffer systems and found that trimethylammonium sulfate, or [(CH3)4N]2SO4, effectively separated rAAV1 particles from VLPs. The rAAV1-GFP chromatographically separated from VLPs induced stronger GFP expression in HEK293 cells than rAAV1-GFP contaminated with VLPs. The transduction of mouse muscles with rAAV1-SEAP (secreted form of alkaline phosphatase) isolated from VLPs also showed higher serum SEAP levels than rAAV1-SEAP with VLPs. These results suggest that chromatographic separation of rAAV1 from empty capsids increased the efficacy of rAAV1.

Genetic design of an optimized packaging cell line for gene vectors transducing human B cells

Viral gene vectors often rely on packaging cell lines, which provide the necessary factors in trans for the formation of virus-like particles. Previously, we reported on a first-generation packaging cell line for gene vectors, which are based on the B-lymphotropic Epstein-Barr virus (EBV), a human gamma-herpesvirus. This 293HEK-derived packaging cell line harbors a helper virus genome with a genetic modification that prevents the release of helper virions, but efficiently packages vector plasmids into virus-like particles with transducing capacity for human B cells. Here, we extended this basic approach towards a non-transforming, virus-free packaging cell line, which harbors an EBV helper virus genome with seven genetic alterations. In addition, we constructed a novel gene vector plasmid, which is devoid of a prokaryotic antibiotic resistance gene, and thus more suitable for in vivo applications in human gene therapy. We demonstrate in this paper that EBV-based gene vectors can be efficiently generated with this much-improved packaging cell line to provide helper virus-free gene vector stocks with transducing capacity for established human B-cell lines and primary B cells.

313. Cell Type Specific Media Optimization for Clinical Retroviral Transduction Protocols

In order to optimize the transduction of CD34+ cells, CD34+ derived Langerhans cells (LCs) and T lymphocytes for upcoming clinical trials, we determined which medium is best suited for each cell type. Our aim is to identify the formulation providing the best cell proliferation, survival and the highest transduction efficiency (TE) while maintaining the biological activity of the cells. To this end, we harvested and screened PG13-derived retroviral vector stocks (VS) collected in various media formulations.

1111. Removal of Empty Particles from Type 1 Adeno-Associated Virus Vector Stocks by Ion Exchange Chromatography Potentiates Transgene Expression

Production of recombinant adeno-associated virus (rAAV) results in substantial quantities of empty capsids or virus-like particles (VLPs), a virus protein shell without the vector genome. The contaminating VLPs would interfere with transduction by competing for cell surface receptors and when administered in vivo, contribute to antigen load, which may elicit a stronger immune response. Density-gradient ultracentrifugation provides a means to separate VLPs from rAAV particles, but is not feasible for large scale preparations of vectors. Since the compositions of the VLP and vector differ by the single-stranded DNA genome, we hypothesized that the isoelectric points of the vector may differ from that of the VLP. In an attempt to separate type 1 rAAV particles from VLPs by ion exchange chromatography, we tested a number of buffer systems and found that trimethylammonium sulfate, or [(CH3)4N] 2SO4, effectively separated rAAV1 particles from VLPs, which was confirmed by electron microscopic observation of samples from pooled fractions. The rAAV1-GFP or -SEAP (secreted form of alkaline phosphatase) chromatographically separated from VLPs induced stronger transgene expression in HEK293 cells than rAAV1 contaminated with VLPs. The transduction of mouse muscles with VLP-free rAAV1-SEAP also showed higher serum SEAP levels than rAAV1-SEAP contaminated with VLPs. These results suggested that chromatographic separation of rAAV1 from empty capsids was possible and increased the efficacy of rAAV1. It will be useful for the purification of large quantities of rAAV1 for large-animal or human applications. Production of recombinant adeno-associated virus (rAAV) results in substantial quantities of empty capsids or virus-like particles (VLPs), a virus protein shell without the vector genome. The contaminating VLPs would interfere with transduction by competing for cell surface receptors and when administered in vivo, contribute to antigen load, which may elicit a stronger immune response. Density-gradient ultracentrifugation provides a means to separate VLPs from rAAV particles, but is not feasible for large scale preparations of vectors. Since the compositions of the VLP and vector differ by the single-stranded DNA genome, we hypothesized that the isoelectric points of the vector may differ from that of the VLP. In an attempt to separate type 1 rAAV particles from VLPs by ion exchange chromatography, we tested a number of buffer systems and found that trimethylammonium sulfate, or [(CH3)4N] 2SO4, effectively separated rAAV1 particles from VLPs, which was confirmed by electron microscopic observation of samples from pooled fractions. The rAAV1-GFP or -SEAP (secreted form of alkaline phosphatase) chromatographically separated from VLPs induced stronger transgene expression in HEK293 cells than rAAV1 contaminated with VLPs. The transduction of mouse muscles with VLP-free rAAV1-SEAP also showed higher serum SEAP levels than rAAV1-SEAP contaminated with VLPs. These results suggested that chromatographic separation of rAAV1 from empty capsids was possible and increased the efficacy of rAAV1. It will be useful for the purification of large quantities of rAAV1 for large-animal or human applications.

469. Optimization of the Lentivirus Vector Containing the MSCV-U3 and EF1α Double Internal Promoters

The double gene vector carrying the expression units of the therapeutic gene and the reporter gene is widely used, since the reporter proteins such as the GFP and the DsRed are useful for measuring the titer of vector stock and the tracing gene modified cells. However, the activities of the double promoters in a single vector are often inhibited by the so called 'promoter interference'. The internal ribosomal entry site (IRES) is being utilized to overcome this problem, but the expression of the second gene is not sufficient for the sensitive detection. In this study, we attempted to optimize the double gene lentivirus vector to achieve a high expression level of both genes.