Effect of temperature, medium composition, and cell passage on production of herpes-based viral vectors

Our work uses replication-defective genomic herpes simplex virus type-1 (HSV-1)-based vectors to transfer therapeutic genes into cells of the central nervous system and other tissues. Obtaining highly purified high-titer vector stocks is one of the major obstacles remaining in the use of these vectors in gene therapy applications. We have examined the effects of temperature and media conditions on the half-life of HSV-1 vectors. The results reveal that HSV stability is 2.5-fold greater at 33 degrees C than at 37 degrees C and is further stabilized at 4 degrees C. Additionally, a significantly higher half-life was measured for the vector in infection culture conditioned serum medium compared to fresh medium with or without serum. Synchronous infections incubated at 33 degrees C produced 2-fold higher amounts of vector than infected cells incubated at 37 degrees C, but with a lag of 16-24 h. Vector production yielded 3-fold higher titers and remained stable at peak levels for a longer period of time in cultures incubated at 33 degrees C than 37 degrees C. A pronounced negative effect of increased cell passage number on vector yield was observed. Vector production at 33 degrees C yielded similar levels regardless of passage number but was reduced at 37 degrees C as passage number increased. Together, these results contribute to improved methods for high-titer HSV vector production.     

Replication-defective genomic herpes simplex vectors: design and production

Herpes simplex virus (HSV) may be engineered to produce flexible and efficient gene delivery vectors. Recent advances in vector design and production have built on increasing understanding of the basic biology of HSV to minimise vector toxicity and exploit viral features that give rise to lifelong latent infection in the nervous system. In addition, the emerging picture of viral cell entry has allowed early steps to be taken towards targeting viral entry to predetermined cellular subsets. Recent work has established sound principles for the straightforward production of large-scale pure preparations of vector stocks for clinical applications.     

Effects of culture parameters on the production of retroviral vectors by a human packaging cell line

The use of retroviral vectors for human gene therapy requires the production of large quantities of high titer vector stocks. Maintaining high titers during the prolonged culture of packaging cells will require that critical parameters be controlled. The aim of this study was to determine which culture parameters critically affect the production/decay of retroviral vectors produced by the human packaging cell line FLYRD18/LNC-hB7. The stability of retroviral vectors released by this cell line was found to be temperature dependent (half-life of 6.9, 11.0, and 64.3 h when incubated at 37, 32, and 0 degrees C, respectively). Titers increased up to 10-fold when the packaging cells were cultured at 32 degrees C, compared to 37 degrees C, despite a decrease in cell yield (cell-specific titers were 20-fold higher). Virus titers were also over 10-fold higher when the packaging cells were cultured in a reduced serum concentration (1%) compared to 5%. Retrovirus production at a range of pH levels revealed a significant decrease in virus titer at pH levels below 6.8 and above 7.2, optimum titers being achieved in cultures at pH 7.2. Dissolved oxygen levels in the range 20-80% did not significantly affect titers under the conditions tested. Finally, a packed bed system containing the packaging cells immobilized on porous microcarriers was shown to sustain the production of active retroviral vectors for over 1 month, in relatively large volumes.     

Effect of genetic background and culture conditions on the production of herpesvirus-based gene therapy vectors.

Herpes simplex virus type-1 (HSV-1) represents a unique vehicle for the introduction of foreign DNA to cells of a variety of tissues. The nature of the vector, the cell line used for propagation of the vector, and the culture conditions will significantly impact vector yield. An ideal vector should be deficient in genes essential for replication as well as those that contribute to its cytotoxicity. Advances in the engineering of replication-defective HSV-1 vectors to reduce vector-associated cytotoxicity and attain sustained expression of target genes make HSV-1 an attractive gene-delivery vehicle. However, the yield of the less-cytotoxic vectors produced in standard tissue-culture systems is at least three order of magnitudes lower than that achieved with wild-type virus. The low overall yield and the complexity involved in the preparation of HSV vectors at high concentrations represent major obstacles in use of replication-defective HSV-derived vectors in gene therapy applications. In this work, the dependence of the vector yield on the genetic background of the virus is examined. In addition, we investigated the production of the least toxic, lowest-yield vector in a CellCube bioreactor system. After initial optimization of the operational parameters of the cellcube system, we were able to attain virus yields similar to or better than those values attained using the tissue culture flask system for vector production with significant savings with respect to time, labor, and cost.     

Bioreactor production of recombinant herpes simplex virus vectors

Serotypical application of herpes simplex virus (HSV) vectors to gene therapy (type 1) and prophylactic vaccines (types 1 and 2) has garnered substantial clinical interest recently. HSV vectors and amplicons have also been employed as helper virus constructs for manufacture of the dependovirus adeno-associated virus (AAV). Large quantities of infectious HSV stocks are requisite for these therapeutic applications, requiring a scalable vector manufacturing and processing platform comprised of unit operations which accommodate the fragility of HSV. In this study, production of a replication deficient rHSV-1 vector bearing the rep and cap genes of AAV-2 (denoted rHSV-rep2/cap2) was investigated. Adaptation of rHSV production from T225 flasks to a packed bed, fed-batch bioreactor permitted an 1100-fold increment in total vector production without a decrease in specific vector yield (pfu/cell). The fed-batch bioreactor system afforded a rHSV-rep2/cap2 vector recovery of 2.8 x 10(12) pfu. The recovered vector was concentrated by tangential flow filtration (TFF), permitting vector stocks to be formulated at greater than 1.5 x 10(9) pfu/mL.     

Gene delivery and gene therapy with herpes simplex virus-based vectors

The development of efficient means of delivery genes in vivo is essential both for testing gene function in the intact animal and for human gene therapy procedures. A number of viral and non-viral gene delivery methods have been developed for this purpose. Of those herpes simplex virus (HSV)-based vectors have particular advantages for gene delivery to the nervous system including their ability to infect non-dividing neurones and establish asymptomatic latent infections. Moreover, considerable progress has been made, firstly, in disabling HSV vectors so as to prevent the damaging effects of wild type virus and secondly, to ensure long-term expression of the inserted transgene(s). These vectors thus offer a valuable tool for testing gene function in neuronal cells in vivo and may ultimately be safe enough for use in human gene therapy procedures.     

Effect of protease inhibitors on yield of HSV-1-based viral vectors

The ability to obtain high titer replication-defective herpes simplex virus (HSV) recombinant vectors will dramatically affect their use in gene therapy clinical trials. A variety of techniques and reagents have been employed to increase the overall yield of the vector. The effects of protease inhibitors on the yield of an HSV-1-based viral vector were examined. Experiments were conducted using a commercial protease inhibitor cocktail typically used in mammalian cell culture for protein production. Contrary to our expectation for enhanced vector yield, the results showed a dramatic reduction in vector yield. Moreover, it was found that AEBSF is the only component in the protease cocktail responsible for the low vector yield. On the basis of our hypothesis regarding the mode of action of AEBSF, we suggest that it should not be included in protease inhibitor cocktails designed for use in cultures aimed at production of viral vectors derived from HSV-1 or possibly several other vectors.     

Comparison of manufacturing techniques for adenovirus production

We have compared three different production methods, which may be suitable for the large scale production of adenovirus vectors for human clinical trials. The procedures compared 293 cells adapted to suspension growth in serum-free medium in a stirred tank bioreactor, 293 cells on microcarriers in serum-containing medium in a stirred tank bioreactor, and 293 cells grown in standard tissue culture plasticware. With a given virus, yields varied between 2000 and 10,000 infectious units/cell. The stirred tank bioreactor routinely produced between 4000 and 7000 infectious units/cell when 293 cells were grown on microcarriers. The 293 cells adapted to suspension growth in serum-free medium in the same stirred tank bioreactor yielded between 2000 and 7000 infectious units/cell. Yields obtained from standard tissue culture plasticware were up to 10,000 infectious units/cell. Cell culture conditions were monitored for glucose consumption, lactate production, and ammonia accumulation. Glucose consumption and lactate accumulation correlated well with the cell growth parameters. Ammonia production does not appear to be significant. Based on virus yields, ease of operation and linear scalability, large-scale adenovirus production seems feasible using 293 cells (adapted to suspension/serum free medium or on microcarriers in serum containing medium) in a stirred tank bioreactor. This revised version was published online in July 2006 with corrections to the Cover Date.     

Gene delivery using herpes simplex virus vectors

Herpes simplex virus (HSV) is a neurotropic DNA virus with many favorable properties as a gene delivery vector. HSV is highly infectious, so HSV vectors are efficient vehicles for the delivery of exogenous genetic material to cells. Viral replication is readily disrupted by null mutations in immediate early genes that in vitro can be complemented in trans, enabling straightforward production of high-titre pure preparations of non-pathogenic vector. The genome is large (152 Kb) and many of the viral genes are dispensable for replication in vitro, allowing their replacement with large or multiple transgenes. Latent infection with wild-type virus results in episomal viral persistence in sensory neuronal nuclei for the duration of the host lifetime. Transduction with replication-defective vectors causes a latent-like infection in both neural and non-neural tissue; the vectors are non-pathogenic, unable to reactivate and persist long-term. The latency active promoter complex can be exploited in vector design to achieve long-term stable transgene expression in the nervous system. HSV vectors transduce a broad range of tissues because of the wide expression pattern of the cellular receptors recognized by the virus. Increasing understanding of the processes involved in cellular entry has allowed preliminary steps to be taken towards targeting the tropism of HSV vectors. Using replication-defective HSV vectors, highly encouraging results have emerged from recent pre-clinical studies on models of neurological disease, including glioma, peripheral neuropathy, chronic pain and neurodegeneration. Consequently, HSV vectors encoding appropriate transgenes to tackle these pathogenic processes are poised to enter clinical trials.     

Development and optimization of herpes simplex virus vectors for multiple long-term gene delivery to the peripheral nervous system

Herpes simplex virus (HSV) has often been suggested as a suitable vector for gene delivery to the peripheral nervous system as it naturally infects sensory nerve terminals before retrograde transport to the cell body in the spinal ganglia where latency is established. HSV vectors might therefore be particularly appropriate for the study and treatment of chronic pain following vector administration by relatively noninvasive peripheral routes. However parameters allowing safe and efficient gene delivery to spinal ganglia following peripheral vector inoculation, or the long-term expression of delivered genes, have not been comprehensively studied. We have identified combinations of deletions from the HSV genome which allow highly efficient gene delivery to spinal dorsal root ganglia (DRGs) following either footpad or sciatic nerve injection. These vectors have ICP34.5 deleted and have inactivating mutations in vmw65. We also report that peripheral replication is probably necessary for the efficient establishment of latency in vivo, as fully replication-incompetent HSV vectors allow efficient gene expression in DRGs only after peripheral inoculation at a high virus dose. Very low transduction efficiencies are otherwise achieved. In parallel, promoters have been developed that allow the long-term expression of individual or pairs of genes in DRGs by using elements from the latently active region of the virus to confer a long-term activity onto a number of promoters which otherwise function only in the short term. This work further defines elements and mechanisms within the latently active region that are necessary for long-term gene expression and for the first time allows multiple inserted genes to be expressed from HSV vectors during latency.     

Defective Herpes Simplex Virus Vectors Expressing the Rat Brain Glucose Transporter Protect Cultured Neurons from Necrotic Insults

Abstract: Because neurons are postmitotic, they are irreplaceable once they succumb to necrotic insults such as hypoglycemia, ischemia, and seizure. A paucity of energy can exacerbate the toxicities of these insults; thus, a plausible route to protect neurons from necrotic injury would be to enhance their glucose uptake capability. We have demonstrated previously that defective herpes simplex virus (HSV) vectors overexpressing the rat brain glucose transporter (GT) gene ( gt ) can enhance glucose uptake in adult rat hippocampus and in hippocampal cultures. Furthermore, we have observed that such vectors can maintain neuronal metabolism during hypoglycemia and reduce kainic acid-induced seizure damage. In this study, we have developed bicistronic vectors that coexpressed gt and Escherichia coli lacZ as a reporter gene, which allows us to identify directly neurons that are infected with the vectors. Overexpression of GT from these vectors protected cultured hippocampal, spinal cord, and septal neurons against various necrotic insults, including hypoglycemia, glutamate, and 3-nitropropionic acid. Our observations demonstrate the feasibility of using HSV vectors to protect neurons from necrotic insults. Although this study has concentrated on the delivery of gt , other genes with therapeutic or protective capability might also be used.     

Purification of recombinant human growth hormone from CHO cell culture supernatant by Gradiflow preparative electrophoresis technology

Purification of recombinant human growth hormone (rhGH) from Chinese hamster ovary (CHO) cell culture supernatant by Gradiflow large-scale electrophoresis is described. Production of rhGH in CHO cells is an alternative to production in Escherichia coli, with the advantage that rhGH is secreted into protein-free production media, facilitating a more simple purification and avoiding resolubilization of inclusion bodies and protein refolding. As an alternative to conventional chromatography, rhGH was purified in a one-step procedure using Gradiflow technology. Clarified culture supernatant containing rhGH was passed through a Gradiflow BF200 and separations were performed over 60 min using three different buffers of varying pH. Using a 50 mM Tris/Hepes buffer at pH 7.5 together with a 50 kDa separation membrane, rhGH was purified to approximately 98% purity with a yield of 90%. This study demonstrates the ability of Gradiflow preparative electrophoresis technology to purify rhGH from mammalian cell culture supernatant in a one-step process with high purity and yield. As the Gradiflow is directly scalable, this study also illustrates the potential for the inclusion of the Gradiflow into bioprocesses for the production of clinical grade rhGH and other therapeutic proteins.     

Transformed lepidopteran cells expressing a protein of the silkmoth fat body display enhanced susceptibility to baculovirus infection and produce high titers of budded virus in serum-free media

Baculovirus vectors constitute important tools for therapeutic protein production and mammalian cell transduction for gene therapy applications. A prerequisite for such applications is that the cell lines in which baculoviruses are propagated be maintained in serum-free media that are devoid of potential human pathogens. However, in serum-free media, the performance of baculovirus-based systems can be significantly reduced. In this report, we show that silkmoth-derived host cell lines for the Bombyx mori-nuclear polyhedrosis virus (BmNPV) that are transformed with the gene for the promoting protein (PP), a silkmoth-derived secreted factor containing a lipid-binding domain, display enhanced susceptibility to BmNPV infection and enhanced budded virus productivity in serum-free media. For transformed silkmoth cells maintained in serum-free media, the rate of BmNPV entry is enhanced by two orders of magnitude relative to the untransformed cells, while the rate of budded virus production is increased five-fold. The infectivity-enhancing effect can be also conferred to normal cells grown in serum-free media by addition of conditioned media from the transformed cells, which contain the secreted recombinant PP. Thus, PP substitutes for serum factors whose presence facilitates baculovirus entry into the cells. However, the effects of silkmoth-derived PP may be specific to the BmNPV-silkmoth system since little or no changes in viral infectivity are obtained by PP expression in Trichoplusia ni-derived High-Fivetrade mark cells grown in serum-free media and infected with a different baculovirus (AcNPV).     

Modeling and optimization of the baculovirus expression vector system in batch suspension culture

A mathematical model has been developed that predicts the cell population dynamics and production of recombinant protein and infective extracellular virus progeny by insect cells after infection with baculovirus in batch suspension culture. Infection in the model is based on the rate of virus attachment to suspended insect cells under culture conditions. The model links the events following infection with the sequence of gene expression in the baculovirus replicative cycle. Substrate depletion is used to account for the decrease in product yield observed when infecting at high cell densities. Model parameters were determined in shaker flasks for two media: serum-supplemented IPL-41 medium and serum free Sf900II medium. There was good agreement between model predictions and the results from an independent series of experiments performed to validate the mode. The model predicted: (1) the optimal time of infection at high multiplicity of infection: (2) the timing and magnitude of recombinant protein production in a 2-L bioreactor; and (3) the timing and magnitude of recombinant protein production at multiplicities of infection from 0.01 to 100 plaque-forming units per cell. Through its ability to predict optimal infection strategies in batch suspension culture, the model has use in the design and optimization of large-scale systems for the production of recombinant products using the baculovirus expression vector system. (c) 1994 John Wiley & Sons, Inc.     

Cancer screening by systemic administration of a gene delivery vector encoding tumor-selective secretable biomarker expression

Cancer biomarkers facilitate screening and early detection but are known for only a few cancer types. We demonstrated the principle of inducing tumors to secrete a serum biomarker using a systemically administered gene delivery vector that targets tumors for selective expression of an engineered cassette. We exploited tumor-selective replication of a conditionally replicative Herpes simplex virus (HSV) combined with a replication-dependent late viral promoter to achieve tumor-selective biomarker expression as an example gene delivery vector. Virus replication, cytotoxicity and biomarker production were low in quiescent normal human foreskin keratinocytes and high in cancer cells in vitro. Following intravenous injection of virus >90% of tumor-bearing mice exhibited higher levels of biomarker than non-tumor-bearing mice and upon necropsy, we detected virus exclusively in tumors. Our strategy of forcing tumors to secrete a serum biomarker could be useful for cancer screening in high-risk patients, and possibly for monitoring response to therapy. In addition, because oncolytic vectors for tumor specific gene delivery are cytotoxic, they may supplement our screening strategy as a "theragnostic" agent. The cancer screening approach presented in this work introduces a paradigm shift in the utility of gene delivery which we foresee being improved by alternative vectors targeting gene delivery and expression to tumors. Refining this approach will usher a new era for clinical cancer screening that may be implemented in the developed and undeveloped world.     

Molecular biology of herpes simplex virus type 1 latency in the nervous system

Herpes simplex virus (HSV) is one of the best studied examples of viral ability to remain latent in the human nervous system and to cause recurrent disease by reactivation. Intensive effort was directed in recent years to unveil the molecular viral mechanisms and the virus-host interactions associated with latent HSV infection. The discovery of the state of the latent viral DNA in nervous tissues and of the presence of latency-associated gene expression during latent infection, both differing from the situation during viral replication, provided important clues relevant to the pathogenesis of latent HSV infection. This review summarizes the current state of knowledge on the site of latent infection, the molecular phenomena of latency, and the mechanisms of the various stages of latency: acute infection, establishment and maintenance of latency, and reactivation. This information paved the way to recent trials aiming to use herpes viruses as vectors to deliver genes into the nervous system, an issue that is also addressed in this review.     

Use of herpes virus amplicon vectors to study brain disorders

There is an enormous initiative to establish the genetic basis for disorders of brain function. Unfortunately, genetic intervention is not accomplished easily in the nervous system. One strategy is to engineer and deliver to neurons specialized viral vectors that carry a gene (or genes) of interest, thereby exploiting the natural ability of viruses to insert genetic material into cells. When delivered to brain cells, these vectors cause infected cells to increase the expression of the genes of interest. The ability to deliver genes into neurons in vitro and in vivo with herpes simplex virus (HSV) amplicon vectors has made it possible to carry out exactly these sorts of experiments. This technology has the potential to offer new insights into the etiology of a wide variety of neuropsychiatric disorders. We describe the use of HSV amplicon vectors to study Alzheimer disease, drug addiction, and depression, and discuss the considerations that enter into the use of these vectors both in vitro and in vivo. The HSV amplicon virus is a user-friendly vector for the delivery of genes into neurons that has come of age for the study of brain function.