Production and Purification of Highly Replication Defective HSV-1 Based Gene Therapy Vectors (original) (raw)
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Evaluation of Infection Parameters in the Production of Replication-Defective HSV-1 Viral Vectors
Biotechnology Progress, 2002
Herpes simplex virus type-1 (HSV-1) is a neurotrophic human pathogen that establishes life-long latency in the nervous system. Our laboratory has extensively engineered this virus to retain the ability to persist in neurons without expression of lytic genes or disease phenotype. Highly defective, replication-incompetent HSV mutants are thus potentially ideal for transfer of therapeutic transgenes to human nerves where long-term therapy of nervous system disease may be provided. A prerequisite for using recombinant HSV vectors for therapeutic gene delivery to humans is the development of methods for large-scale manufacture of HSV vectors.
Generation of Replication-Competent and -Defective HSV Vectors
Cold Spring Harbor Protocols, 2011
INTRODUCTIONEngineering effective vectors has been crucial to the efficient delivery and expression of therapeutic gene products in vivo. Among these, HSV-1 represents an excellent candidate vector for delivery to the peripheral and central nervous systems. The natural biology of HSV-1 includes the establishment of a lifelong latent state in neurons in which the viral genome persists as an episomal molecule. Genomic HSV vectors can be produced that are completely replication-defective, nontoxic, and capable of long-term transgene expression. Herpes simplex virus (HSV) vectors are constructed by using a replication-deficient vector backbone (TOZ.1) for homologous recombination with a shuttle plasmid containing a cassette expressing the gene of interest inserted into the UL41 gene sequence. The TOZ.1 vector expresses a reporter gene (lacZ) in the UL41 locus, such that recombination of the transgenic cassette into the UL41 locus results in the loss of the reporter gene activity. The TO...
Gene Correction Technology and Its Impact on Viral Research and Therapy
Modern Tools for Genetic Engineering, 2016
Aims 1. To explain why gene correction technology is a useful tool for studying chronic/ latent viral infections. 2. To explain how gene editing technology may facilitate or restrict virus replication and impact on future therapy. 3. To cite specific examples of how gene correction technology is being applied to target specific viruses, including HIV, hepatitis B virus (HBV), herpes simplex virus (HSV), and other viruses. Methodology We attempted to identify all scientific publications including basic science, translation research, and any clinical trials involving DNA correction technology [zinc finger endonuclease (ZFN), transcription activator-like endonucleases (TALENs), and CRISPR/Cas-based systems] and persistent viral infections [including but not limited to HIV, hepatitis B, C, and D viruses, herpes viruses, cytomegalovirus, Epstein-Barr virus (EBV), human papillomavirus (HPV), measles virus, and varicella-zoster virus] published on or before December 31, 2015. We conducted searches of MEDLINE, Cochrane Central Register, and EMBASE. The identified papers have been summarized and organized into relevant sections within the chapter. Conclusion Sequence-specific DNA endonucleases target and destroy DNA viruses, with early work describing the use of ZFNs, TALENs, or a third type of endonuclease, called a homing endonuclease (HE), to target HBV, HPV, and HSV-1 with varying degrees of success. The new CRISPR/Cas9 systems do not allow virologists to screen for host genes that affect the replication of pathogenic human viruses but to derive human cell lines that are genetically engineered to either facilitate or suppress viral replication. Scientists now
Development and application of replication-incompetent HSV-1-based vectors
Gene Therapy, 2005
The replication-incompetent HSV-1-based vectors are herpesviruses in which genes that are 'essential' for viral replication have been either mutated or deleted. These deletions have substantially reduced their cytotoxicity by preventing early and late viral gene expression and, together with other deletions involving 'nonessential' genes, have also created space to introduce distinct and independently regulated expression cassettes for different transgenes. Therapeutic effects in gene therapy applications requiring simultaneous and synergic expression of multiple gene products are easily achievable with these vectors. A number of different HSV-1-based nonreplicative vectors for specific gene therapy applications have been developed so far. They have been tested in different gene therapy animal models of neuropathies (Parkinson's disease, chronic pain, spinal cord injury pain) and lysosomal storage disorders. Many replicationincompetent HSV-1-based vectors have also been used either as potential anti-herpes vaccines, as well as vaccine vectors for other pathogens in murine and simian models. Anticancer gene therapy approaches have also been successfully set up; gene therapy to other targets by using these vectors is feasible.
Engineering HSV-1 Vectors for Gene Therapy
Methods in Molecular Biology, 2014
Virus vectors have been employed as gene transfer vehicles for various preclinical and clinical gene therapy applications, and with the approval of Glybera (alipogene tiparvovec) as the fi rst gene therapy product as a standard medical treatment (Yla-Herttuala, Mol Ther 20: 1831-1832, gene therapy has reached the status of being a part of standard patient care. Replication-competent herpes simplex virus (HSV) vectors that replicate specifi cally in actively dividing tumor cells have been used in Phase I-III human trials in patients with glioblastoma multiforme, a fatal form of brain cancer, and in malignant melanoma. In fact, T-VEC (talimogene laherparepvec, formerly known as OncoVex GM-CSF) displayed effi cacy in a recent Phase III trial when compared to standard GM-CSF treatment alone (Andtbacka et al. J Clin Oncol 31: sLBA9008, 2013) and may soon become the second FDA-approved gene therapy product used in standard patient care. In addition to the replication-competent oncolytic HSV vectors like T-VEC, replicationdefective HSV vectors have been employed in Phase I-II human trials and have been explored as delivery vehicles for disorders such as pain, neuropathy, and other neurodegenerative conditions. Research during the last decade on the development of HSV vectors has resulted in the engineering of recombinant vectors that are totally replication defective, nontoxic, and capable of long-term transgene expression in neurons. This chapter describes methods for the construction of recombinant genomic HSV vectors based on the HSV-1 replication-defective vector backbones, steps in their purifi cation, and their small-scale production for use in cell culture experiments as well as preclinical animal studies.
Standardizing viral vector manufacture: maximizing production with the TRiP SystemTM
Cell and Gene Therapy Insights, 2018
The use of viral vectors to create novel gene-and cell-based medicines is now a reality. As gene therapy matures into a new era, the industry will need to adopt improvements to viral vector manufacturing to meet the demand for GMP grade material. The activities of process optimisation/ characterisation with any given viral vector entering into clinical development are considerable. The cell culture (Upstream) and purification/ concentration (Downstream) aspects of this process are multi-faceted. The amount of vector produced during Upstream can vary depending on the transgene encoded, especially if the active protein is expressed in the production cell when constitutive or leaky tissue specific promoters are employed. Oxford BioMedica has developed the Transgene Repression In vector Production (TRiP) System™ to recover vector titres compromised by transgene expression. The system utilises the bacterial protein TRAP and its short RNA binding sequence-inserted within the transgene leader sequence-to repress transgene mRNA translation during vector production only, leaving expression unaffected in target cells. The TRiP System™ has been used to fully recover titres of Lenti, Adeno and AAV based vectors, and is expected to be universally applicable to any viral vector/vaccine platform. We anticipate that the TRiP System™ will enable new gene therapies to be considered, and assist those already in development to become commercially viable. Given that only TRAP and the viral proteins of the vector platform being employed will be expressed during Upstream, the TRiP System™ opens the door to 'plug-and-play' manufacturing, greatly minimising the burden of process development within a given pipeline.
Biotechnology and Bioengineering, 2002
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 signi®cantly impact vector yield. An ideal vector should be de®cient 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 wildtype 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¯ask system for vector production with signi®cant savings with respect to time, labor, and cost. ã