Gene transfer using a disabled herpes virus vector containing the EMCV IRES allows multiple gene expression in vitro and in vivo

Wagstaff, Marcus J. D.; Lilley, Caroline E.; Smith, Jeremy; Robinson, Mark; Rs, Coffin; Latchman, David S.

doi:10.1038/sj.gt.3300754

Cited by 25 publications

(13 citation statements)

References 6 publications

(8 reference statements)

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“…27,[41][42][43][44] In this study, to identify novel IRES sequences that can improve expression vectors, we conducted in vitro and in vivo screening of 13 IRES elements from viral or cellular origin.…”

Section: Discussionmentioning

confidence: 99%

In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression

et al. 2011

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Bicistronic vectors are essential to achieve efficient expression of multiple genes in gene therapy protocols and biomedical applications. Internal ribosome entry site (IRES) elements have been utilized to initiate expression of an additional protein from a bicistronic vector. The IRES element commonly used in current bicistronic vectors originates from the encephalomyocarditis virus (EMCV). As IRES-mediated translation is dependent on availability of IRES trans-acting factors, which vary between cell types and species, adequate gene expression from the EMCV IRES element is not always achieved. To identify a novel IRES element that mediates gene expression consistently with a higher efficiency than the EMCV IRES, we tested 13 bicistronic reporter constructs containing different viral and cellular IRES elements. The in vitro screening in human and mouse fibroblast and hepatocarcinoma cells revealed that the vascular endothelial growth factor and type 1 collagen-inducible protein (VCIP) IRES was the only IRES element that directed translation more efficiently than the EMCV IRES in all cell lines. Furthermore, the VCIP IRES initiated greater reporter expression levels than the EMCV IRES in transfected mouse livers. These results suggest that VCIP-IRES containing vectors improve gene expression compared with those harboring an EMCV-IRES. This could increase the potential benefits of bicistronic vectors for experimental and therapeutic purposes.

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Section: Discussionmentioning

confidence: 99%

In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression

et al. 2011

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“…) Replication-incompetent viruses. Virus strain 17ϩ/27Ϫ/pR19lacZ has previously been described (42) and contains a CMV-lacZ cassette inserted into the LAT region (as shown in Fig. 5) of HSV strain 17ϩ from which ICP27 has been deleted.…”

Section: Methodsmentioning

confidence: 99%

“…tested for gene delivery to DRGs following footpad and sciatic nerve inoculation. These viruses either had ICP27 alone deleted, had ICP27 and ICP34.5 deleted and had the in1814 vmw65 mutation; or had ICP27, ICP4, and ICP34.5 deleted and had the in1814 vmw65 mutation (40,42) (Fig. 10A).…”

Section: Vol 74 2000mentioning

confidence: 99%

Development and Optimization of Herpes Simplex Virus Vectors for Multiple Long-Term Gene Delivery to the Peripheral Nervous System

Palmer

Branston

Lilley

et al. 2000

J Virol

112

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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.

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“…Often, particularly in the study of in situ protein-protein interactions, an experimental protocol depends on the co-expression of two genes in the same cell. While vectors have been described that express multiple gene products either using separate expression cassettes (4) or as multiple genes driven by the same promoter using an internal ribosome entry site (8), these methods do not allow the combinatorial flexibility afforded by multiple infection with separate vectors. An implicit assumption in co-infection experiments is that the expression of each virally introduced gene is independent of the other; the proportion of singly and doubly infected cells would then be expected to fit a binomial distribution.…”

Section: Introductionmentioning

confidence: 99%

Expression of Multiple Proteins within Single Primary Cortical Neurons Using a Replication Deficient HSV Vector

Coopersmith

Neve

1999

BioTechniques

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The study of protein-protein interactions in the nervous system has become dependent on the ability to express foreign proteins (or to overexpress endogenous proteins) within neurons. Often, multiple genes need to be overexpressed in the same cell. To investigate the simultaneous co-expression of more than one virally introduced gene in primary cortical neurons, we infected cultures with two different herpes simplex virus (HSV) vectors and analyzed the proportion of singly and doubly infected cells. The vast majority of neurons expressed both gene products, with a smaller number expressing one or the other protein alone. Increasing the quantity of virus caused an increase in the proportion of doubly labeled cells at the expense of singly labeled cells, which is consistent with a model in which infection with one viral vector is independent of infection with the other. We conclude that co-infection with HSV vectors is an efficient way to obtain expression of multiple gene products within individual primary culture neurons. INTRODUCTIONIncreasingly, infection with recombinant viral vectors is being used for the delivery of genes into primary neuronal cells (1,3,(5)(6)(7). Often, particularly in the study of in situ protein-protein interactions, an experimental protocol depends on the co-expression of two genes in the same cell. While vectors have been described that express multiple gene products either using separate expression cassettes (4) or as multiple genes driven by the same promoter using an internal ribosome entry site (8), these methods do not allow the combinatorial flexibility afforded by multiple infection with separate vectors. An implicit assumption in co-infection experiments is that the expression of each virally introduced gene is independent of the other; the proportion of singly and doubly infected cells would then be expected to fit a binomial distribution. Alternatively, successful infection of a cell with one viral vector might diminish or even preclude infection with the second vector.To distinguish between these possibilities, we infected primary neuronal cultures with replication-defective herpes simplex virus (HSV) vectors expressing either a fusion protein of the neuronal growth-associated protein GAP-43 with sGFP (HSVGFPGAP) and/or Escherichia coli β-galactosidase (β-gal) (HSVlac). Counts of singly and doubly infected cells suggest that successful infection by each of the two vectors is an independent event, yielding a high proportion of double-infected neurons. MATERIALS AND METHODS Generation of Recombinant HSV VectorsReplication-defective HSV vectors expressing sGFPGAP cDNA or β-gal in the plasmid pHSVPrpUC were prepared as previously described (2,5). The titer of the helper virus component of each stock was 1-1.2 × 10 6 plaqueforming units (pfu)/mL on 2-2 cells. The titer of the recombinant virus component of each stock, as assayed by expression of the exogenous gene in PC12 cells, was consistently 3 × 10 7 infectious units (iu)/mL. Primary Neuronal CulturesPrimary cortica...

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Gene transfer using a disabled herpes virus vector containing the EMCV IRES allows multiple gene expression in vitro and in vivo

Cited by 25 publications

References 6 publications

In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression

In vitro and in vivo comparison of viral and cellular internal ribosome entry sites for bicistronic vector expression

Development and Optimization of Herpes Simplex Virus Vectors for Multiple Long-Term Gene Delivery to the Peripheral Nervous System

Expression of Multiple Proteins within Single Primary Cortical Neurons Using a Replication Deficient HSV Vector

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