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A trans-dominant negative and replication defective HSV-1 vector for in vivo and ex vivo gene transfer
DNA VIRUSES: OTHER developed an efficient infectious genomic DNA delivery vector called the infectious bacterial artificial chromosome, or iBAC, based on the herpes simplex virus type 1 (HSV-1) amplicon. We are applying the system to the analysis of two loci involved in genetic susceptibility to neurodegenerative disease, the microtubule associated protein tau (MAPT, or tau) locus and the α-synuclein (SNCA, or α-syn) locus. Three features make the vector well suited: i) the high capacity of HSV-1 amplicons allows us to package the entire tau or SNCA locus; ii) the neurotropism of HSV-1 amplicons ensures efficient gene delivery to primary neuronal cultures; iii) we can use BAC manipulation by homologous recombination to introduce mutations and polymorphisms for analysis. The tau protein is the major component of neurofibrillary tangles found in a range of neurodegenerative disorders, including Alzheimer’s disease, frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). Association studies have established a genetic link between the tau locus and PSP and CBD. The human tau locus lies on 17q21 and consists of 16 exons spanning 135 kb. Alternative splicing of exon 10 leads to a protein containing either three (3R; exon 10-) or four (4R; exon 10+) tandem repeats of a microtubule-binding motif. The regulation of exon 10 splicing control may underlie susceptibility to disease. To study tau splicing control we have constructed an iBAC vector carrying a 143 kb tau locus. The vector is packaged using an improved helper-virus free packaging system to titers of 2-3 x107 transducing units/ml, and we obtain efficient vector delivery to primary neuronal cultures prepared from tau-/- mice. We are now analyzing expression from the iBACtau vector. Parkinson’s Disease (PD) is the second most common neurodegenerative disease and affects about 1% of the Western population over 50 years of age. The specific neuropathological feature of PD is the formation of Lewy bodies, cytoplasmic protein aggregations composed mainly of α-synuclein found within affected neurons of the substantia nigra. SNCA mutations are found in rare forms of inherited autosomal dominant PD. However, the majority of PD cases have no mutations in the α-synuclein gene yet have αsynuclein protein deposits in Lewy bodies. Studies have shown an association of specific alleles of the SNCA locus, which differ in regulatory elements, with sporadic PD, and recent reports have shown triplication of the normal SNCA locus can cause PD. It seems likely that expression control of the SNCA locus will affect susceptibility to sporadic PD. The human SNCA locus contains 6 exons and spans 112 kb. We are building wild-type and recombinant iBAC-SNCA vectors to investigate the effect of sequence variation on the regulation of SNCA expression. The work exploits the high capacity and neurotropism of HSV-1 as a novel tool to study neurodegenerative disease.
797. A Trans-Dominant Negative and Replication Defective HSV-1 Vector for In Vivo and Ex Vivo Gene Transfer Christoph F. P. Theopold,1 Daniela Hoeller,1 Feng Yao.1 1 Laboratory of Tissue Repair and Gene Transfer, Div. of Plastic Surgery, Brigham and Women’s Hospital, Boston, MA. HSV replicates in epithelial cells and establishes life-long latent infection in neuronal cell bodies of the sensory ganglia. This dual life cycle, and its ability to package 30-50kb of transgene, make it an attractive vehicle for gene transfer. However, both replicationdefective and neuroattenuated viruses are replication competent in the context of wild-type virus, raising safety concerns about their use for human gene therapy. To address these concerns, we used the tetracycline repressormediated transcription repression gene switch technology (Yao et S302
al., Human Gene Therapy 9:1939-1950, 1998, T-REx™, Invitrogen Inc. CA) to construct a novel anti-HSV-1-specific HSV-1 recombinant virus, CJ83193, capable of inhibiting its own replication, as well as that of parental wild-type HSV-1 and HSV-2 (Yao and Eriksson, Hum Gene Ther 1999, 10:1811-1818; Antiviral Res 2002, 53:127-33). Notably, unlike the highly toxic single replicationdefective HSV mutants, CJ83193 exhibits little cytotoxicity in vitro: infection of Vero cells with an HSV-1 ICP27 deletion mutant, d27, killed 95% of infected cells (MOI of 30 PFU/cell) at 48 h postinfection, whereas close to 80% of CJ83193-infected cells remained viable for at least 5 days following the same multiplicity of infection. Given these combined unique characteristics, in this report we translated CJ83193 into a new class of HSV-1 viral vector, CJ9lacZ, to deliver therapeutic genes to various tissues or as a safe and effective vaccine vector for immunization. Specifically, we replaced the essential HSV-1 UL9 gene with the lacZ gene under the control of the hCMV immediate-early promoter. Thus, unlike CJ83193, which exhibits a very limited plaque forming ability on Vero cell monolayers with a reduction of a million-fold plaque-forming efficiency compared with its complementing tetR-expressing osteosarcoma cells, CJ9-lacZ is not only dominant-negative but also completely replication-defective. It is estimated that the plaque forming efficiency of CJ9-lacZ on Vero cell monolayers is reduced in a range of 1011- to 1012-fold compared with its complementing tetR- and UL9-expressing cells. Co-infection of Vero cells with wildtype HSV-1 strain KOS at a MOI of 1 PFU/cell and CJ9-lacZ at a MOI of 3 PFU/cell led to a 280-fold reduction in KOS synthesis compared with cells singly infected with KOS. To test whether CJ9-lacZ can function as an efficient vector for gene transfer, mouse footpads were inoculated with CJ9-lacZ followed by X-Gal staining at either 48 h or 5 days post-inoculation. It was observed that direct in vivo delivery of CJ9-lacZ led to strong LacZ expression in fibroblasts, myocytes, and adipocytes. No XGal positive staining cells could be detected in mock-infected footpads. Moreover, X-Gal positive cells could also be observed at day 7 post-inoculation, although it seems that the number of X-Gal positive stained cells was reduced as compared with staining on days 2 and 5. These results demonstrate that CJ9-lacZ can be used as an efficient vector for gene transfer to various tissues in vivo. Experiments to test CJ9lacZ as a vector for gene delivery to the brain are currently being carried out.
798. Intracarotid Delivery of Oncolytic Herpes ∆ to Metastatic Breast Cancer Simplex Virus-1 G47∆ in the Brain Renbin Liu,1 Robert L. Martuza,1 Samuel D. Rabkin.1 1 Neurosurgery, Massachusetts General Hospital, Charlestown, MA. Breast cancer is the most frequently occurring malignant disease in women with a lifetime risk of 1:8 to 1:10 in the United States. Approximately 20% of breast cancer patients have brain metastases, which are the most important cause of the morbidity and mortality. The blood brain barrier (BBB) separates the brain’s interstitial space from the blood and prevents the penetrance of circulating molecules and cells into the brain. This is a significant impediment to systemic delivery in the brain. Oncolytic replication-competent herpes simplex virus (HSV) can infect, replicate and kill tumor cells by a direct cytopathic effect and then spread within the tumor sparing normal cells. G47D is a third generation vector, which has deletions of the g34.5 and a47 genes, and an insertion of E. coli lacZ inactivating the ICP6 gene (ribonucleotide reductase large subunit). As a model for breast cancer metastatic to the brain, we implanted human MDA-MB-435 breast adenocarcinoma cells in the brains of nude mice. These cells are very susceptible to HSV infection and tumors can be treated by oncolytic Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy
797. A Trans-Dominant Negative and Replication Defective HSV-1 Vector for In Vivo and Ex Vivo Gene Transfer Christoph F. P. Theopold,1 Daniela Hoeller,1 Feng Yao.1 1 Laboratory of Tissue Repair and Gene Transfer, Div. of Plastic Surgery, Brigham and Women’s Hospital, Boston, MA. HSV replicates in epithelial cells and establishes life-long latent infection in neuronal cell bodies of the sensory ganglia. This dual life cycle, and its ability to package 30-50kb of transgene, make it an attractive vehicle for gene transfer. However, both replicationdefective and neuroattenuated viruses are replication competent in the context of wild-type virus, raising safety concerns about their use for human gene therapy. To address these concerns, we used the tetracycline repressormediated transcription repression gene switch technology (Yao et S302
al., Human Gene Therapy 9:1939-1950, 1998, T-REx™, Invitrogen Inc. CA) to construct a novel anti-HSV-1-specific HSV-1 recombinant virus, CJ83193, capable of inhibiting its own replication, as well as that of parental wild-type HSV-1 and HSV-2 (Yao and Eriksson, Hum Gene Ther 1999, 10:1811-1818; Antiviral Res 2002, 53:127-33). Notably, unlike the highly toxic single replicationdefective HSV mutants, CJ83193 exhibits little cytotoxicity in vitro: infection of Vero cells with an HSV-1 ICP27 deletion mutant, d27, killed 95% of infected cells (MOI of 30 PFU/cell) at 48 h postinfection, whereas close to 80% of CJ83193-infected cells remained viable for at least 5 days following the same multiplicity of infection. Given these combined unique characteristics, in this report we translated CJ83193 into a new class of HSV-1 viral vector, CJ9lacZ, to deliver therapeutic genes to various tissues or as a safe and effective vaccine vector for immunization. Specifically, we replaced the essential HSV-1 UL9 gene with the lacZ gene under the control of the hCMV immediate-early promoter. Thus, unlike CJ83193, which exhibits a very limited plaque forming ability on Vero cell monolayers with a reduction of a million-fold plaque-forming efficiency compared with its complementing tetR-expressing osteosarcoma cells, CJ9-lacZ is not only dominant-negative but also completely replication-defective. It is estimated that the plaque forming efficiency of CJ9-lacZ on Vero cell monolayers is reduced in a range of 1011- to 1012-fold compared with its complementing tetR- and UL9-expressing cells. Co-infection of Vero cells with wildtype HSV-1 strain KOS at a MOI of 1 PFU/cell and CJ9-lacZ at a MOI of 3 PFU/cell led to a 280-fold reduction in KOS synthesis compared with cells singly infected with KOS. To test whether CJ9-lacZ can function as an efficient vector for gene transfer, mouse footpads were inoculated with CJ9-lacZ followed by X-Gal staining at either 48 h or 5 days post-inoculation. It was observed that direct in vivo delivery of CJ9-lacZ led to strong LacZ expression in fibroblasts, myocytes, and adipocytes. No XGal positive staining cells could be detected in mock-infected footpads. Moreover, X-Gal positive cells could also be observed at day 7 post-inoculation, although it seems that the number of X-Gal positive stained cells was reduced as compared with staining on days 2 and 5. These results demonstrate that CJ9-lacZ can be used as an efficient vector for gene transfer to various tissues in vivo. Experiments to test CJ9lacZ as a vector for gene delivery to the brain are currently being carried out.
798. Intracarotid Delivery of Oncolytic Herpes ∆ to Metastatic Breast Cancer Simplex Virus-1 G47∆ in the Brain Renbin Liu,1 Robert L. Martuza,1 Samuel D. Rabkin.1 1 Neurosurgery, Massachusetts General Hospital, Charlestown, MA. Breast cancer is the most frequently occurring malignant disease in women with a lifetime risk of 1:8 to 1:10 in the United States. Approximately 20% of breast cancer patients have brain metastases, which are the most important cause of the morbidity and mortality. The blood brain barrier (BBB) separates the brain’s interstitial space from the blood and prevents the penetrance of circulating molecules and cells into the brain. This is a significant impediment to systemic delivery in the brain. Oncolytic replication-competent herpes simplex virus (HSV) can infect, replicate and kill tumor cells by a direct cytopathic effect and then spread within the tumor sparing normal cells. G47D is a third generation vector, which has deletions of the g34.5 and a47 genes, and an insertion of E. coli lacZ inactivating the ICP6 gene (ribonucleotide reductase large subunit). As a model for breast cancer metastatic to the brain, we implanted human MDA-MB-435 breast adenocarcinoma cells in the brains of nude mice. These cells are very susceptible to HSV infection and tumors can be treated by oncolytic Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy