Ziv Sandalon - Academia.edu (original) (raw)
Papers by Ziv Sandalon
PubMed, Jun 15, 1997
Tyrphostins are low molecular weight compounds that specifically inhibit protein tyrosine kinases... more Tyrphostins are low molecular weight compounds that specifically inhibit protein tyrosine kinases. We studied the effects of tyrphostins on OCI-Ly8, a cell line derived from a patient with immunoblastic lymphoma that carries the t(14;18) translocation and overexpresses the B-cell lymphoma/leukemia-2 gene (bcl-2). To test the possibility that tyrphostins induce apoptosis in these cells, overcoming the protection rendered by bcl-2, we screened 16 tyrphostins representing different families at a concentration of 0.5-50 microM. We found that AG17 was the most potent in this regard. Cell cycle analysis demonstrated that AG17 induces arrest at the G1 phase followed by apoptosis with general reduction of the intracellular level of tyrosine-phosphorylated proteins. To further elucidate the mechanism of action of AG17, we investigated its effect on some of the key proteins that regulate the cell cycle. Bcl-2 and cdk2 protein levels were not altered with AG17, whereas cdk2 kinase activity, as well as p21 and p16 protein levels, were reduced markedly. These results suggest that the target of AG17 is inactivation of cdk2. Because lymphoma cells with the t(14;18) translocation and bcl-2 overexpression are resistant to chemotherapy, novel drugs selectively able to induce apoptosis in these cells could offer a new approach to the treatment of lymphoma patients.
Journal of Virology, Nov 15, 2000
Mini-adenoviruses (mAd) deleted of all viral coding regions represent an emerging approach for tr... more Mini-adenoviruses (mAd) deleted of all viral coding regions represent an emerging approach for transgene expression. We have exploited the unique features of the adeno-associated virus (AAV) terminal repeats within the context of an adenovirus-adeno-associated hybrid virus (Ad/AAV) as a strategy for rapid and efficient generation of mAd. Excision and generation of mAd from the parental Ad/AAV hybrid vector was achieved in 293 cells through recombination but without selection for mAd production. Analysis of mAd isolated from 293 cells indicated that mAd DNA exists as monomer and dimer forms within the recombinant viral capsid. Formation of recombinant mAd was significantly increased using an AAV Rep78-or Rep68-expressing cell line through Rep-mediated excision utilizing the AAV terminal repeat sequences present in the Ad/AAV hybrid virus genome. The mAd viruses were infectious and able to transfer functional gene to A549 and HeLa cells. This approach is rapid and efficient, thereby providing a simplified methodology for generating mAd with functional transducing capabilities.
Journal of Virology, 1992
Encapsidation of simian virus 40 is a complex biological process involving DNA-protein and protei... more Encapsidation of simian virus 40 is a complex biological process involving DNA-protein and protein-protein interactions in the formation of a unique three-dimensional structure around the viral minichromosome. A pseudoviral system developed in our laboratory, in which the viral early and late gene products are supplied in trans (by helpers), was used to analyze the encapsidation process independent of viral gene expression. With this experimental system we have discovered a requirement for a specific DNA signal for encapsidation, ses (for simian virus 40 encapsidation signal).ses is present within a 200-bp DNA fragment, which includes, in addition to the viral origin of replication (ori), six GGGCGG repeats (GC boxes) and 26 bp of the enhancer element. Deletion of the GC boxes and the enhancer sequences almost abolished encapsidation, while DNA replication was only moderately decreased. The ability to encapsidate was not regained by reinserting a DNA fragment carrying ses in the ses...
Cell and Gene Therapy Insights, 2021
Journal of Molecular Biology, 1994
CANCER RESEARCH, 1997
Page 1. ICANCERRESEARCH57. 2434-2439. June 15. 9971 frequently observed cytogenetic abnormality i... more Page 1. ICANCERRESEARCH57. 2434-2439. June 15. 9971 frequently observed cytogenetic abnormality in malignant lymphoma (found in 90% of cases with follicular lymphoma and in 25â30%of patients with diffuse large cell lymphoma; Ref. 21). ...
Biologicals, 2021
The HIV-1 derived gp145 protein is being investigated by research groups as preclinical studies h... more The HIV-1 derived gp145 protein is being investigated by research groups as preclinical studies have shown high promise for this protein as a vaccine against HIV. However, one of the main challenges with manufacturing this promising protein has been ascribed to the low yield obtained in mammalian cell cultures. Significant improvements in gp145 production are needed to address this issue to test the gp145 protein as a potentially effective, safe, and affordable HIV vaccine. Here we describe the application of a novel expression technology to create GMP-grade CHO cell lines expressing approximately 50 μg/ml in non-optimized fed-batch culture, which is an order of magnitude higher than that obtained in existing processes. Top producing clones show a high degree of similarity in the glycosylation patterns of the purified protein to the reference standard. Conformational integrity and functionality was demonstrated via high-affinity binding to soluble CD4, using a panel of antibodies including VRC01, F105, Hk20, PG9 and 17b. In summary, we were able to generate CHO cell lines expressing HIV gp145 with significantly higher overall expression yields than currently accessible, and high product quality that could potentially be suitable for future studies assessing the efficacy and safety of gp145based HIV vaccines.
Journal of virology, 2004
This study evaluated and compared delivery of the tumor necrosis factor alpha receptor (TNFR)-imm... more This study evaluated and compared delivery of the tumor necrosis factor alpha receptor (TNFR)-immunoglobulin G1 (IgG1) Fc fusion (TNFR:Fc) gene to the lung by single and repeat administrations of multiple pseudotyped adeno-associated virus (AAV) vectors as a means for achieving systemic distribution of the soluble TNFR:Fc protein. A single endotracheal administration of AAV[2/5]cytomegalovirus (CMV)-TNFR:Fc vector (containing the AAV2 inverted terminal repeats and AAV5 capsid) to the rat lung resulted in long-term, high levels of serum TNFR:Fc protein that gradually declined over a period of 8 months. Endotracheal delivery of AAV[2/1]CMV-TNFR:Fc resulted in serum TNFR:Fc protein levels that were detectable for at least 4 months but were 10-fold lower than that of the AAV[2/5] vector. In contrast, secretion of the TNFR:Fc protein following pulmonary delivery of AAV[2/2]CMV-TNFR:Fc vector was very inefficient, and the protein was detected in the blood only when an airway epithelial ce...
Molecular Therapy, 2006
ABSTRACT Molecular Therapy (2006) 13, S223|[ndash]|S223; doi: 10.1016/j.ymthe.2006.08.651 578. Su... more ABSTRACT Molecular Therapy (2006) 13, S223|[ndash]|S223; doi: 10.1016/j.ymthe.2006.08.651 578. Suppression of Inflammation Following Intramuscular Administration of Pseudotyped AAV-TNFR:Fc Vectors in a Rat Model of Arthritis|[ast]| Haim Burstein1, Kurt Lustig1 and Ziv Sandalon11Research, Targeted Genetics Corporation, Seattle, WA|[ast]|This study provides proof of concept for long-term suppression of experimental arthritis in rats, following intramuscular delivery of AAV-TNFR:Fc vectors and represent a novel therapeutic approach for chronic inflammatory arthritis.
SV40 Protocols, 2000
ABSTRACT The use of insect cells and lytic baculovirus for expression of biologically active mamm... more ABSTRACT The use of insect cells and lytic baculovirus for expression of biologically active mammalian proteins has been the method of choice of many investigators. Although prokaryotic expression systems provide higher yields and are technically simpler to use, obtaining biologically active eukaryotic proteins in these systems can be problematic. Posttranslational modifications such as glycosylation and phosphorylation do not occur in prokaryotic systems owing to the lack of enzymatic machinery. On the other hand, insect cells provide an appropriate environment for posttranslational modifications and lead to proper folding and correct assembly of recombinant proteins (1).
Molecular Therapy, 2006
We have presented previously that high dose airway delivery of a pseudotyped AAV2/5 vector to the... more We have presented previously that high dose airway delivery of a pseudotyped AAV2/5 vector to the lungs of Rhesus macaques resulted in enhanced gene transfer and quantitative RNA expression as well as protein expression in non human primates without clinical toxicity. One major limitation to the success of AAV gene therapy for CF is that the large size of the CFTR cDNA insert fills the packaging capacity of AAV viral particles precluding inclusion of a highly efficient promoter. In order to overcome this limitation AAV2/ 5 pseudotyped vectors were generated expressing a truncated CFTR insert (∆264) driven by a chicken beta actin promoter (rAAV-CB-∆264-CFTR). We showed previously that this truncated form was capable of reconstituting physiologic function in vitro and in mouse models.
Virology, 1997
Soluble SV40 capsid proteins were obtained by expression of the three late genes, VP1, VP2, and V... more Soluble SV40 capsid proteins were obtained by expression of the three late genes, VP1, VP2, and VP3, in Sf9 cells using baculovirus expression vectors. Coproduction of the capsid proteins VP1, VP2, and VP3 was achieved by infecting Sf9 cells with the three recombinant baculovirus species at equal multiplicities. All three proteins were found to be localized in the nuclear fraction. Electron microscopy of nuclear extracts of the infected cells showed an abundance of SV40-like capsid structures and heterogeneous aggregates of variable size, mostly 20-45 nm. Under the same staining conditions wild-type SV40 virions are 45 nm. The capsid-like particles sedimented in glycerol gradients similarly to authentic wild-type SV40 virions. Pentamers of the major capsid protein VP1 were also seen. Protein analysis on sucrose gradients demonstrated that the capsid-like particles can be disrupted by treatment with the reducing agent dithiothreitol and the calcium chelator EGTA. The capsid-like particles were found to be significantly less stable than SV40 virions and were partially stabilized by calcium ions. Understanding the complex interactions between the capsid proteins is important for the development of an efficient in vitro packaging system for SV40 virions and pseudovirions. ᭧ 1997 Academic Press
Molecular Therapy, 2004
Phenylketonuria (PKU) is one of the most common single gene disorders in man. The metabolic defec... more Phenylketonuria (PKU) is one of the most common single gene disorders in man. The metabolic defect arises from a deficiency of phenylalanine hydroxylase (PAH). There is a resultant hyperphenylalanemia with subsequent impairment in cognitive abilities, executive functions and motor coordination; these functions are associated with various dopaminergic tracts.
PubMed, Jun 15, 1997
Tyrphostins are low molecular weight compounds that specifically inhibit protein tyrosine kinases... more Tyrphostins are low molecular weight compounds that specifically inhibit protein tyrosine kinases. We studied the effects of tyrphostins on OCI-Ly8, a cell line derived from a patient with immunoblastic lymphoma that carries the t(14;18) translocation and overexpresses the B-cell lymphoma/leukemia-2 gene (bcl-2). To test the possibility that tyrphostins induce apoptosis in these cells, overcoming the protection rendered by bcl-2, we screened 16 tyrphostins representing different families at a concentration of 0.5-50 microM. We found that AG17 was the most potent in this regard. Cell cycle analysis demonstrated that AG17 induces arrest at the G1 phase followed by apoptosis with general reduction of the intracellular level of tyrosine-phosphorylated proteins. To further elucidate the mechanism of action of AG17, we investigated its effect on some of the key proteins that regulate the cell cycle. Bcl-2 and cdk2 protein levels were not altered with AG17, whereas cdk2 kinase activity, as well as p21 and p16 protein levels, were reduced markedly. These results suggest that the target of AG17 is inactivation of cdk2. Because lymphoma cells with the t(14;18) translocation and bcl-2 overexpression are resistant to chemotherapy, novel drugs selectively able to induce apoptosis in these cells could offer a new approach to the treatment of lymphoma patients.
Journal of Virology, Nov 15, 2000
Mini-adenoviruses (mAd) deleted of all viral coding regions represent an emerging approach for tr... more Mini-adenoviruses (mAd) deleted of all viral coding regions represent an emerging approach for transgene expression. We have exploited the unique features of the adeno-associated virus (AAV) terminal repeats within the context of an adenovirus-adeno-associated hybrid virus (Ad/AAV) as a strategy for rapid and efficient generation of mAd. Excision and generation of mAd from the parental Ad/AAV hybrid vector was achieved in 293 cells through recombination but without selection for mAd production. Analysis of mAd isolated from 293 cells indicated that mAd DNA exists as monomer and dimer forms within the recombinant viral capsid. Formation of recombinant mAd was significantly increased using an AAV Rep78-or Rep68-expressing cell line through Rep-mediated excision utilizing the AAV terminal repeat sequences present in the Ad/AAV hybrid virus genome. The mAd viruses were infectious and able to transfer functional gene to A549 and HeLa cells. This approach is rapid and efficient, thereby providing a simplified methodology for generating mAd with functional transducing capabilities.
Journal of Virology, 1992
Encapsidation of simian virus 40 is a complex biological process involving DNA-protein and protei... more Encapsidation of simian virus 40 is a complex biological process involving DNA-protein and protein-protein interactions in the formation of a unique three-dimensional structure around the viral minichromosome. A pseudoviral system developed in our laboratory, in which the viral early and late gene products are supplied in trans (by helpers), was used to analyze the encapsidation process independent of viral gene expression. With this experimental system we have discovered a requirement for a specific DNA signal for encapsidation, ses (for simian virus 40 encapsidation signal).ses is present within a 200-bp DNA fragment, which includes, in addition to the viral origin of replication (ori), six GGGCGG repeats (GC boxes) and 26 bp of the enhancer element. Deletion of the GC boxes and the enhancer sequences almost abolished encapsidation, while DNA replication was only moderately decreased. The ability to encapsidate was not regained by reinserting a DNA fragment carrying ses in the ses...
Cell and Gene Therapy Insights, 2021
Journal of Molecular Biology, 1994
CANCER RESEARCH, 1997
Page 1. ICANCERRESEARCH57. 2434-2439. June 15. 9971 frequently observed cytogenetic abnormality i... more Page 1. ICANCERRESEARCH57. 2434-2439. June 15. 9971 frequently observed cytogenetic abnormality in malignant lymphoma (found in 90% of cases with follicular lymphoma and in 25â30%of patients with diffuse large cell lymphoma; Ref. 21). ...
Biologicals, 2021
The HIV-1 derived gp145 protein is being investigated by research groups as preclinical studies h... more The HIV-1 derived gp145 protein is being investigated by research groups as preclinical studies have shown high promise for this protein as a vaccine against HIV. However, one of the main challenges with manufacturing this promising protein has been ascribed to the low yield obtained in mammalian cell cultures. Significant improvements in gp145 production are needed to address this issue to test the gp145 protein as a potentially effective, safe, and affordable HIV vaccine. Here we describe the application of a novel expression technology to create GMP-grade CHO cell lines expressing approximately 50 μg/ml in non-optimized fed-batch culture, which is an order of magnitude higher than that obtained in existing processes. Top producing clones show a high degree of similarity in the glycosylation patterns of the purified protein to the reference standard. Conformational integrity and functionality was demonstrated via high-affinity binding to soluble CD4, using a panel of antibodies including VRC01, F105, Hk20, PG9 and 17b. In summary, we were able to generate CHO cell lines expressing HIV gp145 with significantly higher overall expression yields than currently accessible, and high product quality that could potentially be suitable for future studies assessing the efficacy and safety of gp145based HIV vaccines.
Journal of virology, 2004
This study evaluated and compared delivery of the tumor necrosis factor alpha receptor (TNFR)-imm... more This study evaluated and compared delivery of the tumor necrosis factor alpha receptor (TNFR)-immunoglobulin G1 (IgG1) Fc fusion (TNFR:Fc) gene to the lung by single and repeat administrations of multiple pseudotyped adeno-associated virus (AAV) vectors as a means for achieving systemic distribution of the soluble TNFR:Fc protein. A single endotracheal administration of AAV[2/5]cytomegalovirus (CMV)-TNFR:Fc vector (containing the AAV2 inverted terminal repeats and AAV5 capsid) to the rat lung resulted in long-term, high levels of serum TNFR:Fc protein that gradually declined over a period of 8 months. Endotracheal delivery of AAV[2/1]CMV-TNFR:Fc resulted in serum TNFR:Fc protein levels that were detectable for at least 4 months but were 10-fold lower than that of the AAV[2/5] vector. In contrast, secretion of the TNFR:Fc protein following pulmonary delivery of AAV[2/2]CMV-TNFR:Fc vector was very inefficient, and the protein was detected in the blood only when an airway epithelial ce...
Molecular Therapy, 2006
ABSTRACT Molecular Therapy (2006) 13, S223|[ndash]|S223; doi: 10.1016/j.ymthe.2006.08.651 578. Su... more ABSTRACT Molecular Therapy (2006) 13, S223|[ndash]|S223; doi: 10.1016/j.ymthe.2006.08.651 578. Suppression of Inflammation Following Intramuscular Administration of Pseudotyped AAV-TNFR:Fc Vectors in a Rat Model of Arthritis|[ast]| Haim Burstein1, Kurt Lustig1 and Ziv Sandalon11Research, Targeted Genetics Corporation, Seattle, WA|[ast]|This study provides proof of concept for long-term suppression of experimental arthritis in rats, following intramuscular delivery of AAV-TNFR:Fc vectors and represent a novel therapeutic approach for chronic inflammatory arthritis.
SV40 Protocols, 2000
ABSTRACT The use of insect cells and lytic baculovirus for expression of biologically active mamm... more ABSTRACT The use of insect cells and lytic baculovirus for expression of biologically active mammalian proteins has been the method of choice of many investigators. Although prokaryotic expression systems provide higher yields and are technically simpler to use, obtaining biologically active eukaryotic proteins in these systems can be problematic. Posttranslational modifications such as glycosylation and phosphorylation do not occur in prokaryotic systems owing to the lack of enzymatic machinery. On the other hand, insect cells provide an appropriate environment for posttranslational modifications and lead to proper folding and correct assembly of recombinant proteins (1).
Molecular Therapy, 2006
We have presented previously that high dose airway delivery of a pseudotyped AAV2/5 vector to the... more We have presented previously that high dose airway delivery of a pseudotyped AAV2/5 vector to the lungs of Rhesus macaques resulted in enhanced gene transfer and quantitative RNA expression as well as protein expression in non human primates without clinical toxicity. One major limitation to the success of AAV gene therapy for CF is that the large size of the CFTR cDNA insert fills the packaging capacity of AAV viral particles precluding inclusion of a highly efficient promoter. In order to overcome this limitation AAV2/ 5 pseudotyped vectors were generated expressing a truncated CFTR insert (∆264) driven by a chicken beta actin promoter (rAAV-CB-∆264-CFTR). We showed previously that this truncated form was capable of reconstituting physiologic function in vitro and in mouse models.
Virology, 1997
Soluble SV40 capsid proteins were obtained by expression of the three late genes, VP1, VP2, and V... more Soluble SV40 capsid proteins were obtained by expression of the three late genes, VP1, VP2, and VP3, in Sf9 cells using baculovirus expression vectors. Coproduction of the capsid proteins VP1, VP2, and VP3 was achieved by infecting Sf9 cells with the three recombinant baculovirus species at equal multiplicities. All three proteins were found to be localized in the nuclear fraction. Electron microscopy of nuclear extracts of the infected cells showed an abundance of SV40-like capsid structures and heterogeneous aggregates of variable size, mostly 20-45 nm. Under the same staining conditions wild-type SV40 virions are 45 nm. The capsid-like particles sedimented in glycerol gradients similarly to authentic wild-type SV40 virions. Pentamers of the major capsid protein VP1 were also seen. Protein analysis on sucrose gradients demonstrated that the capsid-like particles can be disrupted by treatment with the reducing agent dithiothreitol and the calcium chelator EGTA. The capsid-like particles were found to be significantly less stable than SV40 virions and were partially stabilized by calcium ions. Understanding the complex interactions between the capsid proteins is important for the development of an efficient in vitro packaging system for SV40 virions and pseudovirions. ᭧ 1997 Academic Press
Molecular Therapy, 2004
Phenylketonuria (PKU) is one of the most common single gene disorders in man. The metabolic defec... more Phenylketonuria (PKU) is one of the most common single gene disorders in man. The metabolic defect arises from a deficiency of phenylalanine hydroxylase (PAH). There is a resultant hyperphenylalanemia with subsequent impairment in cognitive abilities, executive functions and motor coordination; these functions are associated with various dopaminergic tracts.