Gene therapy of ocular melanoma using a novel episomal vector system

P. W. Chen, T. Uno, T. G. Murray, E. R. Podack, B. R. Ksander

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Purpose: Gene therapy requires a selectable vector that allows the stable expression of specific cDNA in targeted cells. In order to develop an immunotherapeutic treatment for ocular melanoma patients, we attempted to genetically engineer human ocular melanoma cells to either constitutively secrete IL-2, or express the B7.1 costimulatory molecule. The IL-2 and B7.1 genes were chosen because of their ability to activate T cells that specifically eliminate tumor cells. Methods: Human ocular melanoma cell lines were derived from two patients (Mel 202 and 270) by enzymatic digestion of tumor tissue. Tumor cells were transfected with an episomal plasmid vector (PBMG) that uses a replication unit from the bovine papiloma virus. This vector is safer than retroviral vectors, since there is no risk of either virus production, or insertional mutation. Two plasmids were used that contained the cDNA for either B7.1, or interleukin-2. Tumor cells were transfected using calcium phosphate precipitation and selected in media containing geneticin. Expression of B7.1 was determined by FACS analysis using the CTLA-4 Ig fusion protein; secretion of IL-2 was determined using a specific ELISA. Results: Human ocular melanoma cells fail to either secrete IL-2, or express B7.1 on the tumor cell surface. By contrast, Mel 202 and 270 tumor cells transfected with the PBMG vector containing the cDNA for B7.1 expressed high levels of B7.1 as measured by the FACS (100% cells). In addition, tumor cells transfected with the cDNA for IL-2 secreted 696 pg/ml after 2 × 106 cells were cultured for 48 hrs. Conclusions: Ocular melanoma cells transfected with the PBMG episomal vector expressed high levels B7.1 and constitutively secreted IL-2. These results imply that this vector system may be useful in genetically engineering ocular tumor cells to induce specific T cell immunity.

Original languageEnglish
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996
Externally publishedYes

Fingerprint

Genetic Therapy
Melanoma
Interleukin-2
Neoplasms
Complementary DNA
Plasmids
Viruses
T-Lymphocytes
Replicon
Digestion
Cultured Cells
Immunity
Enzyme-Linked Immunosorbent Assay
Cell Line
Mutation

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Chen, P. W., Uno, T., Murray, T. G., Podack, E. R., & Ksander, B. R. (1996). Gene therapy of ocular melanoma using a novel episomal vector system. Investigative Ophthalmology and Visual Science, 37(3).

Gene therapy of ocular melanoma using a novel episomal vector system. / Chen, P. W.; Uno, T.; Murray, T. G.; Podack, E. R.; Ksander, B. R.

In: Investigative Ophthalmology and Visual Science, Vol. 37, No. 3, 15.02.1996.

Research output: Contribution to journalArticle

Chen, PW, Uno, T, Murray, TG, Podack, ER & Ksander, BR 1996, 'Gene therapy of ocular melanoma using a novel episomal vector system', Investigative Ophthalmology and Visual Science, vol. 37, no. 3.
Chen, P. W. ; Uno, T. ; Murray, T. G. ; Podack, E. R. ; Ksander, B. R. / Gene therapy of ocular melanoma using a novel episomal vector system. In: Investigative Ophthalmology and Visual Science. 1996 ; Vol. 37, No. 3.
@article{0ac60fe3493944f2b086b26c3d177a69,
title = "Gene therapy of ocular melanoma using a novel episomal vector system",
abstract = "Purpose: Gene therapy requires a selectable vector that allows the stable expression of specific cDNA in targeted cells. In order to develop an immunotherapeutic treatment for ocular melanoma patients, we attempted to genetically engineer human ocular melanoma cells to either constitutively secrete IL-2, or express the B7.1 costimulatory molecule. The IL-2 and B7.1 genes were chosen because of their ability to activate T cells that specifically eliminate tumor cells. Methods: Human ocular melanoma cell lines were derived from two patients (Mel 202 and 270) by enzymatic digestion of tumor tissue. Tumor cells were transfected with an episomal plasmid vector (PBMG) that uses a replication unit from the bovine papiloma virus. This vector is safer than retroviral vectors, since there is no risk of either virus production, or insertional mutation. Two plasmids were used that contained the cDNA for either B7.1, or interleukin-2. Tumor cells were transfected using calcium phosphate precipitation and selected in media containing geneticin. Expression of B7.1 was determined by FACS analysis using the CTLA-4 Ig fusion protein; secretion of IL-2 was determined using a specific ELISA. Results: Human ocular melanoma cells fail to either secrete IL-2, or express B7.1 on the tumor cell surface. By contrast, Mel 202 and 270 tumor cells transfected with the PBMG vector containing the cDNA for B7.1 expressed high levels of B7.1 as measured by the FACS (100{\%} cells). In addition, tumor cells transfected with the cDNA for IL-2 secreted 696 pg/ml after 2 × 106 cells were cultured for 48 hrs. Conclusions: Ocular melanoma cells transfected with the PBMG episomal vector expressed high levels B7.1 and constitutively secreted IL-2. These results imply that this vector system may be useful in genetically engineering ocular tumor cells to induce specific T cell immunity.",
author = "Chen, {P. W.} and T. Uno and Murray, {T. G.} and Podack, {E. R.} and Ksander, {B. R.}",
year = "1996",
month = "2",
day = "15",
language = "English",
volume = "37",
journal = "Investigative Ophthalmology and Visual Science",
issn = "0146-0404",
publisher = "Association for Research in Vision and Ophthalmology Inc.",
number = "3",

}

TY - JOUR

T1 - Gene therapy of ocular melanoma using a novel episomal vector system

AU - Chen, P. W.

AU - Uno, T.

AU - Murray, T. G.

AU - Podack, E. R.

AU - Ksander, B. R.

PY - 1996/2/15

Y1 - 1996/2/15

N2 - Purpose: Gene therapy requires a selectable vector that allows the stable expression of specific cDNA in targeted cells. In order to develop an immunotherapeutic treatment for ocular melanoma patients, we attempted to genetically engineer human ocular melanoma cells to either constitutively secrete IL-2, or express the B7.1 costimulatory molecule. The IL-2 and B7.1 genes were chosen because of their ability to activate T cells that specifically eliminate tumor cells. Methods: Human ocular melanoma cell lines were derived from two patients (Mel 202 and 270) by enzymatic digestion of tumor tissue. Tumor cells were transfected with an episomal plasmid vector (PBMG) that uses a replication unit from the bovine papiloma virus. This vector is safer than retroviral vectors, since there is no risk of either virus production, or insertional mutation. Two plasmids were used that contained the cDNA for either B7.1, or interleukin-2. Tumor cells were transfected using calcium phosphate precipitation and selected in media containing geneticin. Expression of B7.1 was determined by FACS analysis using the CTLA-4 Ig fusion protein; secretion of IL-2 was determined using a specific ELISA. Results: Human ocular melanoma cells fail to either secrete IL-2, or express B7.1 on the tumor cell surface. By contrast, Mel 202 and 270 tumor cells transfected with the PBMG vector containing the cDNA for B7.1 expressed high levels of B7.1 as measured by the FACS (100% cells). In addition, tumor cells transfected with the cDNA for IL-2 secreted 696 pg/ml after 2 × 106 cells were cultured for 48 hrs. Conclusions: Ocular melanoma cells transfected with the PBMG episomal vector expressed high levels B7.1 and constitutively secreted IL-2. These results imply that this vector system may be useful in genetically engineering ocular tumor cells to induce specific T cell immunity.

AB - Purpose: Gene therapy requires a selectable vector that allows the stable expression of specific cDNA in targeted cells. In order to develop an immunotherapeutic treatment for ocular melanoma patients, we attempted to genetically engineer human ocular melanoma cells to either constitutively secrete IL-2, or express the B7.1 costimulatory molecule. The IL-2 and B7.1 genes were chosen because of their ability to activate T cells that specifically eliminate tumor cells. Methods: Human ocular melanoma cell lines were derived from two patients (Mel 202 and 270) by enzymatic digestion of tumor tissue. Tumor cells were transfected with an episomal plasmid vector (PBMG) that uses a replication unit from the bovine papiloma virus. This vector is safer than retroviral vectors, since there is no risk of either virus production, or insertional mutation. Two plasmids were used that contained the cDNA for either B7.1, or interleukin-2. Tumor cells were transfected using calcium phosphate precipitation and selected in media containing geneticin. Expression of B7.1 was determined by FACS analysis using the CTLA-4 Ig fusion protein; secretion of IL-2 was determined using a specific ELISA. Results: Human ocular melanoma cells fail to either secrete IL-2, or express B7.1 on the tumor cell surface. By contrast, Mel 202 and 270 tumor cells transfected with the PBMG vector containing the cDNA for B7.1 expressed high levels of B7.1 as measured by the FACS (100% cells). In addition, tumor cells transfected with the cDNA for IL-2 secreted 696 pg/ml after 2 × 106 cells were cultured for 48 hrs. Conclusions: Ocular melanoma cells transfected with the PBMG episomal vector expressed high levels B7.1 and constitutively secreted IL-2. These results imply that this vector system may be useful in genetically engineering ocular tumor cells to induce specific T cell immunity.

UR - http://www.scopus.com/inward/record.url?scp=4243869096&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=4243869096&partnerID=8YFLogxK

M3 - Article

AN - SCOPUS:4243869096

VL - 37

JO - Investigative Ophthalmology and Visual Science

JF - Investigative Ophthalmology and Visual Science

SN - 0146-0404

IS - 3

ER -