High gene transfer efficiency utilizing recombinant fibronectin fragments in cd3/c d28-expanded peripheral blood lymphocytes prom an adenosine-deaminase-defioent patient

Karen Pollok, H. Hanenberg, T. W. Nobhrt, W. L. Schroeder, K. Asada, I. Kato, D. Emanuel, D. A. Williaims

Research output: Contribution to journalArticle

Abstract

A highly efficient mode of transierring genes into human primary I" lymphocytes was developed resulting in the targeting of at U-.i-t I om of every 3 T lymphocytes. This protocol was based on our previou-. ohii-rwitions that the efficiency of retroviral-mediated gene transfer was enhanced in hemato pole tic progenitors when genetic transdiiction was performed m the presence of specific fibronectin fragments. The recombinant fragment, CH-296, contains sequences involved in cell adhesion via the integnns, VLA-4 and VI.A-5, separated by type III repeats 12-14 containing the heparin binding domain. Peripheral blood lymphocytes (PBLs) from normal donors and an adenosine-deaminase-deficient (ADA) patient were activated with immobilized anti-CD3 and anti-CD28 monoclonal antibodies and transduced with the LNCmB7-l retrovinis which expresses murine B7-I (mH7-l). Cîene transfer efficiency was monitored by flow cytometnc analysis of mB7-l expression. Activated lymphocytes were transduced for three consecutive days with the mB7-l retrovtrus on CH-2%-coated plates or as a comparison, on BSA-coated plates in the presence of polybrene. At day five post-transduction, PBLs transduced on CH-2%-coated plates, were 75-88% mB7-E', while at 12 days posi-transduction, 40-50% of the PBLs were mB7-U In contrast, PBLs transduced with rnB7-l on BSA-coated plates in the presence of polybrene, were 30-40% mu7-l' on day 5 post-vransduction, while at day 12 post-transduction, only 4-22% were mB7-t'. Since lymphocytes have been used as targets lor gene therapy of adenosine deaminase deficiency, normal and ADA-deficient PBLs were transduced on CH-296 with the pZIP-PGK-mADA retrovinis which expresses murine adenosine deaminase fmADA). At 12 days post-transduction, the level of mADA expression from this patient was 2-3 fold higher than endogenous human ADA expression seen in normal human lymphocytes. The proliferative and cytolytic capacity of these transduced populations as well as the Vb repertoire was maintained. This gene transfer protocol, provides a simple and reliable way of delivering genes at high efficiency to human PBLs and may be a candidate for use in a wide variety of gene therapy protocols designed to target T Ivmphocvtes.

Original languageEnglish
Pages (from-to)880
Number of pages1
JournalExperimental Hematology
Volume25
Issue number8
StatePublished - 1997

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Adenosine Deaminase
Fibronectins
Lymphocytes
Genes
Hexadimethrine Bromide
Genetic Therapy
pX Genes
Integrin alpha4beta1
Tics
Cell Adhesion
Heparin
Monoclonal Antibodies
Tissue Donors
T-Lymphocytes

ASJC Scopus subject areas

  • Cancer Research
  • Cell Biology
  • Genetics
  • Hematology
  • Oncology
  • Transplantation

Cite this

High gene transfer efficiency utilizing recombinant fibronectin fragments in cd3/c d28-expanded peripheral blood lymphocytes prom an adenosine-deaminase-defioent patient. / Pollok, Karen; Hanenberg, H.; Nobhrt, T. W.; Schroeder, W. L.; Asada, K.; Kato, I.; Emanuel, D.; Williaims, D. A.

In: Experimental Hematology, Vol. 25, No. 8, 1997, p. 880.

Research output: Contribution to journalArticle

Pollok, Karen ; Hanenberg, H. ; Nobhrt, T. W. ; Schroeder, W. L. ; Asada, K. ; Kato, I. ; Emanuel, D. ; Williaims, D. A. / High gene transfer efficiency utilizing recombinant fibronectin fragments in cd3/c d28-expanded peripheral blood lymphocytes prom an adenosine-deaminase-defioent patient. In: Experimental Hematology. 1997 ; Vol. 25, No. 8. pp. 880.
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T1 - High gene transfer efficiency utilizing recombinant fibronectin fragments in cd3/c d28-expanded peripheral blood lymphocytes prom an adenosine-deaminase-defioent patient

AU - Pollok, Karen

AU - Hanenberg, H.

AU - Nobhrt, T. W.

AU - Schroeder, W. L.

AU - Asada, K.

AU - Kato, I.

AU - Emanuel, D.

AU - Williaims, D. A.

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N2 - A highly efficient mode of transierring genes into human primary I" lymphocytes was developed resulting in the targeting of at U-.i-t I om of every 3 T lymphocytes. This protocol was based on our previou-. ohii-rwitions that the efficiency of retroviral-mediated gene transfer was enhanced in hemato pole tic progenitors when genetic transdiiction was performed m the presence of specific fibronectin fragments. The recombinant fragment, CH-296, contains sequences involved in cell adhesion via the integnns, VLA-4 and VI.A-5, separated by type III repeats 12-14 containing the heparin binding domain. Peripheral blood lymphocytes (PBLs) from normal donors and an adenosine-deaminase-deficient (ADA) patient were activated with immobilized anti-CD3 and anti-CD28 monoclonal antibodies and transduced with the LNCmB7-l retrovinis which expresses murine B7-I (mH7-l). Cîene transfer efficiency was monitored by flow cytometnc analysis of mB7-l expression. Activated lymphocytes were transduced for three consecutive days with the mB7-l retrovtrus on CH-2%-coated plates or as a comparison, on BSA-coated plates in the presence of polybrene. At day five post-transduction, PBLs transduced on CH-2%-coated plates, were 75-88% mB7-E', while at 12 days posi-transduction, 40-50% of the PBLs were mB7-U In contrast, PBLs transduced with rnB7-l on BSA-coated plates in the presence of polybrene, were 30-40% mu7-l' on day 5 post-vransduction, while at day 12 post-transduction, only 4-22% were mB7-t'. Since lymphocytes have been used as targets lor gene therapy of adenosine deaminase deficiency, normal and ADA-deficient PBLs were transduced on CH-296 with the pZIP-PGK-mADA retrovinis which expresses murine adenosine deaminase fmADA). At 12 days post-transduction, the level of mADA expression from this patient was 2-3 fold higher than endogenous human ADA expression seen in normal human lymphocytes. The proliferative and cytolytic capacity of these transduced populations as well as the Vb repertoire was maintained. This gene transfer protocol, provides a simple and reliable way of delivering genes at high efficiency to human PBLs and may be a candidate for use in a wide variety of gene therapy protocols designed to target T Ivmphocvtes.

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