Genetically engineered intracellular single-chain antibodies in gene therapy |
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Authors: | Bilbao Guadalupe Contreras Juan Luis Curiel David T |
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Institution: | (1) Division of Human Gene Therapy, Department of Medicine, Pathology and Surgery and the Gene therapy Center, The University of Alabama at Birmingham, 1824 Sixth Ave, South, WTI 620, 35294 Birmingham, AL;(2) Division of Human Gene Therapy, Department of Medicine, Pathology and Surgery, and the Gene therapy Center, USA;(3) Department of Surgery, The University of Alabama at Birmingham, Birmingham, AL |
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Abstract: | The delineation of the molecular basis of cancer allows for the possibility of specific intervention at the molecular level
for therapeutic purposes. To a large extent, the genetic lesions associated with malignant transformation and progression
are being identified. Thus, not only in the context of inherited genetic diseases, but also for many acquired disorders, characteristic
aberrancies of patterns of gene expression may be precisely defined. It is therefore clear that elucidation of the genetic
basis of inherited and acquired diseases has rendered gene therapy both a novel and rational approach for these disorders.
To this end, three main strategies have been developed: mutation compensation, molecular chemotherapy, and genetic immunopotentiation.
Mutation compensation relies on strategies to ablate activated oncogenes at the level of DNA (triplex), messenger RNA (antisense
or ribozyme), or protein (intracellular single-chain antibodies), and augment tumor suppressor gene expression. This article
will review in detail practical procedures to generate a single-chain intracellular antibody (scFv). We will emphasize in
this article the different steps in our protocol that we have employed to develop scFvs to a variety of target proteins. |
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Keywords: | Intracellular single-chain antibody (scFv) gene therapy colony lift assay light chain (VL) heavy chain (VH) |
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