Abstract
BackgroundLentiviral gene transfer can provide long-term expression of therapeutic genes such as erythropoietin. Because overexpression of erythropoietin can be toxic, regulated expression is needed. Doxycycline inducible vectors can regulate expression of therapeutic transgenes efficiently. However, because they express an immunogenic transactivator (rtTA), their utility for gene therapy is limited. In addition to immunogenic proteins that are expressed from inducible vectors, injection of the vector itself is likely to elicit an immune response because viral capsid proteins will induce “danger signals” that trigger an innate response and recruit inflammatory cells.Methodology and Principal FindingsWe have developed an autoregulatory lentiviral vector in which basal expression of rtTA is very low. This enabled us to temporally separate the injection of virus and the expression of the therapeutic gene and rtTA. Wistar rats were injected with an autoregulatory rat erythropoietin expression vector. Two or six weeks after injection, erythropoietin expression was induced by doxycycline. This resulted in an increase of the hematocrit, irrespective of the timing of the induction. However, most rats only responded once to doxycycline administration. Antibodies against rtTA were detected in the early and late induction groups.ConclusionsOur results suggest that, even when viral vector capsid proteins have disappeared, expression of foreign proteins in muscle will lead to an immune response.
Highlights
Our results suggest that, even when viral vector capsid proteins have disappeared, expression of foreign proteins in muscle will lead to an immune response
Many forms of gene therapy will require the ability to modulate the expression of therapeutic genes to maintain expression levels within a therapeutic window or adjust expression levels based on disease progression within the patient [1]
The Tet-On system is composed of a chimeric reverse tetracycline transactivator composed of the herpesvirus VP16 transcription activating domain and a mutant tetracycline repressor protein from Escherichia coli
Summary
Many forms of gene therapy will require the ability to modulate the expression of therapeutic genes to maintain expression levels within a therapeutic window or adjust expression levels based on disease progression within the patient [1]. Lentiviral vectors derived from HIV-1 are a well-suited vehicle for the treatment of a variety of inherited and acquired diseases. They can deliver a relatively large therapeutic cassette into both dividing and nondividing cells and integrate into the host cell genome providing life long expression of the therapeutic gene [2,3,4]. In the presence of tetracycline or doxycycline, rtTA binds to the tetO and initiates transcription. Doxycycline inducible vectors can regulate expression of therapeutic transgenes efficiently. Because they express an immunogenic transactivator (rtTA), their utility for gene therapy is limited. In addition to immunogenic proteins that are expressed from inducible vectors, injection of the vector itself is likely to elicit an immune response because viral capsid proteins will induce ‘‘danger signals’’ that trigger an innate response and recruit inflammatory cells
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