Tissue-specific self-inactivating gene therapy vector

Details Tissue-specific self-inactivating gene therapy vector Tissue-specific self-inactivating gene therapy vector

2001-07-17

2004-04-06

Guzo, David (Department: 1636)

Chemistry: molecular biology and microbiology

Vector, per se

C435S456000, C435S462000, C536S023100, C536S024100

Reexamination Certificate

active

06716622

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of gene therapy vector design. More specifically, the present invention relates to a tissue-specific self-inactivating gene therapy vector.
2. Description of the Related Art
Adenoviral vectors possess a number of attributes that render them useful gene delivery vehicles for systemic gene therapy. In particular, the in vivo transduction efficiencies achievable with these agents are greater than with currently available alternative vector systems (1). Ideally, such a system would be designed so that systemically administered vector would home specifically to tumor target cells without ectopic infection of normal cells.
However, a major stumbling block to this approach is the fact that the majority of adenoviral vectors administered systemically are sequestered in the liver. Therefore measures that specifically control the distribution of delivered transgene expression must be superimposed on the basic vector for optimal applicability of adenoviral vectors.
Various approaches to this problem have been proposed, including imparting both transductional and transcriptional targeting properties to Ad vectors (2-7). Although targeting strategies can reduce hepatocyte transduction, the level of sequestration of vector to these cells is so great that this approach alone is insufficient to completely ameliorate ectopic gene expression. Tissue specific promoters may add a further degree of transgene expression selectivity but there are few of these that have been validated in vivo and all are subject to some degree of non-specific activation or “leakiness”. This has several importance consequences, one of which is that many of the genes designed for cancer therapy—“suicide genes” in particular—are of limited practical application due to dose limiting toxicity of the transgene action in the liver.
Hence, even when vectors that have been modified so as not to recognize the native adenovirus receptor are used, ectopic transgene expression in non-target organs remains a potential problem. Additional complementary strategies that can be superimposed upon these systems are needed to afford an even greater measure of control. Thus, the prior art is deficient in a strategy to reduce ectopic transgene expression in adenovirus-mediated gene transfer. The present invention fulfills this longstanding need and desire in the art.
SUMMARY OF THE INVENTION
The present invention provides a strategy that deliberately switches transgene expression off at a site where transgene expression might otherwise be disadvantageous. LoxP sites are strategically placed in an adenovirus vector that carries the transgene. Cre recombinase recognizing these LoxP sites will cut this vector into pieces, thereby de-activates the vector in tissues and organs where the delivered transgene could potentially give rise to toxicity. The Cre recombinase gene operably linked to a tissue-specific promoter can be provided by a separate adenovirus vector or contained in the same vector that carries the transgene. To stop transgene expression in the liver, Cre recombinase is driven by a liver specific promoter.
The object of the present invention is to reduce undesirable transgene expression in non-targeted tissue. In one aspect of the present invention, there is provided a tissue-specific self-inactivating gene expression system comprises of: (i) an adenovirus vector comprising at least one LoxP sequence and a transgene operably linked to a promoter, wherein said transgene and said promoter are flanked by said LoxP sequences; and (ii) an adenovirus vector comprises of a Cre recombinase gene operably linked to a tissue-specific promoter.
In another embodiment of the present invention, there is provided a tissue-specific self-inactivating gene expression vector that contains (i) more than one LoxP sequence, (ii) a transgene operably linked to a promoter wherein the transgene and the promoter are flanked by LoxP sequences, and (iii) a Cre recombinase gene operably linked to a tissue-specific promoter.
In another aspect of the present invention, there are provided methods of reducing transgene expression in non-targeted tissue in an individual by administering a therapeutically effective amount of tissue-specific self-inactivating gene expression vectors disclosed herein.
Other and further aspects, features, and advantages of the present invention will be apparent from the following description of the presently preferred embodiments of the invention. These embodiments are given for the purpose of disclosure.


REFERENCES:
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Dang et al., Gene therapy and translational cancer research, 1999, Clinical Cancer Research, vol. 5, pp. 471-474.*
Xu et al., Strategies for enzyme/prodrug cancer therapy, 2001, Clinical Cancer Research, vol. 7, pp. 3314-3324.*
Romano et al., Latest developments in gene transfer technology: Achievements, perspectives, and controversies over therapeutic applications, 2000, Stem Cells, vol. 18, pp. 19-39.*
Meng et al., Tumor suppressor genes as targets for cancer gene therapy, 1999. In:Gene Therapy of Cancer, Academic Press, pp. 3-20.*
Verma et al., Gene therapy-promises, problems and prospects, 1997, Nature, vol. 389, pp. 239-242.*
Sato et al., Enhanced and specific gene expression via tissue specific production of cre recombinase using adenovirus vector, 1998, Biochemical and Biophysical Research Communications, vol. 244, pp. 455-462.

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