Bifunctional selectable fusion genes

Details Bifunctional selectable fusion genes Bifunctional selectable fusion genes

1995-06-07

2003-04-29

Prouty, Rebecca E. (Department: 1652)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

C536S023200, C536S023400, C435S069700, C435S070300, C435S440000, C435S455000, C435S456000, C435S252300, C435S320100

Reexamination Certificate

active

06555370

ABSTRACT:

BACKGROUND OF THE INVENTION
The present invention relates generally to genes expressing selectable phenotypes. More particularly, the present invention relates to genes capable of co-expressing both dominant positive selectable and negative selectable phenotypes.
Genes which express a selectable phenotype are widely used in recombinant DNA technology as a means for identifying and isolating host cells into which the gene has been introduced. Typically, the gene expressing the selectable phenotype is introduced into the host cell as part of a recombinant expression vector. Positive selectable genes provide a means to identify and/or isolate cells that have retained introduced genes in a stable form, and, in this capacity, have greatly facilitated gene transfer and the analysis of gene function. Negative selectable genes, on the other hand, provide a means for eliminating cells that retain the introduced gene.
A variety of genes are available which confer selectable phenotypes on animal cells. The bacterial neomycin phosphotransferase (neo) (Colbere-Garapin et al.,
J. Mol. Biol
. 150:1, 1981), hygromycin phosphotransferase (hph) (Santerre et al.,
Gene
30:147, 1984), and xanthine-guanine phosphoribosyl transferase (gpt) (Mulligan and Berg,
Proc. Natl. Acad. Sci. USA
78:2072, 1981) genes are widely used dominant positive selectable genes. The Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 11:223, 1977); the cellular adenine phosphoribosyltransferase (APRT) (Wigler et al.,
Proc. Natl. Acad. Sci. USA
76:1373, 1979); and hypoxanthine phosphoribosyltransferase (HPRT) genes (Jolly et al.,
Proc. Natl. Acad. Sci. USA
80:477, 1983) are commonly used recessive positive selectable genes. In general, dominant selectable genes are more versatile than recessive genes, because the use of recessive genes is limited to mutant cells deficient in the selectable function, whereas dominant genes may be used in wild-type cells.
Several genes confer negative as well as positive selectable phenotypes, including the HSV-I TK, HPRT, APRT and gpt genes. These genes encode enzymes which catalyze the conversion of nucleoside or purine analogs to cytotoxic intermediates. The nucleoside analog ganciclovir (GCV) is an efficient substrate for HSV-I TK, but a poor substrate for cellular TK, and therefore may be used for negative selection against the HSV-I TK gene in wild-type cells (St. Clair et al.,
Antimicrob. Agents Chemother
. 31:844, 1987). However, the HSV-I TK gene may only be used effectively for positive selection in mutant cells lacking cellular TK activity. Use of the HPRT and APRT genes for either positive or negative selection is similarly limited to HPRT
−
or APRT
−
cells, respectively (Fenwick, “The HGPRT System”, pp. 333-373, M. Gottesman (ed.),
Molecular Cell Genetics
, John Wiley and Sons, New York, 1985; Taylor et al., “The APRT System”, pp. 311-332, M. Gottesman (ed.),
Molecular Cell Genetics
, John Wiley and Sons, New York, 1985). The gpt gene, on the other hand, may be used for both positive and negative selection in wild-type cells. Negative selection against the gpt gene in wild-type cells is possible using 6-thioxanthine, which is efficiently converted to a cytotoxic nucleotide analog by the bacterial gpt enzyme, but not by the cellular HPRT enzyme (Besnard et al.,
Mol. Cell. Biol
. 7:4139, 1987).
Another negatively selectable gene has recently been reported by Mullen et al.,
Proc. Natl. Acad. Sci. USA
89:33, 1992. The bacterial cytosine deaminase (CD) gene converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU). 5-FU is further metabolized intracellularly to 5-fluoro-uridine-5′-triphosphate and 5-fluoro-2′-deoxy-uridine-5′-monophosphate, which inhibit RNA and DNA synthesis, causing cell death. Thus, 5-FC can effectively ablate cells carrying and expressing the CD gene. The CD gene is not positively selectable in normal cells.
More recently, attention has turned to selectable genes that may be incorporated into gene transfer vectors designed for use in human gene therapy. Gene therapy can be used as a means for augmenting normal gene function, for example, by introducing a heterologous gene capable of modifying cellular activities or cellular phenotype, or alternatively, expressing a drug needed to treat a disease. Gene therapy also is a method for permanently curing a hereditary genetic disease which results from a defect in or absence of one or more genes. Collectively, such diseases result in significant morbidity and mortality. Examples of such genetic diseases include hemophilias A and B (caused by a deficiency of blood coagulation factors VIII and IX, respectively), alpha-1-antitrypsin deficiency, and adenosine deaminase deficiency. In each of these particular cases, the missing gene has been identified and its complementary DNA (cDNA) molecularly cloned (Wood et al.,
Nature
312:330, 1984; Anson et al.,
Nature
315:683, 1984; and Long et al.,
Biochemistry
23:4828, 1984; Daddona et al.,
J. Biol. Chem
. 259:12101, 1984). While palliative therapy is available for some of these genetic diseases, often in the form of administration of blood products or blood transfusions, one way of permanently curing such genetic diseases is to introduce a replacement for the defective or missing gene back into the somatic cells of the patient, a process referred to as “gene therapy” (Anderson,
Science
226:401, 1984).
The process of gene therapy typically involves the steps of (1) removing somatic (non-germ) cells from the patient, (2) introducing into the cells ex vivo a therapeutic or replacement gene via an appropriate vector capable of expressing the therapeutic or replacement gene, and (3) transplanting or transfusing these cells back into the patient, where the therapeutic or replacement gene is expressed to provide some therapeutic benefit. Gene transfer into somatic cells for human gene therapy is presently achieved ex vivo (Kasid et al.,
Proc. Natl. Acad. Sci. USA
87:473, 1990; Rosenberg et al.,
N. Engl. J. Med
. 323:570, 1990), and this relatively inefficient process would be facilitated by the use of a dominant positive selectable gene for identifying and isolating those cells into which the replacement gene has been introduced before they are returned to the patient. The neo gene, for example, has been used to identify genetically modified cells used in human gene therapy.
In some instances, however, it is possible that the introduction of genetically modified cells may actually compromise the health of the patient. The ability to selectively eliminate genetically modified cells in vivo would provide an additional margin of safety for patients undergoing gene therapy, by permitting reversal of the procedure. This might be accomplished by incorporating into the vector a negative selectable (or “suicide”) gene that is capable of functioning in wild-type cells. Incorporation of a gene capable of conferring both dominant positive and negative selectable phenotypes would ensure co-expression and co-regulation of the positive and negative selectable phenotypes, and would minimize the size of the vector. However, positive selection for the gpt gene in some instances requires precise selection conditions which may be difficult to determine. Moreover, the feasibility of using the gpt gene for in vivo negative selection has not yet been clearly established. For these reasons, co-expression of a dominant positive selectable phenotype and a negative selectable phenotype is typically achieved by co-expressing two different genes which separately encode other dominant positive and negative selectable functions, rather than using the gpt gene.
The existing strategies for co-expressing dominant positive and negative selectable phenotypes encoded by different genes often present complex challenges. As indicated above, the most widely used technique is to co-transfect two plasmids separately encoding two phenotypes (Wigler et al.,
Cell
16:777, 1979). However, the efficiency of co-transfer is rarely 100%, and the

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