Engineering Mdr Vectors To Improve Efficiency Of Drug Selection

One of the goals of gene therapy is to modify cells genetically such that they can supply a useful or necessary function to the cell (3). One of the most promising applications of the MDR1 gene in therapeutic vectors as a selectable marker in vivo is the protection of bone marrow cells during intensive chemotherapy. During chemotherapy, the MDR1 gene is transduced or transfected into drug-sensitive bone marrow cells and selected for by exposure to MDR agents. The untransfected/untransduced cells will necessarily be killed and those containing the MDR1 gene will expand. The efficacy of this therapy depends on the interaction between P-gp and the selecting agent employed. Thus, it is important to be able to distinguish between the endogenous P-gp and the exoge-nously introduced molecule. Furthermore, it obviously would be beneficial to create a P-gp molecule that would confer very high levels of resistance to certain drugs, giving an advantage to transduced cells/tissues compared to wild-type P-gp. Studies of a number of mutations made in P-glycoprotein have suggested that it should be possible to construct mutant ''designer'' transporters useful for MDRl-based gene therapy.

One of the hallmark characteristics of the multidrug transporter is its extremely broad substrate specificity. Over the past several years, the identification of specific domains and amino acid residues involved in substrate recognition has contributed to our present understanding of the mechanism of action of P-gp. The major sites of interaction have been shown to reside in transmembrane domains (TM) 5 and 6 in the N-terminal half of the protein and in TMs 11 and 12 in the C-terminal half and the loops that conjoin them (217-221). For the purposes of chemoprotection, the design of a P-gp that has increased resistance to chemotherapeutic agents compared to the endogenous P-gp would be most useful because increased doses of the agent could be administered without harming the bone marrow cells expressing the exogenous Pgp molecule. To date, a number of these types of mutations have been described.

Mutations in TM domains of P-gps from both rodent and human have demonstrated significant alterations in substrate specificity (3,222). An F338A mutation in hamster P-gp enhances resistance to vincristine, colchicine, and daunorubicin but has little impact on resistance to actinomycin D (223,224). An F339P mutation in the same molecule only increases actinomycin D resistance. However, the double F338A/F339P mutant demonstrates an increased level of resistance to actino-mycin D and vincristine but a lowered level of resistance to colchicine and daunorubicin (223,224). Of these mutants, the F338A may prove most useful because it confers increased resistance to a wider range of chemotherapeutic agents. In human P-gp, however, a homologous mutation at F335 confers greater resistance to colchicine and doxorubicin but causes a severe reduction in resistance to vinblastine and acti-nomycin D (225,226) . Additionally, cells expressing a Val> Ala mutation at position 338 also exhibit preferential resistance to colchicine and doxorubicin but are severely impaired for vinblastine (226). Resistance to actinomycin D, however, is unaffected. Alanine scanning of TM 11 in mouse P-gp encoded by mdr1a revealed that two mutants, M944A and F940A, show an increase in resistance to doxorubicin and colchicine while maintaining wild-type levels of resistance to vinblastine and actinomycin D (227). For certain treatment protocols, it is conceivable that increased resistance to certain agents would be desirable, and the reduction in levels of resistance to other compounds would not be problematic, especially if a well-defined chemotherapy regimen was being employed.

Although the majority of residues that increase resistance to various chemotherapeutic agents reside in the TM domains, a number of residues in the putative cytoplasmic loops also have been implicated in defining drug resistance profiles for cytotoxic drugs. The best characterized of these mutations is the G185V mutant that confers an increased resistance to colchicine and etoposide but decreased resistance to actino-mycin D, vinblastine, doxorubicin, vincristine, and taxol

(228-231). Interestingly, and perhaps relevant clinically, when this mutation is made in conjunction with an Asn->Ser mutation at residue 183, increased resistance to actinomycin D, vinblastine, and doxorubicin is achieved without loss of the increase in colchicine resistance (229). Mutations of Gly-141, 187, 288, 812, or 830 to Val in human P-gp increase the relative resistance of NIH3T3 cells to colchicine and doxorubicin but do not alter resistance to vinblastine (232). Only the mutations at positions 187, 288, and 830 confer decreased resistance to actinomycin D to cells in culture.

Due to its broad substrate specificity, P-gp not only interacts with chemotherapeutic compounds but also with reversing agents and inhibitors. In combination chemotherapies, reversing agents increase the efficacy of cytotoxic agents in MDR 1-expressing cancers. Two of the most potent reversing agents currently in use or in clinical trials are cyclosporin A and its nonimmunosuppressive analog PSC833. Recently, a number of mutants have been described that affect sensitivity to these agents. Cells expressing a human P-gp containing a deletion at Phe335 or Phe334 are substantially resistant to cyclosporin A and PSC-833 [(233), Hrycyna, C.A., Pastan, I., and Gottesman, M.M., unpublished data]. A similar phenotype has been observed for a transporter containing 5 mutations in the region including TM 5 and TM6, namely Ile299Met, Thr319Ser, Leu322Ile, Gly324Lys, and Ser351Asn (234). Additionally, in hamster P-gp, the substitution of an alanine at position 339 with proline results in a transporter that confers lowered sensitivity to cyclosporin A (224). From these studies, it appears that TM6 plays an important role in the recognition of cyclosporin A and its analogs. The decreased sensitivity to these reversing agents observed in cells expressing the TM6 mutations could help protect bone marrow stem cells transduced with the mutant MDR1 gene from the toxic effects of chemotherapy given with reversing agents to sensitize MDR 1-expressing tumors.

The cis and trans isomers of flupentixol, a dopamine receptor antagonist, have also been shown to inhibit drug transport and reverse drug resistance mediated by P-gp (235,236). The substitution of a single phenylalanine residue at position 983 with alanine (F983A) in TM 12 affects inhibition of P-gp-mediated drug transport by both isomers of flupentixol (59,60,237). Both isomers were found to be less effective at reversing P-gp mediated drug transport of daunorubicin and bisantrene. However, the inhibitory effects of other reversing agents such as cyclosporin A were not affected. The reduced sensitivity of the F983A mutant to this compound coupled to the apparent lack of clinical toxicity of (trans)-flupentixol (235), suggests that this mutant may be useful in combining MDR1 gene therapy with chemotherapy including trans-flu-pentixol as a chemosensitizer. This approach, in theory, should allow for effective treatment at lower doses of chemotherapeu-tic agents while maintaining bone marrow protection.

The use of MDR1 gene therapy in bone marrow chemopro-tection protocols has undergone preliminary analysis in clinical trials (89,90,238). Results indicate a low efficiency of marking bone marrow cells using retroviral vectors, but some selective advantage manifested as an increased percentage of positive cells after chemotherapy (89). In the future, with the generation of higher resolution structures of human P-gp, it should be feasible to model and synthesize new, more effective cytotoxic drugs or modulators capable of blocking P-gp function clinically. However, until that time, the analysis of spontaneously occurring or engineered mutants, coupled to our knowledge of the current battery of anticancer and reversing agents, offers an opportunity to begin designing second-generation vectors for use in these trials.

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