Cancer Gene Therapy (2006) 13, 335–345 r 2006 Nature Publishing Group All rights reserved 0929-1903/06 $30.00 www.nature.com/cgt

REVIEW Gene therapy with drug resistance genes M Zaboikin1, N Srinivasakumar1 and F Schuening Division of Hematology/Oncology, Department of Medicine, Vanderbilt University, Nashville, TN, USA

A major side effect of cancer is myelosuppression. Expression of drug-resistance genes in hematopoietic stem cells (HSC) using gene transfer methodologies holds the promise of overcoming marrow toxicity in cancer chemotherapy. Adequate protection of marrow cells in cancer patients from myelotoxicity in this way would permit the use of escalating doses of chemotherapy for eradicating residual disease. A second use of drug-resistance genes is for coexpression with a therapeutic gene in HSCs to provide a selection advantage to gene-modified cells. In this review, we discuss several drug resistance genes, which are well suited for in vivo selection as well as other newer candidate genes with potential for use in this manner. Cancer Gene Therapy (2006) 13, 335–345. doi:10.1038/sj.cgt.7700912; published online 7 October 2005 Keywords: hematopoietic stem cell; chemoprotection; drug resistance; gene transfer

Introduction increasing interest due to their demonstrated superiority to g-retroviral vectors for gene delivery into HSCs.4–7 Hematopoietic stem cells (HSCs) are defined by their Retroviral vectors have been shown to be effective in properties of self-renewal, pluripotency, and life-long correcting genetic disorders in small animal models such persistence following transplantation in the host. HSCs as mice. However, success has been meager in large are easily accessible and deliverable back to recipients by animals including humans. Gene therapy has been well-established transplantation methods. Thus transfer and particularly successful wherein the therapeutic gene expression of drug-resistance genes into HSCs would ensure confers a growth advantage to the gene-modified cells. the protection of not only the stem cells but also their This is exemplified by the remarkable success of gene differentiated progeny. The attributes of HSCs also make transfer of common g-chain cytokine receptor to HSCs in 8–10 them an attractive target for correction of genetic defects in children lacking the relevant gene resulting in SCID. different hematological lineages. For example, HSCs can be The success of gene therapy in SCID patients is attributed used for gene therapy of hemoglobinopathies (thalassemia, to not only improved transduction procedures and sickle-cell anemia) or immune-deficiency disorders (e.g. optimized in vitro culture conditions for HSCs using severe combined immune deficiency or SCID). Drug newer combinations of cytokines, but also to the growth resistance genes are being considered in the latter context advantage conferred to gene-modified lymphoid progeny to provide a mechanism for selection or enrichment of gene- by the particular therapeutic gene used (g-chain of T-cell modified cells to improve the efficacy of gene therapy. The receptor). Similar success has not been forthcoming for reader is referred to several recent reviews1–3 for additional other diseases such as mucopolysaccharidosis or thalasse- information not covered in detail here. mia, where there is no growth advantage for gene- modified cells and the level of marked cells in the peripheral blood of transplanted large animals or humans is rather low to begin with. This can be explained by Gene delivery into HSC inefficient gene transfer into HSCs or poor engraftment of transduced long-term repopulating HSCs. In order to permanently mark or modify HSCs, most Engraftment can be improved by eliminating the investigators use retroviral vectors since they have the endogenous, nontransduced HSCs by treatment of the ability to integrate into the host cell genome. g-retroviral recipient with myeloablative or myelosuppressive doses of vectors have been extensively used for this purpose but total body irradiation or chemotherapeutic agents prior to more recently, lentivirus-based vectors have gathered transplantation of the transduced stem cells. This approach has the disadvantage, that it does not affect Correspondence: Professor FG Schuening, Division of Hematology/ the nontransduced stem cells in the transplant. The Oncology, Department of Medicine, Vanderbilt University, 777 PRB, 2220 Pierce Avenue, Nashville, TN 37232, USA. nontransduced HSCs in the transplant, which usually E-mail: [email protected] outnumber the transduced HSCs, have the advantage in 1These authors contributed equally to this review. engraftment. Elimination of nontransduced cells by Received 18 May 2005; revised 8 August 2005; accepted 12 August selection exvivo prior to transplant, although attractive, 2005; published online 7 October 2005 has its disadvantages. Selection of gene-modified cells Gene therapy with drug resistance genes M Zaboikin et al 336 requires further exvivo manipulation of HSCs following for multidrug resistance (MDR1), multidrug resistance- gene transfer by viral vectors, which in itself requires one associated protein 1 (MRP1), dihydrofolate reductase or more days of culture of HSCs. This would include (DHFR), aldehyde-dehydrogenase (ALDH), glutathione- extra time in culture to obtain a level of transgene S-transferase (GST), cytosine deaminase (CDD) and expression high enough for sorting by flow cytometry (if methylguanine-DNA-methyltransferase (MGMT). Of the marker gene is either expressed on the surface and these, the DHFR, MDR1, MGMT and GST genes available for antibody binding or is a fluorescent protein) (Table 1) have been successfully used for selection of or for drug selection and time of exposure to the drug HSCs in vivo in animal models19–30 and are described in itself. Such increased time in culture has been shown to greater detail below. decrease the number of pluripotent HSCs, thereby compromising long-term engraftment capability.11,12 Therefore, selection of gene-modified cells prior to Dihydrofolate reductase transplant is generally not carried out. In vivo selection of HSCs, in contrast to exvivo Dihydrofolate reductase or DHFR, was the first drug selection, allows the selective outgrowth of transduced resistance gene used for retrovirus vector gene transfer HSCs in their natural environment after transplantation. into murine and canine hematopoietic cells.31,32 This For in vivo selection, a selection gene alone or together protein plays an essential role in thymidilate synthesis by with a potentially therapeutic gene, is introduced into regenerating tetrahydrofolate (Figures 1 and 2). The HSCs. The HSCs are transplanted into the recipient and corresponding selective drugs, (MTX) or the appropriate selection agent is administered after the related drug, trimetrexate (TMTX), inhibit the wild- allowing time for the engraftment of the transplant. type enzyme. Several mutants of DHFR have been Engraftment occurs usually around 4 weeks for both identified that are resistant to these drugs.33–35 These murine or canine models. include the leucine to tyrosine substitution at codon 22 The preferential expansion of transduced cells follow- (L22Y) and the phenylalanine to serine substitution at ing transplant can be achieved as follows: (a) by codon 31 (F31S). Double mutations, L22Y and F31S or modifying the HSCs with genes that provide a prolif- L22Y and F31R, of DHFR provide much higher erative or growth advantage; (b) by modifying HSCs with resistance to MTX than each single mutant.36,37 Expres- drug resistance genes that will allow the elimination of sion of mutant DHFR can protect hematopoiesis of nontransduced cells or (c) by using a combination of transplanted mice from the cytotoxic effects of metho- both.13,14 An example of a gene that provides a trexate and trimetrexate.20,21 The advantage of DHFR proliferative advantage is the HoxB4 gene. Expression over other drug-reistance genes, such as MGMT (dis- of this gene increases self-renewal capacity of HSCs and cussed below), is that the drugs used for in vivo selection thereby increases the number of transduced HSCs over of DHFR (MTX and TMTX) are not genotoxic whereas nontransduced HSCs.15–17 Genes that express recombi- the drugs used for selection of MGMT, (BCNU and nant growth factor receptors, containing molecular TMZ) are genotoxic. The major drawback of switches, that can be controlled by pharmacological as selective agents is their inability to kill resting or inducers which bind to the switches, can also be used noncyling cells. However, cycling cells, such as hemato- for providing proliferative advantage to HSCs. The poietic colony forming progenitors, can also avoid being pharmacological inducers bring about dimerization of killed by anitfolates.38 The reason for MTX or TMTX engineered growth factor receptors resulting in selective not killing these cells is that they express transport expansion of gene-modified HSCs.18 These approaches proteins that allow uptake of extracellular nucleosides for using growth factor receptors will not be discussed further nucleotide synthesis by the salvage pathway, thereby, here. bypassing the need for de novo nucleotide synthesis and thus circumventing toxicity.39 This necessitates the use of an inhibitor of the nucleoside transport such as In vivo selection using drug-resistance genes nitrobenzylmercaptopurineriboside phosphate (NBMPR- P), to achieve successful in vivo selection with antifolate The essential requirements for in vivo stem cell selection drugs27. While successful in the murine model, the same using drug-resistance genes are as follows: (1) the drug approach27 was not able to achieve stable in vivo selection resistance gene should not be expressed, or expressed at a with the DHFR in a non-human primate model.40 The low level, in nontransduced HSCs, but at sufficiently high explanation given for this lack of efficacy is that large level in transduced stem cells, (2) the selective cytotoxic animals have fewer cycling HSCs than mice. drug should be able to deplete the majority of non- transduced stem cells, (3) the selective drug should have little nonhematopoietic toxicity and (4) the selective drug Multi-drug resistance gene should preferably be not genotoxic for use in nonmalig- nant conditions. The mutli-drug resistance gene, MDR1, encodes an ATP- Several genes have been identified that confer resistance dependent plasma membrane effluxpump, P-glycoprotein to cytotoxic drugs and can be used for selection of gene- (P-gp) (Figure 1). In contrast to the products of other modified HSCs in vivo (Figure 1). These include the genes drug resistance genes, the P-gp extrudes a broad range of

Cancer Gene Therapy Gene therapy with drug resistance genes M Zaboikin et al 337

Figure 1 Schematic representation of drug-resistance gene products and their mechanisms of action. The numbers 1 through 6 depict the different drug-resistance genes discussed in this review. Details are provided in the text.

human MDR1 in marrow cells has been used to protect hematopoietic cells against myelotoxic drugs. Transgenic mice expressing the human MDR1 gene and normal mice transplanted with marrow from MDR1 expressing transgenic mice, did not develop leukopenia after treatment with cytotoxic drugs42,43 presumably due to chemoprotection of transduced cells by MDR1 gene expression. Similarly, retroviral mediated transfer of the human MDR1 gene into mouse marrow cells has been shown to protect transduced cells from chemotherapeutic toxicity in vivo.22,23,44 Mutants of P-gp can be used to tailor drug resistance profiles. For instance, the wild-type version of human MDR1 (contain- ing Gly at position 185) confers preferential resistance against while the mutant with Val at position 185 confers resistance to colchicine.45 Vinblastine is effective against lymphoma cells in the dog and human Figure 2 Mechanism of action of DHFR. A key ingredient in the and its main side effect is marrow toxicity. In this context, nucleotide synthesis is tetrahydrofolate. DHFR is required for the the wild-type MDR1 would be used to provide protection regeneration of tetrahydrofolate from dihydrofolate. NADPH is used against myelosuppression. MDR1 can be combined with as cofactor in this reaction. Tetrahydrofolate is required for the other drug resistance genes to broaden the spectrum of synthesis of deoxythymidine monophosphate (dTMP) from deoxyur- drugs for combinatorial chemoprotection of transduced idine monophosphate. HSCs.46–49 MDR1 has been successfully used for in vivo selection hydrophobic drugs from cells, including vinca alkaloids, of transduced HSCs in murine and canine models. , epipodophyllotoxins, colchicine, actino- Sugimoto et al.50 used a bicistronic retroviral vector to mycin D, taxol and taxotere.41 Transfer and expression of coexpress MDR1 and gp91 in murine HSCs. They were

Cancer Gene Therapy Gene therapy with drug resistance genes M Zaboikin et al 338 Table 1 Transgenes commonly used for in vivo selection of transduced cells

Transgene Mutants useful Mechanism of Selection agents Advantages Disadvantages for in vivo action selection

DHFR L22Y Provides Methotrexate K Provides high levels K Works on S-phase cells F31S resistance to Trimetrexate of chemoprotection only antifolates K Relative lack of toxicity from antifolate drugs K Drugs used for selection are not genotoxica

MDR1 Wild type Membrane pump Colchicine K Provides high levels K Cryptic splice sites make extrudes drugs Actinomycin D of protection transgene unstable in viral vectors Taxol K Expressed at high levels in Vinblastine hematopoietic cells K Possible myeloproliferative effect on transduced cells G185V K Drugs used for selection are genotoxica

MGMT P140K Removes alkyl BCNU K Expressed at low K Modest levels of P140A groups from the O6 TMZ levels in stem cells chemoprotection G156A position of guanine ACNU K Drugs used for selection and thymidine are genotoxica

GST-pi Wild type Inactivates drugs Adriamycin K Provides resistance K Provides rather low levels by conjugating to different spectrum of protection glutathione of drugs K Drugs used for selection K May be useful for are genotoxica improving the effectiveness of other in vivo selection markers (e.g. MRP1) BCNU: 1,3-bis(2-chloroethyl)-1-nitrosourea; TMZ: ; ACNU: Chloroethylnitrosourea. aGenotoxicity, as defined in ISO 10993-3, refers to gene mutations, chromosomal aberrations, and DNA effects caused by damage to mitotic apparatus.

able to show efficient selection of transduced cells exvivo Despite its potential uses, MDR1 has the following with and in vivo using . In studies disadvantages. It is expressed at relatively high levels in using a canine transplantation model, a bicistronic untransduced HSCs.52 The MDR1 cDNA is prone to retroviral vector construct coexpressing MDR1 and rearrangements because of cryptic RNA splicing sites human IL-2 receptor common g-chain cDNAs was used within the coding sequence.53 Finally, the observation for in vivo selection of HSCs with paclitaxel.51 Two of that expression of MDR1 cDNA in mice is associated three dogs involved in this study died from myelosupres- with the development of a myeloproliferative disorder sion resulting from the administration of paclitaxel at resembling chronic myeloid leukemia54 has diminished dosages of 140 or 175 mg/m2. The only surviving animal enthusiasm for the use of this gene. However, other received paclitaxel three times at a lower dosage of studies in mice using serial transplantation of MDR1 125 mg/m2. In this dog, the transduced genes were found overexpressing marrow cells,44 as well as studies of to be expressed in multiple lineages (granulocytes, transplantation and in vivo selection of MDR1 transduced monocytes and lymphocytes) indicating transduction of marrow cells in cancer patients55–61 have not revealed the pluripotent stem cells. The MDR1 transgene expression development of a malignant phenotype. In a recent study was detectable for more than 1 year after discontinuing Modlich et al.62 demonstrated that leukemogenesis in paclitaxel treatment. There appeared to be no abnormal- mice transplanted with marrow mononuclear cells trans- ities of hematopoiesis following selection of transduced duced with MDR1 containing retroviral vector correlated cells with paclitaxel. with increased number of vector integration sites in

Cancer Gene Therapy Gene therapy with drug resistance genes M Zaboikin et al 339 cellular proto-oncogenes and could not be attributed to viral vectors containing wild-type human MGMT the MDR1 transgene. cDNA, has been shown to increase resistance of The P-gp also has a lipid translocase function.63 This hematopoietic cells to BCNU in vivo.24 Repetitive in vivo function may be exploited as a mechanism for controlling BCNU administration increased the proportion of the growth of MDR1 transduced cells. Madin–Darby hematopoietic cells in mice containing the wild-type canine renal cells, transduced with a retroviral vector MGMT transgene.68 encoding MDR1, showed an accumulation of the glyco- To improve the effectiveness of the use of MGMT for sphingolipid receptor for the Escherichia coli-derived in vivo selection, drugs have been identified that inhibit verotoxin in cellular membranes.64 This rendered the endogenous MGMT/AGT. O6 – benzylguanine (BG), a cells sensitive to verotoxin by approximately a million- pseudosubstrate that inactivates endogenous AGT has fold over unmodified cells. Thus MDR1 can be used been shown to increase the cytotoxic effect of BCNU and for both positive and negative selection of cells over- other chloroethylating agents.69 BG forms an S-benzyl- expressing P-gp. cysteine moiety at the cysteine acceptor site of the wild- type protein, resulting in irreversible inactivation of AGT.70 Several mutants of human MGMT have been identified that show increased resistance to BG while Methyl-guanine methyl transferase retaining the ability to remove alkylator-induced O6 adducts.71,72 These mutants allow the use of BG to Methyl-guanine methyl transferase (MGMT) gene en- inactivate endogenous AGT, thereby increasing the codes O6 – alkylguanine-DNA-alkyltransferase (AGT), cytotoxic effect of nitrosoureas on nontransduced hema- which is a DNA repair protein. AGT removes alkyl topoietic cells while protecting those hematopoietic cells lesions from the O6 position of guanine and to a much transduced with retroviral vectors containing the mutant lesser degree from the O4 position of thymidine (Figures 1 MGMT cDNAs. Human MGMT mutants P140A and and 3). Since the alkyl group remains associated with an G156A have shown a 40- and 240-fold higher resistance active center of the AGT molecule and prevents the to BG compared with wild-type MGMT,72 while P140K recycling of the molecule, MGMT is not a true enzyme in has been shown to be over 1000-fold more resistant to a strict sense. Expression of MGMT provides resistance BG.73 Using a murine stem cell transplant model, Ragg to monofunctional methylating agents such as et al.29 have shown that the P140K mutant is superior to dacarbazine and procarbazine as well as bifunctional the P140A mutant and wild-type MGMT in protecting chloroethylating agents such as the nitrosoureas like 1,3- bone marrow in vivo from BG/BCNU toxicity. They also bis(2-chloroethyl)-1-nitrosourea (BCNU) or , demonstrated by serial marrow transplantations that the ACNU or and temozolomide.65,66 combined use of BG and BCNU leads to improved in vivo AGT expression shows considerable variation in selection of chemoresistant, repopulating HSCs. Persons mammalian tissues. For instance, liver cells contain and coinvestigators convincingly demonstrated, in a high AGT activity while HSCs possess low activity, murine model of beta-thalassemia, that coexpression of explaining the prolonged myelosuppression after MGMT with the g-globulin gene, followed by in vivo nitrosourea treatment.67 For this reason, gene transfer selection using three cycles of BG and BCNU, lead to of MGMT genes into hematopoietic stem cells is correction of the disease phenotype. This study showed of considerable interest for in vivo selection of HSCs. for the first time that in vivo selection can overcome the Transplantation of marrow cells, transduced with retro- low levels of engraftment of genetically marked HSCs

Figure 3 Mechanism of action of AGT. Alkylating agents add methyl or ethyl groups to DNA bases. Alkylation of the O6 position of guanine results in formation of O6-alkylated guanine. AGT restores guanine from the O6-alkylated guanine.

Cancer Gene Therapy Gene therapy with drug resistance genes M Zaboikin et al 340 seen when using nonmyeloablative conditioning regimens transplants of marrow from the primary marrow recipi- for marrow transplant.30 ents28 providing evidence of the utility of GST for We recently described the cloning of canine MGMT myeloprotection. In other studies, several bicistronic gene and the creation of a P144K mutant (P144K) that vectors were developed to coexpress GST transgene renders the canine MGMT more resistant to BG than the together with a second drug resistance marker with the wild-type protein.74 The canine MGMT was shown to be aim to broaden the scope of chemoprotection.83,84 as efficient as the human MGMT for selection of gene Additional studies with GST are warranted to determine modified cells in vitro. These studies pave the way for the usefulness of GST for in vivo selection and myelopro- testing the canine MGMTs in canine models of genetic tection. disorders or cancer. The canine MGMT has the advantage of decreased likelihood of elimination of gene marked cells by an immune response in the dog. Neff Newer candidates for in vivo selection 75 et al. demonstrated successful in vivo selection of human Aldehyde dehydrogenase MGMT transduced allogeneic donor canine marrow cells Aldehyde dehydrogenase (ALDH) catalyzes the detox- in dogs. In other studies, Neff et al.76 also demonstrated ification of cyclophosphamide, 4-hydroperoxycyclophos- the use of MGMT for in vivo selection of transduced cells phamide (active metabolite of cyclophosphamide), in dogs after autologous transplantation of canine CD34 , and 4-hydroperoxyfosfamide. positive cells transduced with a retroviral vector expres- Two classes of ALDH have been described: ALDH1 sing the P140K mutant of human MGMT. Marking of and ALDH3. Both classes are capable of detoxification of peripheral blood cells was multilineage with predominant the above mentioned drugs.85 Detoxification of cyclophos- marking of granulocytes. The transplanted animals phamide is achieved by conversion of aldophosphamide, a tolerated escalating doses of temozolomide without signs metabolite of cyclophosphamide, to the inactive com- of thrombocytopenia and other toxicities related to drug pound, carboxyphosphamide, thereby preventing the treatment. This augurs well for the evaluation of MGMT decomposition of aldophosphamide to its cytotoxic in clinical trials to overcome marrow toxicity associated derivative, phosphoramide mustard. Human hematopoie- with high-dose cancer chemotherapy. tic progenitor cells have increased amounts of ALDH.86 This can be exploited for the isolation of HSCs by flow cytometry, using a fluorescent substrate for ALDH.87 The Glutathione S-transferase overexpression of either ALDH188,89 or ALDH390 has been shown to provide increased resistance to 4-hydro- Glutathione S-transferase (GST) represents a family of peroxycyclophosphamide or mafosfamide in vitro, after proteins that protect cells from oxidative injury. GST retroviral transduction using a number of different cell proteins can detoxify metabolites containing carbonyl, lines. Bicistronic retroviral vectors encoding ALDH1 and peroxide and epoxide groups resulting from oxidative a mutant DHFR can be used to provide dual resistance to injury. GSTs can also detoxify a wide range of xenobiotics 4-hydroperoxycyclophosphamide and MTX in NIH 3T3 and are therefore useful as drug-resistance markers. GST fibroblasts, CD34-enriched human peripheral blood proteins provide chemoprotection by conjugating the progenitor cells, and murine marrow cells.91 High levels thiol group in the reduced tripeptide glutathione with of expression of ALDH in HSCs, and short half-life of the drugs containing an electrophilic center (Figure 1). They ALDH1 mRNA92 are potential drawbacks to the use of also bind directly to nonsubstrate ligands and can this drug-resistance gene. detoxify oxidative damage that results from drug ex- posure. GST proteins provide resistance to many drugs Cytidine deaminase including alkylating agents such as melphalan, cis-platin, Nucleoside analogs such as cytosine arabinoside (ara-C) adriamycin and etoposide. GST proteins are either and 2020-difluorodeoxycytidine are important drugs for cytosolic, mitochondrial or membrane bound, and only the treatment of acute myeloid leukemia, but can cause the cytosolic forms have been used for chemoprotection.77 severe myelosuppression. Increased resistance to ara-C Several cytosolic isoforms of GST are known: a, m, p, s was initially observed in human myeloblasts and cell lines and y families. Rat GST alpha5 (also referred to as Yc2) overexpressing cytidine deaminase (CDD).93,94 CDD provides the greatest level of resistance to most commonly deaminates cytosine nucleosides and their analogs such used drugs.78 Gene transfer of GST genes into HSCs is of as ara-C and thereby prevents the accumulation of interest to increase resistance to cytotoxic drugs because intracellular ara-CTP, the active metabolite of ara-C primitive hematopoietic cells do not express GST, (Figures 1 and 4).95 Overexpression of CDD in mouse particularly the alpha version.79 The efficacy of GST in fibroblasts, hematopoietic cell lines and primary hemato- providing drug-resistance was first demonstrated in in poietic cells provides resistance to ara-C in vitro.96–100 vitro experiments in NIH 3T3 fibroblasts,80 followed by Thus CDD shows great promise for myeloprotection in experiments in primary hematopoietic cells.81,82 Transfer situations that warrant the use of ara-C. of GST p into murine marrow cells allowed in vivo selection of hematopoietic stem cells using cyclophos- Multidrug-resistance-associated protein phamide both in the primary recipient of the gene The multidrug-resistance-associated protein (MRP1) is a modified transplant as well as in mice after serial member of the ATP-binding cassette transporter super-

Cancer Gene Therapy Gene therapy with drug resistance genes M Zaboikin et al 341 Clinical trials of drug resistance genes Very few of the above mentioned drug resistance genes have been tested in clinical trials. Patients with breast cancer, ovarian cancer, lymphoma or germ cell tumors have received autologous CD34 þ hematopoietic cells marked with MDR1. In all these trials, CD34 þ cells derived from the bone marrow or peripheral blood were marked with retroviral vectors encoding MDR1.55–61 In one trial,59 an expansion of MDR1 transduced hemato- poietic cells, relative to control neo transduced cells, was observed in some patients after chemotherapy with drugs that could be extruded by P-gp. In another study of patients with germ cell tumors, Abonour et al.,61 using an improved gene transfer protocol that used CH-296 Figure 4 Mechanism of action of cytidine deaminase. Cytosine fragment of fibronectin during exvivo transfer of arabinoside (ara-C) inhibits nuclear DNA synthesis by interfering with MDR1 gene into HSCs, observed persistence of the DNA polymerase function. Cytidine deaminase deaminates ara-C to transgene for more than a year after autologous a much less active compound, arabinosyluracil (ara-U) that is then transplantation in four out of nine patients. However, excreted. the utility of MDR1 for myeloprotection was not evaluated. MGMT(G156A) is being evaluated for mye- loprotective effect against BG and BCNU in patients with advanced solid tumors and non-Hodgkin’s lymphoma.113 family and a transmembrane effluxtransporter similar to The utility and safety of MGMT, MDR1 and other P-gp (Figure 1). MRPs are organic anion transporters.101 potential transgenes for correction of genetic defects or They transport anionic drugs, such as methotrexate, myeloprotection in cancer chemotherapy requires further and neutral drugs conjugated to glutathione, glucuronate pre-clinical and clinical studies. or sulfate. Of the 10 known members of the MRP family, only MRP1 has received the most scrutiny and may be useful clinically.41 The cDNA of MRP1 was cloned Conclusions and future directions from a small-cell lung carcinoma cell line (H69/AR) that was resistant to multiple drugs, but did not The variety of selective markers with different drug overexpress P-gp.102 The MRP1 gene was subsequently resistance profiles that are now available, make it possible found to be overexpressed in several other P-gp negative for gene therapy to be used to overcome myelotoxicity in cancer cell lines103–105 that were also resistant to multiple cancer chemotherapy. As with any form of new treatment, drugs. MRP1 overexpressing cells show increased the use of drug resistance genes for myeloprotection in resistance to podophyllins, certain anthracyclines and cancer chemotherapy has to undergo rigorous testing in vinca alkaloids but, unlike P-gp-overexpressing cells, large animal models and in clinical trials, to establish the have little or no increased resistance to colchicine, efficacy and long-term safety of this approach. Future or taxol.103–107 Transfection of MRP1 clinical trials also need to consider the mutagenic risk of cDNA in human cells is sufficient to confer multidrug retroviral gene transfer that has been recently highlighted resistance similar to that of the MRP-overexpressing in mice and humans.114–116 In addition, obstacles to gene cancer cell lines.107,108 NIH 3T3 fibroblasts, transduced therapy in large animal models and in humans, such as with MRP1 expressing retroviral vectors, have increased poor efficiency of gene transfer and lack of sustained resistance against , etoposide and vincris- transgene expression, need to be addressed to make this tine.109 Overexpression of MRP1 in long-term repopulat- form of treatment a reality for cancer patients. ing HSCs in mice, provides protection against doxorubicin induced leukopenia.110 Since MRP1 prefers glutathione conjugated substrates,111 coexpression of References MRP1 with GST might provide enhanced protection in transduced cells. 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