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2 Selectable Markers for Gene Therapy

3 Michael M. Gottesman*, Thomas Licht1, Chava Kimchi-Sarfaty, Yi Zhou2, Caroline Lee3, 4 Tzipora Shoshani4, Peter Hafkemeyer5, Christine A. Hrycyna6 and Ira Pastan 5 National Cancer Institute, National Institutes of Health, Bethesda, Maryland

6 I. INTRODUCTION The resistance of many cancers to anticancer drugs is due, 30 in many cases, to the overexpression of several different ATP- 31 7 A. The Use and Choice of Selectable dependent transporters (ABC transporters), including the 32 8 Markers human multidrug resistance gene MDR1 (ABC B1) (3,5,6), 33 MRP1 (ABC C1), the multidrug resistance-associated protein 34 9 One of the major problems with current approaches to gene (7) and other MRP family members (8), and MXR (ABC G2) 35 10 therapy is the instability of expression of genes transferred into (9). MDR1 encodes the multidrug transporter, or P-glycopro- 36 11 recipient cells. Although in theory, homologous recombination tein (P-gp). P-gp is a 12 transmembrane domain glycoprotein 37 12 or use of artificial chromosomes can stabilize sequences with composed of 2 homologous halves, each containing 6 trans- 38 13 wild-type regulatory regions, such approaches to gene therapy membrane (TM) domains and one ATP binding/utilization 39 14 are not yet feasible and may not be efficient for some time to site. P-gp recognizes a large number of structurally unrelated 40 15 come. In most high efficiency DNA transfer in current use in hydrophobic and amphipathic molecules, including many 41 16 intact organisms, selectable markers must be used to maintain chemotherapeutic agents, and removes them from the cell via 42 17 transferred sequences; in the absence of selection the trans- an ATP-dependent transport process (see Fig. 1). 43AQ2 18 ferred DNAs or their expression is rapidly lost. MDR1 has many obvious advantages for use as a selectable 44 F1 19 There are several different selectable markers that might marker in gene therapy. It is a cell surface protein that can 45 20 be used for in vivo selection, including genes whose expres- be easily detected by FACS or immunohistochemistry. Cells 46 21 sion has been associated with resistance of cancers to antican- expressing P-gp on their surfaces can be enriched using cell 47 22 cer drugs. Examples include: (a) methotrexate resistance due sorting or magnetic bead panning technologies. The very 48 23 to mutant dihydrofolate reductase [DHFR] (1); (b) alkylating broad range of cytotoxic substrates recognized by P-gp makes 49 24 agent resistance due to expression of methylguanine methyl- it a pharmacologically flexible system, allowing the investiga- 50 25 transferase [MGMT] (2); and (c) the expression of the multid- tor to choose among many different selection regimens with 51 26 rug transporting proteins P-glycoprotein (P-gp, the product of differential toxicity for different tissues and different pharma- 52 27 the MDR1 gene) (3) and MRP (multidrug resistance associated cokinetic properties. Furthermore, as will be discussed in de- 53 28 protein) (4). In this chapter, we will detail our experience with tail in this chapter, P-gp can be mutationally modified to in- 54 AQ1 29 the MDR1 gene. crease resistance to specific substrates and alter inhibitor 55 sensitivity. Hematopoietic cells initially appeared to tolerate 56 Current affiliation: relatively high levels of P-gp expression without major effects 57 * To whom correspondence should be addressed: on differentiated function (10). 58 1 Technical University of Munich, Munich, Germany. 2 Cellular Genomics, Inc., Branford, Connecticut. B. Lessons from Transgenic and Knockout 59 3 National University of Singapore, Singapore. Mice 60 4 QBI Enterprises Ltd., New Ziona, Israel. 5 University Hospital Freiburg, Freiburg, Germany. Two lines of evidence support the concept of using MDR1as 61 6 Purdue University, West Lafayette, Indiana. a selectable marker in human gene therapy. Transgenic mice 62

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Figure 1 Multidrug transporters such as MDR1, MRP1, and MXR confer resistance on transduced cells. The triangles are cytotoxic drugs; the large, bold arrows are transporters.

63 expressing the MDR1 gene in their bone marrow are resistant DNA methyltransferase cDNA were protected from metho- 98 64 to the cytotoxic effects of many different anticancer drugs trexate or 1,3-bis (2-chloroethyl) nitrosourea (BCNU) toxic- 99 65 (10–12). MDR1 transgenic bone marrow can be transplanted ity, respectively (26–24). 100 66 into drug sensitive mice, and the transplanted marrow is resis- Upon overexpression in target cells, drug resistance genes 101 67 tant to cytotoxic drugs (13). Mice transplanted with bone mar- may also protect them from environmental toxins such as car- 102 68 row transduced with the human MDR1 cDNA and exposed cinogens in addition to amelioration of anticancer chemother- 103 69 to taxol show specific enrichment of the MDR1-transduced apy (27). For instance, transfer of O6-methylguanine methyl- 104 70 cells (14–16), and this transduced marrow can be serially transferase increases repair of DNA damage in sensitive cells. 105 71 transplanted and remains drug resistant (16). Recently, this In vitro and in vivo studies confirmed this aspect of the func- 106 72 ability to select transduced bone marrow with taxol has been tion of drug resistance genes (28–29). Liu et al. (30) showed 107 73 demonstrated in a canine bone marrow transplantation model that rapid repair of O6-methylguanine-DNA adducts in 108 74 (17). transgenic mice protected them from N-methyl-nitrosourea- 109 75 The mouse mdr1a and mdr1b genes have been insertionally induced thymic lymphomas. This protection from carcinogens 110 76 inactivated in mice (18–21). These animals, although other- can be targeted to other organs like liver or skin by suitable 111 77 wise normal, are hypersensitive to cytotoxic substrates of P- promoter systems (31,32). 112 78 gp. This hypersensitivity is due in part to the abrogation of Chemoprotection exerted by overexpression of chemore- 113 AQ3 79 the mdr1a-based blood brain barrier (22), and to enhanced sistance genes in hematopoietic organs of transgenic animals 114 80 absorption and decreased excretion of mdr1 substrates (23). could be transferred by transplantation of bone marrow to 115 81 These studies demonstrate the critical role that P-gp plays in normal recipients (13,33). These experiments provided a basis 116 82 drug distribution and pharmacokinetics, and argue that spe- for gene therapy approaches with drug resistance genes. 117 83 cific targeting of P-gp to tissues that do not ordinarily express Hence, drug resistance genes that were initially studied be- 118 84 it (as in gene therapy), will protect such tissues from cytotoxic cause of their association with failure of anticancer chemo- 119 85 mdr1 substrates. therapy are expected to serve as useful tools for gene therapy 120 of cancer by protecting patients from the toxic side effects of 121 chemotherapy. Protection of chemosensitive cells from toxic 122 86 II. SELECTABLE MARKERS IN compounds may be particularly helpful in the case of the he- 123 87 HEMATOPOIETIC SYSTEMS AND IN THE matopoietic system because most cells in blood and bone mar- 124 88 SKIN row are highly susceptible to antineoplastic compounds. 125 ם 89 As noted above, studies on mice transgenic for human MDR1 CD34 hematopoietic progenitor cells do not express gluta- 126 90 established that constitutive overexpression of this gene pro- thione-S-transferases (34), and only very low levels of endog- 127 91 tects animals from antineoplastic agents. Drugs could be ad- enous MDR1 gene are expressed in myeloid and erythroid 128 AQ4 92 ministered safely at dose-levels several-fold higher than to progenitor cells (35,36). These low expression levels are not 129 93 mice of the respective background strains (10,11). To demon- capable of providing protection from the cytotoxicity of anti- 130 94 strate the specificity of this protection, verapamil, an inhibitor cancer drugs. Conversely, the high susceptibility of normal 131 95 of P-glycoprotein, was coadministered, resulting in reversal of hematopoietic cells to cytotoxic agents allows selection strate- 132 96 drug resistance (12). Similarly, mice transgenic for a mutated gies exploiting drug resistance genes if sufficient levels of 133 97 dihydrofolate reductase (DHFR) or an O6-methylguanine resistance can be conferred. 134 4104-8—Templeton—Ch21—R1—07-24-03 16:17:03—

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135 A variety of different genes have been used to confer drug resistance to neomycin, puromycin, or hygromycin. Because 175 T1 136 resistance on bone marrow cells (see Table 1). Retroviral of their pharmacology or pharmacokinetics such compounds 176 137 transduction with a full-length MDR1 cDNA promoted by cannot be used for selection in vivo. 177 138 Long-Terminal Repeats (LTRs) of Harvey sarcoma virus pro- Further support for the potential usefulness of drug resis- 178 139 tected normal, clonogenic hematopoietic precursors or eryth- tance genes for selection in vivo was provided by experiments 179 140 roleukemia cells from anticancer drugs (37,38). Transduced in which MDR1-transduced bone marrow was first trans- 180 141 cells were found to be resistant to multiple drugs including planted into recipient mice (16). After taxol treatment of recip- 181 142 taxol, colchicine, and daunomycin. Murine hematopoietic ient mice, their bone marrow was then retransplanted into 182 143 stem cells originating from fetal liver (39), peripheral blood a second generation of recipient mice. In several cycles of 183 144 following mobilization with the use of growth factors (40), retransplantation and taxol treatment of recipient animals, in- 184 145 or from bone marrow (41), were efficiently transduced with creasingly high levels of drug resistance were generated in 185 146 retroviral MDR1 vectors. In the latter study, it was shown that vivo. Mice of the fifth and sixth generation survived doses of 186 147 transplantation of transduced hematopoietic stem cells results taxol that were lethal for mice that had not undergone bone 187 148 in efficient expression of functional human P-glycoprotein in marrow transplantation. 188 149 recipient mice. In spite of generally lower transduction fre- Bunting et al. (51) reported that transduction of murine 189 ם 150 quencies, CD34 human progenitor cells could also be trans- bone marrow cells with pHaMDR1 retroviral vector enables 190 151 duced with retroviruses conveying the multidrug resistance ex vivo stem cell expansion, which might help account for 191 152 gene (42,43). Similarly, vectors containing MRP1 (44) or mu- the ability of transduced cells to survive multiple cycles of 192 153 tated DHFR cDNAs are highly efficient in rendering bone transplantation. However, the biological safety of expansion 193 154 marrow cells resistant to methotrexate or trimetrexate, respec- of transduced stem cells is currently under scrutiny. When 194AQ5 155 tively (45,46). MDR1-transduced progenitor cells are expanded with growth 195 156 Pluripotent human hematopoietic stem cells or early pro- factors for extended periods (up to 12 days), uncontrolled 196 157 genitors, respectively, are difficult to transduce with ampho- proliferation occurs, as has been observed in one study (51). 197 158 tropic retroviruses (47). Fruehauf et al. (48) targeted imma- The authors concluded that the finding was related to the 198 159 ture, cobblestone area-forming progenitor cells. However, in MDR1 transgene because the development of a myeloprolifer- 199 160 this study significant vincristine resistance was achieved only ative syndrome was not observed following transfer of DHFR. 200 161 in a small minority of the immature cell population. This might Other groups have not observed this adverse effect after 201 162 be due to endogenously high MDR1 expression in hematopoi- MDR1 transfer to murine hematopoietic cells. More impor- 202 163 etic stem cells (49), which can make it difficult to analyze the tantly, comparable studies in nonhuman primates did not re- 203 164 function of the transgene. DHFR might be a better marker for veal perturbations of myelopoiesis (52). It appears possible 204 165 selection at the level of long-term culture-initiating cells (50). that these differences are related to the recent finding that the 205 166 Transplantation of MDR1-transduced murine bone marrow integration sites of retroviral vectors reveal predilections for 206 167 cells into W/Wv mice (14) or lethally irradiated normal synge- certain chromosomes and are not randomly distributed in the 207 168 neic mice (15) resulted in significant gene expression in the genome of bone marrow repopulating cells (53). 208 169 bone marrow of recipient animals. Both investigators detected These concerns have prompted recent investigations with 209 170 elevated levels of MDR1 expression after treatment of recipi- vector systems other than retroviruses. For instance, SV40 210 171 ent mice with taxol, favoring the idea of a selective advantage pseudovirions allow for highly efficient MDR1 gene transfer 211 172 in vivo of hematopoietic cells overexpressing the MDR1 to hematopoietic cells (54,55). Alternatively, Epstein-Barr 212 173 transgene. This observation was in marked contrast to previ- virus-based vectors can be designed that contain the MDR1 213 174 ous studies with selectable markers such as genes conferring cDNA episomally in target cells (56). With such vector sys- 214

Table 1 Use of Drug-Resistance Genes to Confer Resistance on Bone Marrow

Gene Selection Reference

Multidrug resistance gene 1 (MDR1) Multiple cytotoxic natural product drugs Gottesman et al., 1995 (3) Multidrug associated protein 1 (MRP1) Multiple cytotoxic natural product drugs Omori et al., 1999 (44) Dihydrofolate reductase (DHFR) Methotrexate and trimethotrexate Flasshove et al., 1998 (1) Warlick et al., 2002 (70) Cytidine deaminase Cytosine arabinoside Momparler et al., 1996 (74) Glutathione transferase Yc Melphalan, mechlorethamine, chlorambucil Letourneau et al., 1996 (75) Aldehyde dehydrogenase Cyclophosphamide Magni et al., 1996 (76) Moreb et al., 1996 (77) O6-methylguanine methyltransferase Nitrosourea (BCNU) Allay et al., 1995 (78) (O6-MGMT) 4104-8—Templeton—Ch21—R1—07-24-03 16:17:03—

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215 tems life-long expression cannot be achieved because they tate increased levels of resistance after several cycles of trans- 271 216 fail to integrate into the genome. Conversely, for protection plantation and drug treatment of recipient animals. 272 217 of hematopoietic cells during a series of chemotherapy cycles, In vivo selection of retrovirally transduced hematopoietic 273 218 sustained expression may not be required. Loss of MDR1 cells has convincingly been demonstrated with the DHFR as 274 219 expression after the period of chemotherapy might in fact in- a selectable marker (73). In this study mice were transplanted 275 220 crease the safety of MDR1 gene therapy. with DHFR-transduced bone marrow cells. Drug treatment 276 221 With the use of retroviral vectors, functional drug resis- resulted in significantly increased expression in granulocytes, 277 222 tance of hematopoietic progenitor cells transduced by MDR1 erythrocytes, platelets, and T- and B-lymphocytes. Secondary 278 223 vectors was first demonstrated in tissue culture (37) and re- recipients revealed that selection had occurred at the stem cell 279 224 cently in mice transplanted with transduced human progenitor level. 280 225 cells (57). As a result, recipient animals can be treated safely Resistance to another antimetabolite drug, cytosine arabi- 281 226 with intensified chemotherapy following reinfusion of MDR1- noside, which is a major component of treatment for acute 282 227 transduced cells (58). leukemias, is conferred by cytidine deaminase. Hematopoietic 283 228 The efficiency of chemoprotection during antineoplastic cells were rendered resistant to cytosine arabinoside by trans- 284 229 chemotherapy is questionable if the cancers themselves ex- fer of this gene (74). 285 230 press high amounts of P-glycoprotein. To further enhance the Different patterns of chemoresistance can be attributed to 286 231 efficiency of this approach a vector has been designed that various drug resistance genes. For instance, the MRP1 gene 287 232 contains a mutant MDR1 cDNA (59). A point mutation in is genetically and functionally related to MDR1. Retroviral 288 233 amino acid position 938 by which phenylalanine is replaced transfer of MRP1 resulted in resistance to doxorubicin, etopo- 289 234 by alanine has little effect on the substrate specificity of P- side, and vincristine (4). However, since binding and transport 290 235 glycoprotein but abolishes the reversing effect of a chemosen- of inhibitors to MDR1 may be different from MRP, transfer 291 of this gene may be useful if naturally occurring resistance due 292 236 sitizing agent, trans(E)-flupenthixol (60). Such vectors can be to MDR1 overexpression in cancer cells has to be overcome 293 237 useful to overcome multidrug resistance in cancer cells, which to allow for effective chemotherapy of an MDR1-expressing 294 238 are protected by wild-type P-glycoprotein, whereas hemato- cancer. 295 239 poietic cells are protected by the mutant gene even in the Resistance to alkylating agents is multicausative, and sev- 296 240 presence of anticancer drug and chemosensitizer. eral genes may be useful as selectable markers. Retroviral 297 241 Treatment of MDR1-transduced hematopoietic cells with transfer of a rat glutathione S-transferase Yc cDNA to hemato- 298 242 anticancer drugs in tissue culture increases expression of poietic cells conveyed moderate resistance to melphalan, 299 243 MDR1 mRNA and P-glycoprotein in the surviving cell popu- mechlorethamine, and chlorambucil (75). Resistance to cyclo- 300 244 lation as a result of clonal selection (61). Moreover, MDR1 phosphamide or 4-hydroperoxycyclophosphamide, respec- 301 245 transduced hematopoietic cells exposed to high concentrations tively, could be conferred on hematopoietic cells by transfer 302 246 of anthracyclines or colchicine prior to transplantation main- of aldehyde dehydrogenase with the use of retroviral vectors 303 247 tain their ability to engraft and rescue recipient mice from (76,77). Leukemic or primary hematopoietic cells were ren- 304 248 otherwise lethal irradiation (62). These animal studies provide dered resistant to BCNU by retroviral transfer of a human O6- 305 249 useful models for the optimization of conditions for clinical alkylguanine-DNA alkyltransferase cDNA (2,78,79). Trans- 306 250 applications. plantation of transduced bone marrow cells rescued recipient 307 251 Several of the drug resistance genes have been used to animals from the toxicity of nitrosoureas (80). In particular, 308 252 protect hematopoietic cells from drugs used in anticancer nitrosourea-induced severe immunodeficiency can be over- 309 253 treatment. As has been seen with MDR1, chemoprotection of come by transduction of immature progenitor cells (80,81). 310 254 hematopoietic progenitor cells and a selective advantage in Furthermore, resistance to nitrosoureas in combination with 311 255 vitro were demonstrated following transduction by mutated an inhibitor of O6-alkylguanine-DNA alkyltransferase, a key 312 256 DHFR cDNAs that confer resistance to methotrexate and tri- enzyme involved in naturally occurring resistance to nitro- 313 257 metrexate (1,63–66). Williams et al. (67), Cline et al. (68), soureas, could be conferred by retroviral transfer of a mutated 314 258 and Vinh et al. (69) demonstrated protection of recipient ani- O6-methylguanine DNA methyltransferase cDNA (26). This 315 ם 259 mals from lethal doses of methotrexate. Human CD34 cells approach protected mice from lethal drug doses and allowed 316 260 can be transduced efficiently with a DHFR vector (1). selection of transduced hematopoietic progenitor cells. 317 261 Mice transplanted with DHFR-transduced hematopoietic To widen the range of anticancer drugs to be inactivated 318 262 cells display resistance against high levels of trimetrexate, by gene therapy, vectors have been constructed for coexpres- 319 263 which can be transferred to a second generation of transplant sion of two different drug resistance genes. For instance, a 320 264 recipients (70). Interestingly, mice can be transplanted with vector containing MDR1 and O6-alkylguanine-DNA-alkyl- 321 265 low amounts of DHFR-transduced cells following mild total transferase rendered human erythroleukemia cells resistant to 322 AQ6 266 body irradiation at a reduced dose of 1_Gy and acquire metho- the MDR1 substrates, colchicine and doxorubicin, as well as to 323 267 trexate resistance by treatment with this drug for 60 days fol- alkylating agents, N-Methyl-N-nitrosourea and temozolomide 324 268 lowing transplantation (71). Retransplantation experiments (82). 325 269 performed with dihydrofolate reductase (72) gave results com- A different approach to utilize vectors that allow for coex- 326 270 parable to those obtained with MDR1 (16); both genes facili- pression of two genes is to include a second gene that may 327 4104-8—Templeton—Ch21—R1—07-24-03 16:17:03—

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328 enhance the efficacy of a selectable marker gene. This has from immature progenitor cells. In accord with these findings, 328 329 been shown by construction of a vector that contained an Schiedlmeier et al. (99) reported that IL-3-stimulated hemato- 329 330 MRP1-cDNA and a cDNA encoding ␥-glutamyl-cysteine syn- poietic cells engrafted more poorly than cells grown in the 330 331 thetase, the rate-limiting enzyme of glutathione biosynthesis presence of other growth factor combinations. Both studies 331 332 (83). Resistance to substrates of MRP1 was thereby increased resulted in efficient retroviral MDR1 transfer to primitive 332 333 due to elevated glutathione levels in transduced cells. The human progenitor/stem cells. 333 334 MRP1 transporter extrudes glutathione-conjugated com- A different approach to improve the utility of selectable 334 335 pounds from the cell; hence, elevated concentrations of gluta- markers is to coexpress two drug resistance genes, thereby 335 336 thione increase the concentrations of potential substrates. conferring resistance to a broad range of cytotoxic agents. To 336 337 Based on experiments in tissue culture and animal models, this end, mutated dihydrofolate reductase has been coex- 337 338 early clinical trials on transfer of the MDR1 gene to hemato- pressed with MDR1orwith thymidylate synthase (100,101), 338 339 poietic progenitor cells have been conducted (84–86). Bone and MDR1 has also been expressed with O6-methylguanine- 339 340 marrow or peripheral blood progenitor cells from patients suf- DNA-methyltransferase (102,103). Coexpression of glutathi- 340 341 fering from advanced neoplastic diseases were retrovirally one S-transferase and cytidine deaminase rendered cells resis- 341 342 transduced and reinfused after high-dose chemotherapy tant to cytosine arabinoside and alkylating agents such as mel- 342 343 (87–89). These studies revealed that transduction efficiencies phalan and chlorambucil (104). These compounds are used in 343 344 using MDR1 vectors as detected in bone marrow or peripheral chemotherapy of malignant lymphomas. 344 345 blood of patients tended to be low, and varied from one patient Alternatively, a dominant-positive selectable marker gene 345 346 to another. Notably, in two recent studies enrichment of can be coexpressed with a negative selectable marker such 346 347 MDR1-transduced cells was observed following treatment as thymidine kinase from Herpes simplex virus (HSV-TK) 347 348 with etoposide or paclitaxel, respectively (90,91). The studies (105,106). The latter approach allows selective elimination of 348 349 confirm the concept that the human multidrug resistance gene transduced cells. Such an approach may increase the safety 349 350 can serve as a drug-selectable marker gene in vivo in the of gene transfer if cancer cells contaminating hematopoietic 350 351 hematopoietic system. However, gene transfer procedures and cell preparations are inadvertently rendered drug-resistant, or 351 352 selection strategies need to be improved to efficiently protect if transduced cells become malignant (51,107). Selective kill- 352 353 human hematopoietic cells from the cytotoxicity of drug treat- ing of MDR1-HSV-TK transduced cells in vivo has been dem- 353 354 ment. In particular, clinical studies should be conducted with onstrated (106). Thymidine kinase may not only facilitate se- 354 355 novel vector constructs and improved culture conditions that lective killing of cancer cells but instead increase the efficacy 355 356 allow for increased transduction rates. of certain selectable marker genes. A bicistronic vector in 356 357 Improvements in vector design have been suggested by which thymidine kinase was combined with dihydrofolate re- 357 358 several groups. Using the multidrug resistance gene, Metz et ductase displayed enhanced resistance as compared to a con- 358 359 al. (92) showed that retroviral vectors derived from Harvey struct that contained a neomycin phosphotransferase instead of 359 360 viruses can be substantially shortened without reduction of thymidine kinase (108). The authors concluded that thymidine 360 361 gene transfer efficiency, thereby increasing the maximum size kinase may be useful to salvage thymidine. 361 362 of the packaged gene of interest. By systematic analysis of To increase the safety of gene therapy of cancer, drug resis- 362 363 the U3-region of various 5′-long-terminal repeats, Baum et tance genes may be combined with cDNAs that specifically 363AQ7 364 al. (93) optimized MDR1 transfer to hematopoietic cells. Nota- eliminate cancer cells. This has been demonstrated for chronic 364 365 bly, transfer to immature hematopoietic progenitor cells, myeloid leukemia (CML), which is characterized by a specific 365 366 which are generally difficult to transduce, was improved (94). molecular marker, the BCR/ABL gene fusion. A vector has 366 367 More recent improvements of posttranscriptional processing been constructed that combined a methotrexate-resistant dihy- 367 368 led to a vector that reliably ensured MDR1 expression and drofolate reductase with an antiBCR/ABL antisense sequence 368 369 drug efflux in human hematopoietic cells following an in vivo (109). Transfer of this vector to CML cells led to the restora- 369 cells 370 םpassage in immunodeficient mice (95). Other vector systems tion of normal cellular function of BCR/ABL cDNA 370 371 used for chemoresistance gene transfer to hematopoietic cells due to reduced levels of transcripts while conferring drug re- 371 372 include adeno-associated virus vectors (96) or liposomes (97) sistance. 372 373 (see Sections IV and V.). In addition to improvement of gene therapy of cancer, drug 373 374 New vector constructs increase the efficiency of gene resistance genes may be helpful for gene therapy of nonmalig- 374 375 transfer to hematopoietic cells but do not necessarily ensure nant diseases if increased gene expression is desired. In fact, 375 376 gene expression for sustained periods. A major obstacle to there is considerable interest in using drug selectable marker 376 377 long-term gene expression is the limited lifespan of some genes to introduce and enrich otherwise nonselectable genes 377 378 transduced cell clones. Since only hematopoietic stem cells in target organs. Gene therapy, although thought to bear the 378 379 have the capability of self-renewal, the lifespan of progeny potential of curing genetically determined diseases, is fre- 379 380 generated by more differentiated progenitor cells is limited. quently hampered by low gene expression in target organs. 380 381 Berger et al. (98) have shown that expansion of cells with This is particularly true for hematopoietic disorders because 381 382 cytokines, particularly with interleukin-3 (IL-3), reduces the the efficiency of gene transfer is often limited, and stable 382 383 frequency of long-term culture-initiating cells (LTC-IC), expression of transgenes in bone marrow has been found diffi- 383 384 which correlated with reduction of Rhodamine-123 efflux cult to accomplish. 384 4104-8—Templeton—Ch21—R1—07-24-03 16:17:03—

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442 For instance, Gaucher disease is characterized by accumu- other nonselectable genes via bicistronic vectors to serve as 499 443 lation of a glucosylceramide in glucocerebrosidase-deficient a source of protein to treat a genetic defect in the skin or 500 444 hematopoietic cells. These patients suffer from skeletal le- elsewhere in the host (122). 501 445 sions, severe hepatosplenomegaly, anemia, and disorders of While MDR vectors are well characterized in vitro, their 502 446 the central nervous system. While it is possible to efficiently usefulness in vivo has still to be established. We have recently 503 447 transduce a glucocerebrosidase cDNA to hematopoietic pro- demonstrated in a large-animal model that expression of a 504 448 genitor cells (110,111), expression levels tend to decrease after nonselectable gene that had been undetectable for more than 505 449 several weeks or months in vivo because of silencing or lim- one year can be recovered by coexpression with a drug-se- 506 450 ited lifespan of the transduced cells’ progeny. To increase lectable marker, MDR1 (17). In this study, high levels of the 507 451 expression of glucocerebrosidase in vivo, Aran et al. (112) nonselectable, therapeutic gene were achieved in peripheral 508 452 constructed a transcriptional fusion between MDR1 and the blood and bone marrow following treatment of the animal 509 453 glucocerebrosidase gene. Increased expression of the latter with paclitaxel. The toxicity of this treatment, however, was 510 454 gene was achieved by selection with cytotoxic substrates of considerable. Thus, further investigations in animal models 511 455 P-glycoprotein. Appropriate selection strategies allowed com- are needed to optimize selection strategies in live animals. 512 456 plete restoration of the underlying genetic defect in cells from Detection of the function of transferred genes may be diffi- 513 457 Gaucher patients (113). Transduction of such bicistronic vec- cult if normal animals are utilized because of the activity of 514 458 tors into hematopoietic stem cells might allow treatment of the respective endogenous gene product. To circumvent this 515 459 patients by chemotherapeutical elimination of non-transduced difficulty, ‘‘knock-out’’ animals whose gene has been inacti- 516 460 cells that continue to synthesize or store glucosylceramide. vated by targeted disruption can serve as useful models. For 517 461 Moreover, following chemotherapy, the numbers of geneti- instance, mice whose ␣–galactosidase gene has been dis- 518 462 cally corrected hematopoietic progenitor cells should increase rupted may be helpful to characterize a bicistronic vector in 519 463 in bone marrow to maintain physiological numbers of mature which MDR1iscombined with the respective human gene 520 464 granulocytes, monocytes, and lymphocytes in peripheral for correction of Fabry disease (123). Another alternative is 521 465 blood. Recently, in vivo selection for cells expressing glucoc- to use marking genes that are not physiologically expressed 522 466 erebrosidase was demonstrated with a vector containing the at high levels in normal tissues. To characterize bicistronic 523 467 selectable marker gene, DHFR (114). vectors containing MDR1, this gene has been coexpressed 524 468 Similarly, bicistronic vectors that facilitate coexpression with a green fluorescent protein or ␤–galactosidase (124). 525 469 of MDR1 and ␣–galactosidase A have been engineered (115). These model systems should help to improve protocols for 526 470 Defects of ␣–galactosidase A are the cause of Fabry disease, efficient drug selection and to identify strategies for selection 527 471 a globotriaosylceramide storage disorder that affects the skin, at limited systemic toxicity. For instance, addition of P-glyco- 528 472 kidneys, heart, and nervous system. Other applications for protein inhibitors at low concentration to cytotoxic drugs may 529 473 bicistronic fusions include immunological disorders such as increase the stringency of drug selection, thereby allowing 530 474 chronic granulomatous disease and X-linked or adenosine de- use of anticancer drugs at low concentrations for selection 531 475 aminase (ADA) deficiency-related severe combined immuno- (113). 532 476 deficiency (SCID) syndromes. For treatment of these diseases, 477 vectors have been constructed that contain a gp91phox or an 478 ADA cDNA (116–119). DHFR was used as a selectable III. BICISTRONIC VECTORS CONTAINING 533 ␣ 479 marker gene in a bicistronic vector for correction of 1-anti- SELECTABLE MARKERS 534 480 trypsin deficiency (120). Further discussion of the use of bicis- 481 tronic vectors is found in Section III. Although coexpression of two proteins can be achieved 535 482 A different strategy to exploit the MDR1 gene as a drug- through the use of separate promoters, the coexpression is 536 483 selectable marker for correction of ADA deficiency was de- frequently uncoupled due to promoter interference or shutoff 537 484 scribed by Germann et al. (121). In this study, both genes of gene expression from one of the promoters, which causes 538 485 were fused to a single cDNA encoding a bifunctional chimeric the selected cells not to express the desired protein. To over- 539 486 protein. This approach, however, cannot be used if the two come this problem, the selectable marker may be expressed 540 487 proteins are physiologically located in different cellular com- with the therapeutic gene as a translational or transcriptional 541 488 partments. fusion. A therapeutic protein can be directly linked to the 542 489 Another system in which selectable markers may be useful carboxylterminus of the multidrug transporter P-glycoprotein 543 490 is in the skin. It is possible to grow keratinocytes in culture (P-gp). The resulting fusion protein possesses functions of 544 491 and introduce the MDR1 gene via retroviral vectors. Such both P-gp and the target protein (125). Since P-gp is an integral 545 492 keratinocytes are resistant to MDR drugs in vitro, and when membrane protein that functions on the cell plasma mem- 546 493 transplanted on keratinocyte ‘‘rafts’’ to recipient animals, they brane, unless two proteins can be separated by a posttransla- 547 494 remain resistant to colchicine, which can be applied as an tional proteolytic modification, the expressed target protein 548 495 ointment. If colchicine is withdrawn, transplanted keratino- will be associated with the plasma membrane regardless of 549 496 cytes are gradually replaced by nontransduced host skin; in its normal cellular location. Thus, even though translational 550 497 the presence of selection, the transplanted keratinocyte graft is fusions guarantee protein coexpression, their potential is lim- 551 498 maintained. It should be possible in such a system to introduce ited. On the other hand, transcriptional fusions, e.g., using 552 4104-8—Templeton—Ch21—R1—07-24-03 16:17:03—

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553 bicistronic or polycistronic mRNA to encode more than one correct intracellular protein trafficking (112). The demonstra- 553 554 cDNA, may prove to be more generally applicable. tion that a noncoding RNA, such as a hammerhead ribozyme, 554 can function even though tethered to an mRNA encoding a 555 555 A. MDR1 Bicistronic Vectors Containing functional MDR1 provides an additional powerful way to use 556 557 556 Internal Ribosome Entry Sites (IRES) bicistronic vectors (130). Another approach to the use of MDR1-based bicistronic 558 557 A DNA segment corresponding to one polypeptide chain plus vectors is to develop ‘‘suicide’’ vectors for cancer gene ther- 559 558 the translational start and stop signals for protein synthesis apy. Using MDR1toprotect bone marrow cells from cytotoxic 560 559 can be loosely defined as a cistron. An mRNA encoding only drugs represents a promising approach to improve cancer 561 560 a single polypeptide is called monocistronic mRNA; if it en- chemotherapy. However, contaminating cancer cells may be 562 561 codes two or more polypeptide chains, it may be called bicis- inadvertently transduced with MDR1, or transduced bone mar- 563 562 tronic or polycistronic mRNA. Almost all eukaryotic mRNA row cells may accidentally develop new tumors. In those 564 563 molecules are monocistronic. Initiation of translation of eukar- cases, overexpression of P-gp could cause multidrug resis- 565 564 yotic mRNA is mediated by cap-binding protein that recog- tance in inadvertently transduced tumor cells that contaminate 566 565 nizes a methylated guanosine cap at the 5′ terminus of mRNA. bone marrow, or in any transduced cells that later become 567 566 However, some viral mRNA molecules transcribed in eukary- malignant. A bicistronic ‘‘suicide’’ vector developed in this 568 567 otic cells are polycistronic. They can use a cap-independent laboratory links P-gp expression with herpes simplex virus 569 568 mechanism to initiate translation in the middle of mRNA mol- thymidine kinase (TK) expression (105,106). Thus the cells 570 569 ecules. For picornavirus, this cap-independent internal initia- containing this vector can be eliminated through ganciclovir 571 570 tion of translation is mediated through a unique internal ribo- treatment. 572 571 some entry site (IRES) within the mRNA molecule (126,127). A third approach is to link two drug resistance genes to- 573 572 Identification of IRES sequences has led to the develop- gether using a bicistronic vector system to extend the ability 574 573 ment of bicistronic vectors that allow coexpression of two of the vector to confer drug resistance. Examples include the 575 574 different polypeptides from a single mRNA molecule in euk- use of MDR1 with dihydrofolate reductase that confers metho- 576 575 aryotic cells (128,129). Using a bicistronic vector containing trexate resistance (132), MDR1 plus methylguanine methyl- 577 576 an IRES to coexpress a target gene and a selectable marker transferase (MGMT) that confers resistance to certain alkylat- 578 577 has several advantages. First, since two polypeptides are trans- ing agents (102,103,133), and MRP1 plus gamma- 579AQ8 578 lated from the same mRNA molecule, the bicistronic vector glutamylcysteine synthetase that confers resistance to alkylat- 580 579 guarantees coexpression of a selectable marker and a second ing agents as well (83). 581 580 protein. Secondly, bicistronic mRNA allows two polypeptides Finally, bicistronic vectors can be used to introduce marker 582 581 to be translated separately. Thus, this system does not compro- genes into selected cells. For example, MDR1 vectors contain- 583 582 mise the correct intracellular trafficking of proteins directed ing green fluorescent protein or ␤-galactosidase have been 584 583 to different subcellular compartments. In addition, using a constructed to determine the efficiency of expression of the 585 584 bicistronic vector, expression of a target gene is proportionate target gene in transduced and MDR1 selected cells (124). 586 585 to the expression of a selectable marker. Hence, expression 586 of a target protein can be achieved quantitatively by applying B. Efficiency of IRES-Dependent 587 587 selections of different stringencies. Translation 588 588 To demonstrate coexpression of a dominant selectable 589 marker with a therapeutic gene using a bicistronic vector, our Using an IRES to generate a bicistronic mRNA ensures coex- 589 590 laboratory has coexpressed P-gp with glucocerebrosidase pression of two different proteins. However, IRES-dependent 590 591 (112,113), ␤-galactosidase (115), adenosine deaminase (118), mRNA translation (or cap-independent translation) is less effi- 591 592 a subunit of the NAPH oxidase complex (116,117), the shared cient than cap-dependent translation, so that the two proteins 592 593 gamma chain of the interleukin receptors (119), and a ham- are not expressed in equal amounts. It has been shown that 593 594 merhead ribozyme targeted to the U5 region of HIV-1 LTR in a monocistronic vector, insertion of an IRES upstream from 594 595 (130). In those experiments, MDR1 served as a selectable an open reading frame of either P-gp or dihydrofolate re- 595 596 marker linked to the target gene by an IRES from encephalo- ductase (DHFR) reduces the translation efficiency by 2- to 596 597 myocarditis virus (EMCV) and constructed in a retroviral vec- 10-fold (129,134). Using a bicistronic vector, expression of 597 598 tor containing Harvey sarcoma virus LTR (131). Two configu- neo in the position downstream from the IRES is 25% to 50% 598 599 rations, in which MDR1isplaced either before or after the of that observed when neo is in the upstream position (128). 599 600 IRES, have been examined in some cases. As demonstrated The asymmetric expression pattern of the bicistronic vector 600 601 in those experiments, P-gp and the target gene are coexpressed results in a significant difference in MDR1 transducing titer 601 602 in the cells selected using cytotoxic P-gp substrates, such as between a configuration with P-gp placed before the IRES 602 603 colchicine or vincristine; the expressed target proteins are and a configuration in which P-gp is placed after the IRES. 603 604 functional as detected using in vitro, or ex vivo analysis. In We have found that the apparent titer of a bicistronic vector 604 605 one case, using subcellular fractionation, we have demon- containing ADA-IRES-MDR1 was only 7% of the titer of a 605 606 strated that P-gp and glucocerebrosidase are translocated sepa- bicistronic vector containing MDR1-IRES-ADA (118). Similar 606 607 rately to the cell plasma membrane and lysosomes, indicating reductions in MDR1 transducing titer and in expression of 607 4104-8—Templeton—Ch21—R1—07-24-03 16:17:03—

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663 the nonselected downstream gene was seen with MDR1-␤- C. Flexibility Using Bicistronic Vectors in 719 664 galactosidase bicistronic vectors too (115). The apparent Coordinating Expression of Selectable 720 665 MDR1 transducing titer of the retrovirus is based on the drug Markers and a Therapeutic Gene 721 666 resistance conferred by expression of P-gp as the result of 667 retroviral infection; thus the viral titer is proportional to the Selectable bicistronic vectors provide great flexibility in coor- 722 668 P-gp expression level. Insufficient expression of P-gp is una- dinating expression of a selectable marker, such as P-gp, and 723 669 ble to protect the cells from cytotoxic drug selection. To a therapeutic gene. The low translation efficiency of the IRES 724 670 achieve P-gp expression at the same level, the lower efficiency results in asymmetric expression of genes positioned before 725 671 of translation would have to be compensated for by a higher and after the IRES. This asymmetric expression pattern makes 726 672 level of transcription, which can occur only in a minority of it possible to alter the relative expression level of a therapeutic 727 673 the cells in the transduced population. This may account for gene and P-gp to achieve maximum therapeutic effects while 728 674 the apparent lower MDR1-transducing titer of bicistronic vec- applying minimal selective pressure using a cytotoxic drug. 729 675 tors with a configuration of P-gp placed after the IRES. On By choosing different configurations, i.e., placing MDR1 be- 730 676 the other hand, when cells express P-gp at the same level (i.e., fore or after the IRES, we can select cells expressing a thera- 731 677 the cells survived vincristine or colchicine selection at the peutic gene at either a low level (MDR1 before the IRES) or 732 678 same concentration), ADA expressed from ADA-IRES-MDR1 a high level (MDR1 after IRES). 733 In addition, expression of a therapeutic gene can also be 734 679 is 15-fold higher than the ADA expressed from MDR1-IRES- achieved at a desired level by altering the selection conditions. 735 680 ADA. This difference is probably due to a combination of the The degree of multidrug resistance conferred by P-gp corre- 736 681 lower translation efficiency of ADA located downstream from sponds to the amount of P-gp expressed on the plasma mem- 737 682 the IRES and the high transcription level of ADA-IRES-MDR1 brane. Using a bicistronic vector, the expression of a target 738 683 as the result of vincristine selection. A similar asymmetric gene is proportional to the expression of P-gp, which is di- 739 684 expression of P-gp and human ␤–galactosidase A is also ob- rectly linked to the selection conditions. In a highly stringent 740 685 served in NIH3T3 cells, where the difference is about 8-fold. selection, instead of increasing the concentration of cytotoxic 741 686 IRES-dependent translation is a complex process, in which drug, P-gp reversing agents can also be applied in combination 742 687 mRNA containing IRES interacts with various cellular pro- with low concentrations of cytotoxic drugs (143). P-gp revers- 743 688 teins, including IRES transacting factors [reviewed in Hellen ing agents, also known as chemosensitizers, are noncytotoxic 744 689 and Sarnow (135)]. The efficiency of IRES-dependent transla- hydrophobic compounds that interact with P-gp and cause a 745 690 tion can be affected by the cell type (136), IRES origin direct inhibition of P-gp function. In the presence of a P-gp 746 691 (137,138), and the size and structure of a particular mRNA reversing agent, most P-gp-expressing cells are killed by the 747 692 molecule. We have found that the titer of retrovirus containing cytotoxic drug unless they express a large amount of P-gp 748 693 pHa-MDR1 was higher than pHa-MDR1-IRES-ADA, even to overcome the inhibitory effects. Using a combination of 749 694 though P-gp translation was cap-dependent in both cases. P- cytotoxic drug and chemosensitizer allows selection of cells 750 695 gp expressed from pHa-MDR1 was also at a higher level in expressing the therapeutic gene at a high level without need 751 696 a vincristine resistant cell population than the P-gp expressed for a high concentration of cytotoxic drug. This strategy is 752 697 from pHa-MDR1-IRES-ADA.Apossible explanation for the especially desirable for an in vivo selection in which avoiding 753 698 relatively low retroviral titers observed is RNA instability or systemic toxicity is essential. 754 699 alternative splicing, since no DNA rearrangement was de- High expression of the target gene can be selected using 755 700 tected by Southern blot analysis of the transduced cells using cytotoxic drugs, cytotoxic drugs combined with chemosensi- 756 701 an MDR1 probe. tizers, or the vector configured to place the target gene placed 757 702 In addition to IRESes derived from viruses, several IRES before the IRES. However, those approaches also reduce the 758 703 elements have been identified in human genes. Those IRESes overall number of cells that can survive the selection. Never- 759 704 play important roles in cell cycle-dependent or stress-response theless, using a minimum concentration of drug, the selectable 760 705 translation regulation [reviewed in Sachs (139)]. In contrast bicistronic vector provides options for selecting a large popu- 761 706 to viral IRESes, human IRESes are shorter and are comple- lation of cells with low expression of the target gene, or a 762 707 mentary to 18s rRNA [reviewed in Mauro and Edelman small population of cells with high expression of the target 763 708 (140)]. It has been found that a 9-nt sequence from the 5′- gene. Both options may be useful for gene therapy. For in- 764 709 UTR of the mRNA encoding the Gtx homeodomain protein stance, ADA levels in normal individuals occur over a very 765 710 can function as an IRES. Ten linked copies of the 9-nt se- broad range. Heterozygous carriers can be immunologically 766 711 quence are 3- to 63-fold more active than the classical EMCV normal even with as little as 10% of the normal amount of 767 712 IRES in all 11 cell lines tested (141). Similarly, an IRES ADA [reviewed by Blaese (144)]. Expression of ADA at a 768 713 isolated from the human EIF 4G gene also exhibits 100-fold low level in a large number of cells may prove sufficient to 769 714 more IRES activity than EMCV IRES in 4 different cell lines treat SCID. On the other hand, high ADA-expressing 770 715 (142). In addition to higher efficiency and smaller size, trans- lymphoid cells, even through present as a small percentage 771 716 lation from a human IRES can be regulated by cellular events of total cells, are also able to correct the SCID syndrome 772 717 (142), which may be advantageous for certain cancer gene due to a beneficial by-stander effect (145). In gene therapy 773 718 therapies. applications, the choice of the approach depends on the thera- 774 4104-8—Templeton—Ch21—R1—07-24-03 16:17:03—

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775 peutic strategy for a specific disease. Experiments on animal into a variety of tissues and persistence of transgene expres- 775 776 models are essential to prove the concepts that underlie gene sion in these nondividing tissues, was reported (149–154). 776 777 therapy using selectable markers such as MDR1. Baudard et al. (96) demonstrated that in rapidly dividing cells, 777 continuous selective pressure is necessary to sustain gene 778 expression in cells. MDR1 was used as the selectable marker 779 778 IV. NONRETROVIRAL AND EPISOMAL in this study. Being among the smallest DNA animal viruses 780 ϳ 779 VECTORS EXPRESSING SELECTABLE ( 20 nm in diameter), another disadvantage of the AAV sys- 781 780 MARKERS: AAV, SV40, AND EBV tem is its limited packaging capacity since it can accomodate 782 only aproximately 4.7 kb of the gene of interest. As such, a 783 781 Efficient delivery of a therapeutic gene to the appropriate tar- small and efficient promoter would be required to drive the 784 782 get cells and its subsequent maintenance and expression are expression of large genes. One such promoter is the AAV p5 785 783 important steps for successful gene therapy. Genes introduced promoter, which, together with the ITR, forms a 263-base pair 786 784 into cells are rapidly lost unless there is a mechanism to retain cassette capable of mediating efficient expression in a CF 787 785 these genes within the nucleus and to ensure that the genes bronchial epithelial cell line (149,150). Baudard et al. further 788 786 are also replicated and partitioned into daughter cells during demonstrated that the reduction of the p5 promoter-ITR cas- 789 787 cell division. Long-term expression of the transgene within sette to 234 bp was also able to promote efficient gene expres- 790 788 cells can be achieved either via the integration of the trans- sion (96). 791 789 ferred DNA into the host genome or maintenance of the intro- 790 duced DNA as an autonomously replicating extrachromo- B. SV40 792 791 somal element or episome. In either case, inclusion of a drug- 792 selectable marker, like the MDR1 gene, in the construct would Vectors that facilitate extrachomosomal replication have some 793 793 ensure that rapidly dividing cells containing the transgene are advantages. High gene expression is often observed in such 794 794 given a selective growth advantage. vectors. This could be a result of vector amplification, promo- 795 795 Delivery modalities can be viral or nonviral. Retroviral tion of nuclear localization and retention, as well as transcrip- 796 796 gene transfer, one of the most exploited systems for gene tional activation by viral genes involved in episomal replica- 797 797 transfer into actively dividing cells, has been discussed earlier tion. Selective pressure using selectable markers like the 798 798 in this chapter while liposomal gene delivery will be discussed MDR1 gene, however, is necessary to maintain these episomes 799 799 later in the chapter. In this section, nonretroviral and/or epi- in actively dividing cells. Thus, another potential advantage 800 800 somal vectors expressing selectable markers will be described. of using episomally replicating vectors is that since they are 801 not integrated into the cells, one could potentially extinguish 802 801 A. AAV expression at will by withdrawing selective pressure to repli- 803 cating cells. Episomally replicating vectors can be easily cre- 804 802 In addition to retroviruses, adeno-associated virus (AAV) can ated by the inclusion into the vector design of replicons that 805 803 also facilitate integration of the transgene into the host ge- can be derived from DNA viruses like the Simian Virus 40 806 804 nome. Unlike retroviruses, AAV was found to integrate pref- (SV40) (155), Epstein-Barr virus (EBV) (156) and the (BK) 807AQ10 805 erentially into a specific site on chromosome 19 (146). AAV virus (157–159). Such replicons usually comprise a viral ori- 808 806 is a naturally defective, nonpathogenic, single-strand human gin of replication as well as a viral gene product that is impor- 809 807 DNA parvovirus. For productive infection and viral replica- tant for maintaining extrachromosomal replication. 810 AQ9 808 tion, coinfection with helper viruses, e.g., adenovirus, herpes- SV40 is a 5.2 kb DNA papovavirus that was discovered 811 809 virus, or vaccinia virus are required. In the absence of a helper as a harmless contaminant in early preparations of the Salk 812 810 virus, AAV establishes latency in the host by integrating itself polio vaccine (160,161). SV40 is a double-stranded circular 813 811 into the host genome. AAV has a broad host range and is also 5.2 kb DNA simian virus. It is a nonenveloped virus that 814 812 able to infect both dividing and nondividing cells (147). Hence belongs to the papovavirus family. The SV40 capsid is com- 815 813 recombinant AAV (rAAV) vectors have been exploited as posed of 72 pentamers of the major capsid protein VP1, which 816 814 alternative vehicles for gene therapy. are tied together through their carboxy-termini. VP2 and VP3, 817 815 AAV-based vectors (148) are simple to construct, requiring which share 234 amino acids at their carboxy-termini, connect 818 816 only that the viral inverted terminal repeat (ITR) (which are the minichromosome core to the axial cavities of VP1. It has 819 817 145 nucleotides each) is upstream from the gene of interest. been suggested that correct interpentamer bonding is facili- 820 818 Other important viral genes like rep (involved in replication tated by host chaperones (162,163). SV40 infection begins 821 819 and integration) and cap (encoding structrual genes) can then with the virus binding to its primary receptor, the major histo- 822 820 be supplied in trans. One disadvantage with such rAAV vec- compatibility complex class I (MHC class I), without internal- 823 821 tors is that site-specific integration of the gene of interest into ization of the receptors. The entry is mediated by caveolae and 824 822 the host genome is not observed (96). This is probably because the virus is transported to the Golgi membranes. Its pathway 825 823 the rep gene, which is important for mediating site specific extends to the endoplasmic reticulum (ER), where it is disas- 826 824 integration in the absence of helper viruses, is not included sembled. The mechanism by which the virus reaches the nu- 827 825 in the construct with the gene of interest. Nonetheless, rAAV cleus is not yet known (164–167). The wild-type virus is una- 828 826 has been successfully applied to the delivery of various genes ble to replicate its DNA in rodent cells; therefore no progeny 829 4104-8—Templeton—Ch21—R1—07-24-03 16:17:03—

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882 virions can be produced in these cells (168). Infection of SV40 sitivity of these two viruses. Strayer et al. (169) found evi- 939 883 wild-type virus in cells can result in the integration of viral dence for integration of the recombinant gene or parts of it, 940 884 DNA into the host chromosome, permitting transmission of a few days after transduction in random sites, which might 941 885 expression to daughter cells (169). Some reports associate explain the long-term expression of this system. 942 886 SV40 DNA (specifically the T-antigen) with human tumors, Pseudovirions can transfer the gene of interest to a variety 943 887 mainly based on the presence of sequences from SV40 wild- of cells (including hematopoietic cells) with high efficiency, 944 888 type in some brain tumors and melanomas (170,171). but their clinical applicability is currently limited by the pres- 945 889 The two major SV40 delivery systems are vectors that use ence of wild-type SV40 sequences. The in vitro, method of 946 890 SV40 sequences or the wild-type virus as a helper, and vectors preparing helper-free SV40 vectors utilizes the SV40 viral late 947 891 that are packaged in vitro, with no SV40 sequences and in proteins, VP1, VP2, VP3 and agno or VP1 only (183–186). 948 892 which the wild-type virus is not present. SV40 has numerous Nuclear extracts of baculovirus-transduced Spodopterafrugi- 949 893 advantages as a gene-delivery vehicle (54,172,173): it is able perda (Sf9) insect cells that include these proteins are incu- 950 894 to infect a wide variety of mammalian cells, including human bated with supercoiled plasmid DNA in the presence of 8 mM 951 895 cells, and to express its genes in these cells; the vector system MgCl2,1mM CaCl2, and 5 mM ATP to form the SV40 in 952 896 has an ability to deliver untranslated RNA products; the gene vitro, packaged vectors (55). Such in vitro, assembly allows 953 897 expression may be transient or stable in cell lines, depending larger DNA plasmids (up to 17.6 kb) to be packaged very 954 898 on the specific SV40 system that is used; and episomal replica- efficiently, with no need for SV40 sequences. We have dem- 955 899 tion in SV40 virus requires the SV40 replication origin as onstrated very efficient delivery of the MDR1(ABC B1), MXR 956 900 well as the large T antigen (T-Ag), which activates the replica- (ABC G2), and MRP1(ABC C1) genes, which can confer 957 901 tion origin . Such episomal replication can generate more than multidrug resistance on virtually all cell types (human, mu- 958 902 105 copies per cell of recombinant plasmids (174). Using the rine, and monkey cell lines), in addition to delivery of the 959 903 SV40 delivery systems, no immune response is expected, as GFP gene as a reporter (187). The expression of both MDR1 960 904 well as no inflammatory reaction. and GFP genes is dose dependent. The alteration in the level 961 905 Replacing the late or early region with a foreign gene can of expression suggests that MHC class I receptors play an 962 906 generate SV40 recombinant viral particles important role in determining the efficiency of transduction. 963 907 (169,172,175–181). These are then propagated using either MDR1 constructs that carried a promoter with an intron dem- 964 908 wild-type, or a temperature-sensitive mutant of SV40 as onstrated higher expression than those without the intron. In 965 909 helper, or via a viral producer cell line, COS7, that stably low-expressing MHC class I cell lines, the CMV promoter 966 910 expresses an origin-defective SV40 mutant and is capable of produced more P-gp expression compared with the SV40 pro- 967 911 supporting the lytic cycle of SV40. Multiple infections result moter. In vitro packaged-GFP vectors that carried the CMV 968 912 in higher titers of the virus — up to 1010 infectious units/ml. promoter consistently confirmed higher expression than those 969 913 It has been demonstrated that when the large T-antigen (T- that carried the SV40 promoter. Expression was transient up 970 914 Ag) gene is replaced with a reporter gene, replication-deficient to 21 days, but did last for 3 months under colchicine selection 971 915 recombinant SV40 viruses can be produced and can mediate for MDR1 and was lost soon after selection was withdrawn 972 916 gene transfer in vivo. Reporter gene expression was detectable (55). 973 917 for about 3 months without selection. Present SV40 vectors of The short-term expression of the SV40/MDR1invitro, vec- 974 918 the first type have most of the viral coding sequences removed, tors may be an advantage for use in chemoprotection. Long- 975 919 retaining only the packaging sequences, the polyadenylation term expression beyond the chemotherapy period is undesira- 976 920 signal, and the early promoter of the virus, thus increasing ble, and may put patients at risk for treatment-induced myelo- 977 921 the capacity for DNA to ϳ5.3 kb. The DNA from these vectors dysplasia or secondary leukemia. The SV40/MDR1 vectors 978 922 integrates into the genome of the target cells. that are prepared in vitro, may provide not only a safe vehicle 979 923 Rund et al. (54) demonstrated very efficient delivery (Ͼ for gene delivery but will also potentially avoid the problem 980 924 95%)ofthe drug-selectable marker, MDR1, into various mu- of persistent bone marrow drug resistance in cancer patients. 981 925 rine and human cell types including primary human bone mar- 926 row cells (54). SV40 vectors efficiently deliver HIV-1-inhibi- C. EBV and Other Episomal Vectors 982 927 tory RNAs using pol II or III promoters. Other vectors, which 928 encode a variable fragment antibody recognizing HIV-1 integ- Episomal vectors based on EBV are also being developed 983 929 rase, inhibited HIV-1 infection in SCID mice. This system for gene therapy purposes. EBV is a human B-lymphotropic 984 930 may prove to be useful in antiHIV-1 therapeutics. Fang et al. herpesvirus that resides asymptomatically in more than 90% 985AQ11 931 (182) reported a different packaging system for SV40 vectors of the adult human population by establishing latency and 986 932 where the vector carrying the gene of interest contains only maintaining its genome episomally (188). The life cycle of 987 933 the SV40 origin of replication (182). Instead of using wild- EBV comprises two phases, a lytic and a latent phase. During 988 934 type SV40 viruses as helpers to package the recombinant vec- the lytic phase, EBV DNA replicates via a rolling circle inter- 989 935 tor, recombinant adenoviruses expressing SV40 capsids were mediate to achieve a 1000-fold increase in copy number. The 990 936 used in COS7 cells. The helper adenovirus can be effectively origin of replication, Ori Lyt, and the transacting element 991 937 heat-inactivated without adverse effect on the infectivity of ZEBRA are required for the lytic replication. Rolling circle 992 938 the recombinant SV40 viruses due to the differential heat sen- replication results in the formation of linear head-to-tail con- 993 4104-8—Templeton—Ch21—R1—07-24-03 16:17:03—

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ם 994 catamers. The presence of the EBV terminal repeat (TR) se- as well as human CD34 stem cells. MDR1 was found to be 1051 995 quence causes cleavage of the concatemerized DNA to mole- expressed at a higher level in the episomal vector compared 1052 996 cules of about 150–200 kbp, which are then packaged into to its nonepisomal counterpart and more drug colonies were 1053 997 virions. Upon infection into a permissive cell, the viral DNA obtained upon selection. Episomal plasmids could be re- 1054 998 circularizes by ligation of TR. Latency is established in the covered in drug selected cells for many weeks (56). 1055 999 cells by episomal replication of the circular DNA. Other episomally replicating vectors can be derived from 1056 1000 Episomal replication in EBV is maintained by two ele- BPV viruses (196) or BK virus (158). Unfortunately, BPV 1057AQ14 1001 ments interacting to ensure that the viral genome is retained vectors cannot be reliably maintained as episomes as they 1058 1002 within the nucleus, efficiently replicated and partitioned into exhibit high spontaneous mutation rate (ϳ1%), frequently 1059 1003 daughter cells. Although the copy numbers of episomal viral undergoing integration, deletion, recombination, and rear- 1060 1004 DNA varies from 1–800, only between 4–10 episomal copies rangements (197). Furthermore, BPV has a limited host range 1061 1005 per cell are usually observed using vectors containing EBV and BPV vectors cannot be efficiently maintained in human 1062 1006 OriP and EBNA-1 (189). Unlike other episomal vector sys- cells. Not too much is known about BK-virus-derived epi- 1063 1007 tems, very low rates of spontaneous mutation have been ob- somal vectors. Nonetheless, successful stable maintenance of 1064 1008 served with EBV-based episomal vectors (190). The cis-acting episomal gene expression was reported in human transitional 1065 1009 element responsible for episomal replication is a 1.8 kb OriP carcinoma cells using BK-based vectors but not EBV-based 1066 1010 while the transacting element is EBNA-1. OriP comprises two vectors, probably due to the differential tropism of BK and 1067 1011 distinct sequence motifs, the dyad symmetry motif (DS) from EBV viruses for human uroepithelial cells (157). 1068 1012 which replication is initiated and the family of repeats (FR) Various chimeric viruses have been developed to improve 1069 1013 that serves as a replication fork barrier. Interaction of EBNA- the efficiency of gene transfer as well as the maintenance 1070 1014 1 with DS initiates bidirectional replication, while binding of of gene expression within target cells. These chimeric virus 1071 1015 EBNA-1 to FR enhances transcription from the episome and systems attempt to combine the favorable attributes of each 1072 1016 terminates DNA replication. EBNA is reported not to be onco- vector system and overcome the limitations associated with 1073 1017 genic nor immunogenic. It evades the host immune system each system. The episomal replication ability of EBV was 1074 1018 via the presence of the repeat motif, Gly-Ala, which was found exploited to produce both rapid and long-term high-titer re- 1075 1019 to interfere with antigen processing and MHC class I-re- combinant retroviruses (up to 107 TU/ml) for efficient gene 1076 1020 stricted presentation (191). These EBV episomal vectors repli- transfer into human hematopoietic progenitor cells (198,199). 1077 1021 cate once per cell cycle (192) and are capable of stably main- A novel adenoviral/retroviral chimeric vector was also re- 1078 1022 taining human genomic inserts of sizes between 60–330 kb ported in which an adenoviral delivery system was utilized to 1079 1023 for at least 60 generations (193). efficiently deliver both the retroviral vector and its packaging 1080 AQ12 1024 Vos and colleagues (194) developed a helper-dependent components, thereby inducing the target cells to function as 1081 1025 infectious recombinant EBV to evaluate the feasibility of transient retroviral producers capable of infecting neighboring 1082 1026 using such a vector system to correct hereditary syndromes cells. This system capitalizes on the superior efficiency of 1083 1027 in B-lymphocytes already harboring the EBV virus latently. adenoviruses to deliver genes in vivo and the integrative abil- 1084 1028 The EBV-containing target B-lymphocytes will supply ity of retroviruses to achieve stable gene expression (200). 1085 1029 EBNA-1 in trans for the episomal maintenance of the An EBV/HSV-1 amplicon vector system was also described 1086 AQ13 1030 transgene. Hence only minimal cis-EBV elements for epi- that combines the efficiency of HSV-1 virus to transfer DNA 1087 1031 somal replication (OriP), viral amplification (Ori Lyt), and into various mammalian cells, including the postmitotic neu- 1088 1032 packaging (TR) are included in their construct. The hygro- ronal cells and the ability of EBV to maintain genes episom- 1089 1033 mycin resistance gene was included as a selectable marker in ally. This vector system contains the HSV-1 origin of DNA 1090 1034 their vector. Infectious virions are generated by the producer replication (oriS) and a packaging signal, which allow replica- 1091 1035 cell line HH514. They demonstrated successful transfer of tion and packaging of the amplicon into HSV-1 virions in the 1092 1036 such infectious virions carrying the therapeutic gene, Fanconi presence of HSV-1 helper functions as well as EBV oriP and 1093 1037 anemia group C (FA-C) cDNA, into HSC536, a FA-C patient EBNA-1 (201). Another report describes the use of a similar 1094 1038 cell line. Upon selection with hygromycin, long-term (at least HSV-1 amplicon system for efficient gene transfer, but AAV 1095 1039 6 months) correction of the Fanconi phenotype in vitro, was was included in their vector to achieve stable expression. This 1096 1040 observed, as determined by cellular resistance to the cross- HSV/AAV hybrid vector contains OriS and packaging se- 1097 1041 linking agent, diepoxybutane. They also observed that in the quences from HSV-1, a transgene cassette that is flanked by 1098 1042 absence of selective pressure, their episomal vector is retained AAV ITRs as well as an AAV rep gene residing outside the 1099 1043 in rapidly dividing cells at a rate of 98% per cell division transgene cassette to mediate amplification and genomic inte- 1100 1044 translating to a half-life of 30 days in cells doubling every 20 gration of ITR-flanked sequences (202). An HVJ-liposome 1101 1045 hours. vector system reported by Dzau et al. (203) was utilized to 1102 1046 Our laboratory has been exploring the use of EBV episomal improve the efficiency of liposome-mediated transfer of an 1103 1047 vectors containing only the OriP and EBNA-1 and carrying EBV-episomally maintained transgene (204,205). This system 1104 1048 the selectable marker MDR1aspotential gene therapy vectors. exploits the fusigenic properties of the hemagglutinating virus 1105 1049 Using the liposome formulation, DOGS/DOPE (1:1) (195), of Japan (HVJ or Sendai virus) since envelope proteins of 1106 1050 we successfully delivered the vector to various cultured cells inactivated HVJ were found to mediate liposome-cell mem- 1107 4104-8—Templeton—Ch21—R1—07-24-03 16:17:03—

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1108 brane fusion and facilitate cellular uptake of packaged plasmid tion of human artificial microchromosomes (HAC) by creating 1165 1109 DNA, bypassing endocytosis and lysosomal degradation. synthetic alpha satellite arrays ϳ1Mbinsize (214). They 1166AQ16 1110 One of the limitations with using viral episomal systems found that such an HAC which is about 6–10 Mb is size is 1167 1111 is the limited host range of such vectors. Although EBV epi- mitotically and cytogenetically stable for up to 6 months in 1168 1112 somal vectors replicate well in various human and primate culture in the absence of selective pressure. Nonetheless, the 1169 1113 cells, they are unable to replicate in rodent cells, limiting their technical challenge of assembling a mammalian artificial 1170 1114 utility in gene therapy since testing of these vectors in rodent chromosome is still formidable as cloning and manipulating 1171 1115 models is not easy. Nonetheless, it was found that large frag- such large constructs are not trivial using conventional bacte- 1172 1116 ments of human genomic DNA (between 10–15 kb) can me- rial cloning systems, and transfer to mammalian cells is diffi- 1173 1117 diate autonomous replication if there is also a mechanism to cult. 1174 1118 retain them in the nucleus (206). Such vectors based on a 1119 human origin of replication were also found to be capable of 1120 replicating in rodent cells (207), probably due to the common V. USE OF LIPOSOMES TO DELIVER 1175 1121 host factors that drive their replication. A hybrid class of vec- 1122 tors was thus developed, which employs a human origin of VECTORS WITH SELECTABLE 1176 1123 replication to mediate vector replication as well as the EBV MARKERS 1177 1124 FR and EBNA-1 gene product to provide nuclear retention Liposome-mediated gene transfer appears to be a safe and 1178 1125 functions. [see Calos (208)]. EBNA-1 binding to the FR of noninvasive method of DNA delivery into cells. Since high 1179 1126 the vector DNA causes the adherence of this complex to the efficiency and stable expression have not yet been achieved 1180 1127 chromosomal scaffold in a noncovalent fashion, thus retaining using liposomal methods, the use of the human MDR1 gene 1181 1128 the vector DNA in the nucleus (209). These vectors were as a selectable marker may allow for the selection and enrich- 1182 1129 reported to replicate somewhat in synchrony with chromo- ment of the recipient cells and may be useful in the future 1183 1130 somal DNA once per cell cycle. Maintenance of these vectors for the long-term maintenance of the cationic liposome:DNA 1184 1131 within cells is related to the frequency of cell division (208). complex. 1185 1132 Such vectors have been reported to persist in cells for at least Previous studies in our laboratory have shown that a liposo- 1186 1133 2 months under no selective pressure (206,210). mal delivery system can mediate successful MDR1 transfec- 1187 1134 Ultimately, the development of a true mammalian artificial tion of mouse bone marrow cells and in vivo expression of 1188 1135 chromosome (MAC) without dependence on viral elements functional P-gp in hematopoietic cells (97). The introduction 1189 1136 will be the key to obtaining stable episomal replication without via liposomes into hematopoietic cells of an MDR1 gene 1190 1137 dependence on selective pressure. Functional elements in driven by Harvey murine sarcoma virus long-terminal repeat 1191 1138 mammalian cells important for maintaining DNA episomally sequences (Ha-MSV-LTR) was achieved either ‘‘directly’’ 1192 1139 as a minichromosome include a replication origin to promote by intravenous administration into mice, or ‘‘indirectly’’ by 1193 1140 autonomous replication, telomeres to protect ends of linear adoptive transplantation of previously in vitro-transfected 1194 1141 DNA and replicate DNA termini, and a centromere to facilitate bone marrow cells. In these studies, using a cationic liposome 1195 1142 correct segregation of the construct during mitotic division. complex consisting of dioctadecylamidoglycyl spermidine 1196 1143 Various mammalian chromosomal DNA replication initiation 1197 1144 sites have been identified [reviewed in DePamphilis (211)] (DOGS) and dioleoylphosphosphatidyl ethanolamine % 1145 and found to comprise a 0.5–11 kb primary origin of bidirec- (DOPE), MDR1 transfection was detected in up to 30 of 1198 % 1146 tional replication (OBR) flanked by an initiation zone of about unselected and 66 of vincristine preselected murine bone 1199 1147 6–55 kb. These sequences show characteristics of DNA un- marrow cells as demonstrated by drug resistance in an in vitro, 1200 1148 winding, a densely methylated island, attachment sites to the colony-forming unit assay. Although transfection into human 1201 1149 nuclear matrix, and some palindromic sequences. bone marrow cells is likely to be much less efficient, the poten- 1202 1150 Vectors utilizing human genomic sequences that promote tial of obtaining drug-selectable mouse bone marrow progeni- 1203 1151 extrachromosomal vector replication have already been suc- tor cells after gene transfer using such a liposome delivery 1204 1152 cessfully applied as mentioned above. Telomeres that are re- system may eventually make it possible to protect cancer pa- 1205 1153 quired for the stability and integrity of the eukaryotic chromo- tients undergoing chemotherapy from bone marrow toxicity 1206 1154 some have been well characterized. In mammalian cells, the of anticancer drugs. 1207 1155 telomeric tracts comprise 2–50 kb of tandem TTAGGG re- Liposome-mediated gene transfer can also be used for in 1208 1156 peats. Human centromeres, necessary for proper chromosome vivo delivery of Adeno-Associated-Vectors (AAV)-MDR1- 1209 1157 segregation at mitosis and meiosis, have been localized cyto- based vectors. Recently, drug-selected coexpression of both 1210 1158 genetically as primary constrictions of the chromosomes. P-gp and glucocerebrosidase (GC) was achieved with an AAV 1211 1159 They are thought to consist of up to several megabases of vector containing the MDR1-IRES-GC fusion delivered to 1212 1160 highly repetitive DNA belonging to the alpha satellite DNA NIH 3T3 cells by lipofection (96). Moreover, a single intrave- 1213 1161 family (212) and are attached to microtubules (213). Until nous injection of this bicistronic vector complexed with ca- 1214 1162 recently, the functional isolation of the centromere has been tionic liposomes into recipient mice allowed detection of GC 1215 1163 a great hurdle in the progress towards the construction of an and MDR1 sequences by PCR in all organs tested 7 weeks 1216 AQ15 1164 MAC. The group of Willard et al. developed the first genera- later. 1217 4104-8—Templeton—Ch21—R1—07-24-03 16:17:03—

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1218 For nonintegrating DNA vectors such as EBV-based sys- to transduced cells/tissues compared to wild-type P-gp. Stud- 1272 1219 tems (see Section IV) and the AAV system (96), liposome- ies of a number of mutations made in P-glycoprotein have 1273 1220 based gene delivery usually results in transient transgene suggested that it should be possible to construct mutant ‘‘de- 1274 1221 expression due to the episomal nature of the transfected signer’’ transporters useful for MDR1-based gene therapy. 1275 1222 plasmid and loss of the plasmid when the cells proliferate One of the hallmark characteristics of the multidrug trans- 1276 1223 (215,216). Use of a selectable marker such as MDR1 may porter is its extremely broad substrate specificity. Over the 1277 1224 make it possible to maintain nonintegrated episomal forms in past several years, the identification of specific domains and 1278 1225 proliferating cells (see Section IV). Since only cells carrying amino acid residues involved in substrate recognition has con- 1279 1226 such episomal MDR1-based vectors would survive the selec- tributed to our present understanding of the mechanism of 1280 1227 tion, this advantage should be useful for gene therapy with action of P-gp. The major sites of interaction have been shown 1281 1228 episomal MDR1 vectors in vivo. Combining liposomes with to reside in transmembrane domains (TM) 5 and 6 in the N- 1282 1229 AAV- or EBV-based vectors and MDR1asaselectable marker terminal half of the protein and in TMs 11 and 12 in the C- 1283 1230 may make it possible to expand the population of expressing terminal half and the loops that conjoin them (217–221). For 1284 1231 cells by MDR1-drug selection. the purposes of chemoprotection, the design of a P-gp that 1285 1232 We are developing a gene therapy model to treat Fabry has increased resistance to chemotherapeutic agents compared 1286 1233 disease (123) using intravenous injections of a pHa-aGal- to the endogenous P-gp would be most useful because in- 1287 1234 IRES-MDR bicistronic vector complexed to cationic lipo- creased doses of the agent could be administered without 1288 1235 somes into ␣-galactosidase A deficient mice (T. Shoshani and harming the bone marrow cells expressing the exogenous P- 1289 1236 M. M. Gottesman, unpublished results). Both human ␣-Gal gp molecule. To date, a number of these types of mutations 1290 1237 and MDR1 were detectable in the lungs of the recipient Fabry have been described. 1291 1238 mice by Southern blot analysis 7 days after injection. Reverse Mutations in TM domains of P-gps from both rodent and 1292 1239 transcriptase polymerase chain reaction (RT-PCR) analysis of human have demonstrated significant alterations in substrate 1293 1240 total RNA extracted from the kidneys of recipient Fabry mice specificity (3,222). An F338A mutation in hamster P-gp en- 1294 1241 showed the presence of both human ␣-Gal and MDR1 mRNA. hances resistance to vincristine, colchicine, and daunorubicin 1295 1242 The expression in the kidneys was specific to the ␣–galactosi- but has little impact on resistance to actinomycin D (223,224). 1296 1243 dase-A-deficient mice, where renal tubule cells may be dam- An F339P mutation in the same molecule only increases acti- 1297 1244 aged by an accumulation of glycosphingolipids. In situ hybrid- nomycin D resistance. However, the double F338A/F339P 1298 1245 ization analysis localized the mRNA expression to the renal mutant demonstrates an increased level of resistance to actino- 1299 1246 distal tubule epithelial cells. Higher RNA expression was ob- mycin D and vincristine but a lowered level of resistance to 1300 1247 tained in Fabry mice that were injected 3 times every third colchicine and daunorubicin (223,224). Of these mutants, the 1301 1248 day. The repeated administration is tolerated by the recipient F338A may prove most useful because it confers increased 1302 1249 mice and no toxic effects were obtained. It remains to be resistance to a wider range of chemotherapeutic agents. In 1303 1250 determined whether selection in vivo will allow expansion of human P-gp, however, a homologous mutation at F335 con- 1304 1251 cell populations expressing human ␣-Gal by repeated adminis- fers greater resistance to colchicine and doxorubicin but 1305 1252 tration of cytotoxic MDR1 substrates. causes a severe reduction in resistance to vinblastine and acti- 1306 nomycin D (225,226) . Additionally, cells expressing a Val- 1307 ϾAla mutation at position 338 also exhibit preferential resis- 1308 1253 VI. ENGINEERING MDR VECTORS TO tance to colchicine and doxorubicin but are severely impaired 1309 1254 IMPROVE EFFICIENCY OF DRUG for vinblastine (226). Resistance to actinomycin D, however, 1310 1255 SELECTION is unaffected. Alanine scanning of TM 11 in mouse P-gp en- 1311 coded by mdr1a revealed that two mutants, M944A and 1312 1256 One of the goals of gene therapy is to modify cells genetically F940A, show an increase in resistance to doxorubicin and 1313 1257 such that they can supply a useful or necessary function to colchicine while maintaining wild-type levels of resistance to 1314 1258 the cell (3). One of the most promising applications of the vinblastine and actinomycin D (227). For certain treatment 1315 1259 MDR1 gene in therapeutic vectors as a selectable marker in protocols, it is conceivable that increased resistance to certain 1316 1260 vivo is the protection of bone marrow cells during intensive agents would be desirable, and the reduction in levels of resis- 1317 1261 chemotherapy. During chemotherapy, the MDR1 gene is tance to other compounds would not be problematic, espe- 1318 1262 transduced or transfected into drug-sensitive bone marrow cially if a well-defined chemotherapy regimen was being em- 1319 1263 cells and selected for by exposure to MDR agents. The ployed. 1320 1264 untransfected/untransduced cells will necessarily be killed and Although the majority of residues that increase resistance 1321 1265 those containing the MDR1 gene will expand. The efficacy to various chemotherapeutic agents reside in the TM domains, 1322 1266 of this therapy depends on the interaction between P-gp and a number of residues in the putative cytoplasmic loops also 1323 1267 the selecting agent employed. Thus, it is important to be able have been implicated in defining drug resistance profiles for 1324 1268 to distinguish between the endogenous P-gp and the exoge- cytotoxic drugs. The best characterized of these mutations 1325 1269 nously introduced molecule. Furthermore, it obviously would is the G185V mutant that confers an increased resistance to 1326 1270 be beneficial to create a P-gp molecule that would confer very colchicine and etoposide but decreased resistance to actino- 1327 1271 high levels of resistance to certain drugs, giving an advantage mycin D, vinblastine, doxorubicin, vincristine, and taxol 1328 4104-8—Templeton—Ch21—R1—07-24-03 16:17:03—

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1329 (228–231). Interestingly, and perhaps relevant clinically, positive cells after chemotherapy (89). In the future, with the 1329 1330 when this mutation is made in conjunction with an Asn-ϾSer generation of higher resolution structures of human P-gp, it 1330 1331 mutation at residue 183, increased resistance to actinomycin should be feasible to model and synthesize new, more effec- 1331 1332 D, vinblastine, and doxorubicin is achieved without loss of tive cytotoxic drugs or modulators capable of blocking P-gp 1332 1333 the increase in colchicine resistance (229). Mutations of Gly- function clinically. However, until that time, the analysis of 1333 1334 141, 187, 288, 812, or 830 to Val in human P-gp increase the spontaneously occurring or engineered mutants, coupled to 1334 1335 relative resistance of NIH3T3 cells to colchicine and doxoru- our knowledge of the current battery of anticancer and revers- 1335 1336 bicin but do not alter resistance to vinblastine (232). Only the ing agents, offers an opportunity to begin designing second- 1336 1337 mutations at positions 187, 288, and 830 confer decreased generation vectors for use in these trials. 1337 1338 resistance to actinomycin D to cells in culture. 1339 Due to its broad substrate specificity, P-gp not only inter- 1340 acts with chemotherapeutic compounds but also with revers- VII. CONCLUSIONS AND FUTURE 1338 1341 ing agents and inhibitors. In combination chemotherapies, re- PROSPECTS 1339 1342 versing agents increase the efficacy of cytotoxic agents in We have argued in this review that drug-selectable marker 1340 1343 MDR1-expressing cancers. Two of the most potent reversing genes may be helpful for gene therapy in two ways: first, to 1341 1344 agents currently in use or in clinical trials are cyclosporin A protect bone marrow progenitor cells (and other sensitive 1342 1345 and its nonimmunosuppressive analog PSC833. Recently, a cells) from the cytotoxicity of anticancer drugs, thereby allow- 1343 1346 number of mutants have been described that affect sensitivity ing safe chemotherapeutic treatment at reduced risk of severe 1344 1347 to these agents. Cells expressing a human P-gp containing a side effects, and second, to enrich the expression of otherwise 1345 1348 deletion at Phe335 or Phe334 are substantially resistant to nonselectable genes in drug-sensitive cells to overcome low 1346 1349 cyclosporin A and PSC-833 [(233), Hrycyna, C.A., Pastan, I., or unstable gene expression in vivo. Given the current instabil- 1347 1350 and Gottesman, M.M., unpublished data]. A similar phenotype ity of expression of genes from existing vectors, especially 1348 1351 has been observed for a transporter containing 5 mutations episomal vectors, such selectable markers may be an essential 1349 1352 in the region including TM 5 and TM6, namely Ile299Met, component of gene therapy protocols. 1350 1353 Thr319Ser, Leu322Ile, Gly324Lys, and Ser351Asn (234). Ad- We are still in the early stages of vector development, and 1351 1354 ditionally, in hamster P-gp, the substitution of an alanine at until transduction efficiencies into human tissues such as bone 1352 1355 position 339 with proline results in a transporter that confers marrow are improved and shown to be safe, long-term human 1353 1356 lowered sensitivity to cyclosporin A (224). From these studies, gene therapy will not be feasible. The combination of more 1354 1357 it appears that TM6 plays an important role in the recognition efficient gene transfer targeted vector systems, and effective, 1355 1358 of cyclosporin A and its analogs. The decreased sensitivity to relatively nontoxic selection systems to maintain gene expres- 1356 1359 these reversing agents observed in cells expressing the TM6 sion may make long-term correction of human genetic defects 1357 1360 mutations could help protect bone marrow stem cells trans- feasible and safe. 1358 1361 duced with the mutant MDR1 gene from the toxic effects of 1362 chemotherapy given with reversing agents to sensitize MDR1- 1363 expressing tumors. REFERENCES 1359 1364 The cis and trans isomers of flupentixol, a dopamine recep- 1365 tor antagonist, have also been shown to inhibit drug transport 1366 and reverse drug resistance mediated by P-gp (235,236). The 1. 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