No evidence of clonal dominance in primates up to 4 years following transplantation of multidrug resistance 1 retrovirally transduced long-term repopulating cells.

Previous murine studies have suggested that retroviral multidrug resistance 1 (MDR1) gene transfer may be associated with a myeloproliferative disorder. Analyses at a clonal level and prolonged long-term follow-up in a model with more direct relevance to human biology were lacking. In this study, we analyzed the contribution of individual CD34-selected peripheral blood progenitor cells to long-term rhesus macaque hematopoiesis after transduction with a retroviral vector either expressing the multidrug resistance 1 gene (HaMDR1 vector) or expressing the neomycin resistance (NeoR) gene (G1Na vector). We found a total of 122 contributing clones from 8 weeks up to 4 years after transplantation. One hundred two clones contained the G1Na vector, whereas only 20 clones contained the HaMDR1 vector. Here, we show for the first time real-time polymerase chain reaction based quantification of individual transduced cell clones constituting 0.0008% +/- 0.0003% to 0.0041% +/- 0.00032% of primate peripheral blood cells. No clonal dominance was observed. Disclosure of potential conflicts of interest is found at the end of this article.

Modulation of homo- and heterodimerization of Harvey sarcoma virus RNA by GACG tetraloops and point mutations in palindromic sequences.

Retroviruses harbour a diploid genome of two plus-strand RNAs linked non-covalently at the dimer linkage structure. Co-packaging of two parental RNAs is a prerequisite for recombination in retroviruses, but formation of heterodimers has not been demonstrated directly in vivo. Here, we explore elements in Harvey sarcoma virus (HaSV) RNA involved in homodimerization and heterodimerization with RNA of Moloney (Mo) and Akv murine leukemia viruses (MLV). By an in vitro assay, we found that HaSV dimerization specificity could be modulated by mutations in a decanucleotide palindrome (Pal) probably folded into a kissing-loop. Autocomplementary and non-autocomplementary sequences introduced into the putative loop directed the specificity towards formation of homodimers and heterodimers, respectively. Two stem-loop (SL) structures, both exposing a GACG tetraloop, enhanced the formation of stable HaSV dimers.A similar decanucleotide palindrome has been implicated in homodimerization of MLVs. Heterodimers between HaSV RNA and Mo- or Akv MLV were unstable, but could be stabilized by introduction of two point mutations in the putative HaSV kissing-loop, creating exact complementarity with Mo/Akv MLV palindromes. Moreover, such changes increased the HaSV RNA affinity for the two MLV RNAs. Similar to HaSV RNA homodimers, formation of heterodimers with Mo- or Akv MLV RNAs was induced by the presence of GACG loops. On the basis of these results, we propose that palindromic sequences act as variable determinants of specificity and GACG tetraloops as conserved determinants in the formation of homodimers and heterodimers of gamma-retrovirus retroviral RNAs in vivo. The complementarity of loop sequences in the packaging signal upstream of the GACG tetraloops might therefore determine homo- and heterodimerization specificity and recombination activity of these viruses.

Transduction of murine bone marrow cells with an MDR1 vector enables ex vivo stem cell expansion, but these expanded grafts cause a myeloproliferative syndrome in transplanted mice.

Attempts to expand repopulating hematopoietic cells ex vivo have yielded only modest amplification in stem cell numbers. We now report that expression of an exogenous human multi-drug resistance 1 (MDR1) gene enables dramatic ex vivo stem cell expansion in the presence of early acting hematopoietic cytokines. Bone marrow cells were transduced with retroviral vectors expressing either the MDR1 gene or a variant of human dihydrofolate reductase (DHFR), and then expanded for 12 days in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor. When these cells were injected into nonirradiated mice, high levels of long-term engraftment were only seen with MDR1-transduced grafts. To verify that expansion of MDR1-transduced repopulating cells had occurred, competitive repopulation assays were performed using MDR1 expanded grafts. These experiments showed progressive expansion of MDR1-transduced repopulating cells over the expansion period, with a 13-fold overall increase in stem cells after 12 days. In all of the experiments, mice transplanted with expanded MDR1-transduced stem cells developed a myeloproliferative disorder characterized by high peripheral white blood cell counts and splenomegaly. These results show that MDR1-transduced stem cells can be expanded in vitro using hematopoietic cytokines without any drug selection, but enforced stem cell self-renewal divisions can have adverse consequences.

Retroviral coexpression of thymidylate synthase and dihydrofolate reductase confers fluoropyrimidine and antifolate resistance.

Retroviral gene transfer of dominant selectable markers into hematopoietic cells can be used to select genetically modified cells in vivo or to attenuate the toxic effects of chemotherapeutic agents. We show that retroviral gene transfer of thymidylate synthase (TS) confers resistance to TS directed anticancer agents and that co-expression of TS and dihydrofolate reductase (DHFR) confers resistance to TS and DHFR cytotoxic agents. Retroviral vectors encoding Escherichia coli TS, human TS, and the Tyr-to-His at residue 33 variant of human TS (Y33HhTS) were constructed and fibroblasts transfected with these vectors conferred comparable resistance to the TS-directed agent fluorodeoxyuridine (FdUrd, approximately 4-fold). Retroviral vectors that encode dual expression of Y33HhTS and the human L22Y DHFR (L22YhDHFR) variants conferred resistance to FdUrd (3- to 5-fold) and trimetrexate (30- to 140-fold). A L22YhDHFR-Y33HhTS chimeric retroviral vector was also constructed and transduced cells were resistant to FdUrd (3-fold), AG337 (3-fold), trimetrexate (100-fold) and methotrexate (5-fold). These results show that recombinant retroviruses can be used to transfer the cDNA that encodes both TS and DHFR and dual expression in transduced cells is sufficiently high to confer resistance to TS and DHFR directed anticancer agents.

Retroviral vectors containing a variant dihydrofolate reductase gene for drug protection and in vivo selection of hematopoietic cells.

Transfer of drug resistance genes to hematopoietic cells is being studied as a means to protect against the myelosuppression associated with cancer chemotherapy and as a strategy for the in vivo selection and amplification of genetically modified cells. The goal of this study was to test if retroviral-mediated gene transfer of a dihydrofolate reductase (DHFR) variant (L22Y) could be used for in vivo selection of transduced myeloid cells and to determine what proportion of transduced cells was required for protection from myelosuppression. Based on previous work suggesting that selection with antifolates may also require inhibition of nucleoside transport mechanisms, mice transplanted with DHFR-transduced bone marrow cells were treated with trimetrexate and the nucleoside transport inhibitor prodrug nitrobenzylmercaptopurine riboside phosphate. In vivo selection of transduced myeloid progenitors was seen in the bone marrow and in circulating mature peripheral blood cells following drug treatment. These results show that the novel combination of the L22Y-DHFR cDNA, trimetrexate and nitrobenzylmercaptopurine riboside phosphate can be used to select for transduced myeloid cells, and that this approach warrants further study in large animal models. A bicistronic vector containing a human CD24 reporter gene was used to determine the number of modified cells needed for chemoprotection. Partial protection from neutropenia was seen when greater than 10% of myeloid cells expressed the vector, and high levels of protection were obtained when the proportion exceeded 30%. These results suggest that gene transfer may be useful for myeloprotection in certain pediatric cancers, but that more efficient gene transfer will be required to apply this approach to adult cancer patients.

Efficient long-term coexpression of a hammerhead ribozyme targeted to the U5 region of HIV-1 LTR by linkage to the multidrug-resistance gene.

Ribozymes as anti-HIV-1 agents hold promise for the treatment of AIDS. They can be delivered into cells either exogenously or through an expression system. For effective protection against HIV-1, sufficient and sustained amounts of the antiviral ribozymes must be delivered into target cells. The coexpression of a dominant selectable marker with ribozymes would serve to enrich for cells containing the molecular antiviral and facilitate prolonged expression of these ribozymes. The multidrug resistance gene (MDR1) is a potential clinically relevant selectable marker and offers many advantages over other known dominant selectable markers, including the use of diverse pharmacologically characterized drug or drug combinations for selection. Harvey sarcoma-based retroviral vectors encoding the MDR1 multidrug transporter with a hammerhead ribozyme targeted to highly conserved sequences within the HIV-1 U5 LTR segment have been constructed in a bicistronic format. The internal ribosome entry site (IRES) from encephalomyocarditis virus was used to initiate translation of the MDR1 mRNA. The ribozyme remained functional despite being tethered to MDR1. Long-term, high-level expression of both the ribozyme and MDR1, as evident by RT-PCR and FACS analysis, was observed in a human T cell line containing the construct selected with vincristine, a cytotoxic substrate for the multidrug transporter.

Cytokine-facilitated transduction leads to low-level engraftment in nonablated hosts.

Using a murine bone marrow transplantation model, we evaluated the long-term engraftment of retrovirally transduced bone marrow cells in nonmyeloablated hosts. Male bone marrow was stimulated in a cocktail of interleukin-3 (IL-3), IL-6, IL-11, and stem cell factor (SCF) for 48 hours, then cocultured on the retroviral producer line MDR18.1 for an additional 24 hours. Functional transduction of hematopoietic progenitors was detected in vitro by reverse transcriptase-polymerase chain reaction (RT-PCR) amplification of multiple drug resistance 1 (MDR1) mRNA from high proliferative potential-colony forming cell (HPP-CFC) colonies. After retroviral transduction, male bone marrow cells were injected into nonablated female mice. Transplant recipients received three TAXOL (Bristol-Myers, Princeton, NJ) injections (10 mg/kg) over a 14-month period. Transplant recipient tissues were analyzed by Southern blot and fluorescence in situ hybridization for Y-chromosome-specific sequences and showed donor cell engraftment of approximately 9%. However, polymerase chain reaction amplification of DNAs from bone marrow, spleen, and peripheral blood showed no evidence of the transduced MDR1 gene. RT-PCR analysis of total bone marrow RNA showed that transcripts from the MDR1 gene were present in a fraction of the engrafted donor cells. These data show functional transfer of the MDR1 gene into nonmyeloablated murine hosts. However, the high rates of in vitro transduction into HPP-CFC, coupled with the low in vivo engraftment rate of donor cells containing the MDR1 gene, suggest that the majority of stem cells that incorporated the retroviral construct did not stably engraft in the host. Based on additional studies that indicate that ex vivo culture of bone marrow induces an engraftment defect concomitantly with progression of cells through S phase, we propose that the cell cycle transit required for proviral integration reduces or impairs the ability of transduced cells to stably engraft.

In vivo drug-selectable markers in gene therapy.

Two-gene vectors with positive or positive-negative drug-selectable markers enable the expansion or elimination of gentetically modified cells in vivo. We have established a bicistronic retroviral vector system which utilizes an internal ribosome entry site (IRES) to co-express two independent genes with high efficiency. As a positive-negative (suicide) marker, Herpes simplex virus thymidine kinase was co-expressed with the human multidrug resistance gene, MDR1. Using this vector, almost all the MDR1-transduced cells showed hypersensitivity to a nucleoside analog, ganciclovir. As a dominant selectable marker, the MDR1 gene was co-expressed with alpha-galactosidase A for the model of gene therapy of Fabry disease. Vincristine selection efficiently enhanced the population of transduced cells expressing the second non-selectable genes. These drug-selectable retroviral vectors could be applicable to the therapy of many diseases.

ATP-dependent choline phosphate-induced mitogenesis in fibroblasts involves activation of pp70 S6 kinase and phosphatidylinositol 3'-kinase through an extracellular site. Synergistic mitogenic effects of choline phosphate and sphingosine 1-phosphate.

In serum-starved NIH 3T3 clone 7 fibroblasts, choline phosphate (ChoP) (0.5-1 mM) and insulin synergistically stimulate DNA synthesis. Here we report that ATP also greatly enhanced the mitogenic effects of ChoP (0.1-1 mM) both in the absence and presence of insulin; maximal potentiating effects required 50-100 microM ATP. The co-mitogenic effects of ATP were mimicked by adenosine 5'-O-(3-thiotriphosphate), adenosine 5'-O-(2-thiodiphosphate), ADP, and UTP, but not by AMP or adenosine, indicating the mediatory role of a purinergic P2 receptor. Externally added ChoP acted on DNA synthesis without its detectable uptake into fibroblasts, indicating that ChoP can be a mitogen only if it is released from cells. Extracellular ATP (10-100 microM) induced extensive release of ChoP from fibroblasts. ChoP had negligible effects, even in the presence of ATP or insulin, on the activity state of p42/p44 mitogen-activated protein kinases, while in combination these agents stimulated the activity of phosphatidylinositol 3'-kinase (PI 3'-kinase). Expression of a dominant negative mutant of the p85 subunit of PI 3'-kinase or treatments with the PI 3'-kinase inhibitor wortmannin only partially (approximately 40-50%) reduced the combined effects of ChoP, ATP, and insulin on DNA synthesis; in contrast, the pp70 S6 kinase inhibitor rapamycin almost completely inhibited these effects. ATP and insulin also potentiated, while rapamycin strongly inhibited, the mitogenic effects of sphingosine 1-phosphate (S1P). Furthermore, even maximally effective concentrations of ChoP and S1P synergistically stimulated DNA synthesis. The results indicate that in the presence of extracellular ATP and/or S1P, ChoP induces mitogenesis through an extracellular site by mechanisms involving the activation of pp70 S6 kinase and, to a lesser extent, PI 3'-kinase.

Ectopic expression of murine TPO receptor (c-mpl) in mice is pathogenic and induces erythroblastic proliferation.

c-mpl, the cellular homologue of the v-mpl oncogene transduced in the myeloproliferative leukemia virus (MPLV), encodes the receptor for thrombopoietin, a cytokine involved in the proliferation and differentiation of cells of the megakaryocytic lineage. Here, we show that a retrovirus containing murine c-mpl cDNA (HSFmmpl) is pathogenic in vivo when inoculated in adult mice. All mice developed hepatosplenomegaly and died within 9 to 12 weeks after infection. Histological analysis showed that spleen, liver, and peripheral blood were invaded by erythroblasts at every stage of differentiation. In contrast to the myeloproliferative syndrome induced by MPLV, we did not observe an infiltration of these organs with cells from the granulocytic lineage nor a thrombocytosis. In fact, the platelet count of HSFmmpl mice progressively decreased and a severe thrombocytopenia was observed late in the course of the disease. Further characterization of the target progenitor of HSFmmpl virus in the spleen and bone marrow of diseased animals was accomplished using in vitro clonogenic progenitor cell assays. This analysis indicated that both late and early erythroid compartment (colony-forming unit-erythroid and burst-forming unit-erythroid) were largely increased in the spleens. The colony-forming unit-granulocyte-macrophage compartment was also increased but to a lesser extent. This study shows for the first time that ectopic expression of a member of the cytokine receptor superfamily promotes hematopoietic progenitor cell proliferation and could play a role in leukemogenesis.

HIV-1 nucleocapsid protein induces "maturation" of dimeric retroviral RNA in vitro.

After a retrovirus particle is released from the cell, the dimeric genomic RNA undergoes a change in conformation. We have previously proposed that this change, termed maturation of the dimer, is due to the action of nucleocapsid (NC) protein on the RNA within the virus particle. We now report that treatment of a 345-base synthetic fragment of Harvey sarcoma virus RNA with recombinant or synthetic HIV-1 NC protein converts a less stable form of dimeric RNA to a more stable form. This phenomenon thus appears to reproduce the maturation of dimeric retroviral RNA in a completely defined system in vitro. To our knowledge, maturation of dimeric RNA within a retrovirus particle is the first example of action of an "RNA chaperone" protein in vivo. Studies with mutant NC proteins suggest that the activity depends upon basic amino acid residues flanking the N-terminal zinc finger and upon residues within the N-terminal finger, including an aromatic amino acid, but do not require the zinc finger structures themselves.

Expression of the human multidrug resistance and glucocerebrosidase cDNAs from adeno-associated vectors: efficient promoter activity of AAV sequences and in vivo delivery via liposomes.

Recombinant adeno-associated viruses (rAAV) are attractive tools for gene therapy. We designed plasmids in which the human multidrug resistance gene (hMDR1) cDNA was placed downstream from portions of the 5' end of AAV including either a 234-bp cassette or the entire AAV p5 promoter. The drug-resistant phenotype conferred by the P-glycoprotein (Pgp) efflux pump encoded by the hMDR1 cDNA was used to select NIH-3T3 cells transfected with these plasmids. The 234-bp region alone showed promoter activity similar in strength to that of the entire p5 promoter or the retroviral Harvey murine sarcoma virus long terminal repeat (LTR); this result demonstrates that the 234-bp cassette might be used as a small and efficient promoter in rAAV designed to express large genes approaching the packaging limit of AAV particles. After transfection of AAV-MDR1 vectors, the integration of MDR1 sequences into the host cell genome was demonstrated by fluorescent in situ hybridization (FISH). In addition, Southern analysis of low-molecular-weight DNA extracted from drug-resistant cells grown under continuous selection pressure indicated the persistence of nonintegrated AAV-MDR1 plasmids. Coordinate expression of Pgp and human glucocerebrosidase (hGC) was observed in drug-selected NIH-3T3 cells transfected with a bicistronic vector in which MDR1 cDNA was linked to hGC cDNA via the encephalomyocarditis internal ribosome entry site sequence. Moreover, following a single intravenous injection of the bicistronic vector complexed to cationic liposomes into recipient mice, delivery of MDR1 and GC cDNAs was achieved in all the organs we tested. Our results demonstrate that the efficiency of liposomes as vehicles for in vitro and in vivo gene delivery, the advantages of AAV-vectors, and the use of MDR1 as a selectable marker might be successfully combined in gene therapy protocols.

Viral interference during simultaneous transduction with two independent helper-free retroviral vectors.

The ability to stably transduce a single cell with two independent retroviral vectors would have distinct advantages for gene therapy. We determined that cells can be transduced with two distinct retroviral vectors and have quantitated transduction efficiencies in cells infected sequentially and simultaneously. Two amphotropic, helper virus-free, retroviral vectors, a murine Moloney sarcoma virus-based vector containing the nuclear beta-galactosidase and neomycin resistance genes (MMSVn beta-gal/neoR) and a Harvey virus-derived vector containing the human multidrug resistance gene (HaMDR) were introduced into NIH-3T3 cells, pig keratinocytes, and primary pig fibroblasts simultaneously and sequentially. Analytical flow cytometry was utilized to determine retroviral transduction efficiency by assessing the percentage of cells transduced by either one or both retroviruses, in the absence of selection. Simultaneous retroviral transductions were infrequent events. In addition, transduction of previously infected cells (sequential transductions) occurred at lower than expected frequencies. Our data suggest that there is quantifiable viral interference in sequential retroviral transductions. This interference occurs by a mechanism that appears to be independent of the amphotropic retroviral receptor. Thus, such dual transductions will likely require in vitro selection or the use of a single retrovirus which contains both desired genes on the same genome.

Bicistronic and two-gene retroviral vectors for using MDR1 as a selectable marker and a therapeutic gene.

We describe a series of two-gene and bicistronic retroviral vectors that use the human MDR1 gene as a selectable marker for the overexpression of a second heterologous gene in transduced cells. The vectors use Harvey murine sarcoma virus sequences for viral expression and packaging functions and include sites for cloning foreign genes of interest under the control of either an internal promoter (two-gene vectors) or an internal ribosome entry site (bicistronic vectors). To characterize these vectors, we used neo as a reporter gene for foreign gene expression and as an independently selectable marker for comparison with MDR1. Each of the vector constructions supported high-titer retrovirus production and transduction of mouse and human cell lines. Using MDR1-neo virus supernatants in parallel titering assays, we found that titers based on colchicine resistance were 10- to 20-fold lower than titers based on G418 resistance, suggesting that MDR1 is a more stringent selectable marker than neo in NIH 3T3 and KB-3-1 cell lines. Whereas neo gene expression with the two-gene vectors was subject to host-specific limitations on internal promoter activity, the bicistronic vectors were highly active in three cell lines tested. In K562 cells, using the bicistronic vector, selection with colchicine led to at least 20-fold higher expression of the MDR1 gene product than did selection with G418, suggesting that the stringent MDR1 selection system is very efficient for obtaining overexpression of foreign genes. Retroviral vectors carrying MDR1 as a selectable marker plus a second, heterologous gene of interest could have widespread utility for in vitro and in vivo applications of gene transfer technology, including gene therapy.

Suppression of the metastatic phenotype of a mouse skin carcinoma cell line independent of E-cadherin expression and correlated with reduced Ha-ras oncogene products.

The HaCa4 cell line, derived from a mouse skin carcinoma induced by Harvey murine sarcoma virus, is highly tumorigenic when injected into nude mice and produces multiple metastases in the lungs. HaCa4 cells express high levels of viral Ha-ras oncogene products, anomalously synthesize the embryonic/simple epithelial keratin K8, and have lost the expression of the cell-cell adhesion receptor E-cadherin (E-CD). E-CD(+) cell clones (E62 and E24), obtained by transfection of an exogenous E-CD cDNA into HaCa4 cells, had a decreased ability to migrate through type IV collagen matrices. However, the E-CD (+) E62 clone remained as metastatic as the parental cell line, whereas the E24 clone, which does not take up the exogenous cDNA but spontaneously switches on the endogenous E-CD gene, suppressed the metastatic phenotype although it maintained its tumorigenicity. E24 cells had fivefold to sixfold lower levels of viral Ha-ras mRNA and p21 protein than the other cell lines. In addition, they did not synthesize K8 but rather switched on keratin K19. The comparison of E-CD proteins synthesized by E62 and E24 cell lines revealed no structural or functional differences because both localized at cell-cell contacts and associated with alpha-catenin, beta-catenin, and plakoglobin. Furthermore, E-CD was still expressed in metastatic lung nodules produced by E62 cells. These results suggest that suppression of the metastatic phenotype in E24 cells occurs independently of E-CD expression and correlates with decreased levels of the oncogenic ras p21 protein.

An internal ribosomal entry signal in the rat VL30 region of the Harvey murine sarcoma virus leader and its use in dicistronic retroviral vectors.

The genetic organization of the 5' genomic RNA domain of the highly oncogenic Harvey murine sarcoma virus appears to be unusual in that a multifunctional untranslated leader precedes the v-ras oncogene. This 5' leader is 1,076 nucleotides in length and is formed of independent regions involved in key steps of the viral life cycle: (i) the Moloney murine leukemia virus 5' repeat, untranslated 5' region, and primer binding site sequences necessary for the first steps of proviral DNA synthesis, (ii) the virus-like 30S (VL30)-derived sequence containing a functional dimerization-packaging signal (E/DLS) directing viral RNA dimerization and packaging into MLV virions, and (iii) an Alu-like sequence preceding the 5' untranslated sequence of v-rasH which contains the initiation codon of the p21ras oncoprotein. These functional features, the unusual length of this leader (1,076 nucleotides), and the presence of stable secondary structures between the cap and the v-ras initiation codon might well cause a premature stop of the scanning ribosomes and thus inhibit v-ras translation. In order to understand how Harvey murine sarcoma virus achieves a high level of expression of the ras oncogene, we asked whether the rat VL30 sequence, 5' to v-ras, could contribute to an efficient synthesis of the ras oncoprotein. The implications of the VL30 sequence in the translation initiation of Ha-ras were investigated in the rabbit reticulocyte lysate system and in murine cells. Results show that the rat VL30 sequence allows a cap-independent translation of a downstream reporter gene both in vitro and in murine cells. Additional experiments performed with dicistronic neo.VL30.lacZ mRNAs indicate that the 5' VL30 sequence (positions 380 to 794) contains an internal ribosomal entry signal. This finding led us to construct a new dicistronic retroviral vector with which the rat VL30 sequence was able to direct the efficient expression of a 3' cistron and packaging of recombinant dicistronic RNA into murine leukemia virus virions.

Efficient expression of functional human MDR1 gene in murine bone marrow after retroviral transduction of purified hematopoietic stem cells.

A procedure for efficient transfer of the human MDR1 (multi-drug resistance) gene into murine hematopoietic stem cells was developed. Cells expressing Sca-1 but no lineage-specific or major histocompatibility complex (MHC) class II antigens (Lin-MHC II-Sca-1+) were enriched from 5-fluorouracil-pretreated bone marrow by Ficoll density-gradient and immunomagnetic sorting. Purified cells were cocultured with growth factors and fibroblasts producing replication-deficient retroviruses containing human MDR1 cDNA. Fluorescence-activated cell sorter analysis and rhodamine-123 efflux experiments showed that greater than 60% of cocultured hematopoietic cells expressed functional human P-glycoprotein. After 6 to 8 days, hematopoietic cells were injected intravenously into sublethally irradiated SCID mice. Stem cell properties of the isolated population were confirmed by sustained expression of MDR1 marker cDNA for greater than 4 to 6 months after transplantation, multilineage engraftment, and presence of MDR1 cDNA in bone marrow of secondary recipient mice after retransplantation. Reconstitution of H-2K-mismatched SCID mice showed high engraftment capacity of Lin-MHC II-Sca-1+ cells. MDR1 cDNA was detected in blood of 78% of recipients. P-glycoprotein was expressed in bone marrow of 71% of mice, in both lymphocytes and myelomonocytoid progenitors. P-glycoprotein function in host marrow was confirmed by rhodamine-123 efflux. Transduction of P-glycoprotein may be useful for gene therapy in two ways: to protect bone marrow from myelosuppression after chemotherapy and as a selectable marker in vivo for the introduction of otherwise nonselectable genes.

Expression of retroviral vectors containing the human multidrug resistance 1 cDNA in hematopoietic cells of transplanted mice.

Transfer of the human multidrug resistance 1 (MDR1) gene to hematopoietic stem cells offers an approach to overcome the myelosuppression caused by a number of antineoplastic drugs. This study was designed to determine the effect of MDR1 gene transfer on overall P-glycoprotein (P-gp) expression in murine hematopoietic cells. Mice were transplanted with bone marrow cells infected with either of two different MDR1 retroviral vectors. A reverse-transcriptase polymerase chain reaction-based assay was used to quantify expression levels of both endogenous and vector-derived P-gp encoding transcripts in hematopoietic cells of transplanted mice. Expression of both a truncated and full-length MDR1 mRNA species was noted in bone marrow and spleen colony cells. The truncated message resulted from cryptic mRNA splice sites within the MDR1 cDNA and was detected with both vectors. Full-length message levels exceeded those from the endogenous genes in all but one case and roughly approximated that seen in the modestly drug-resistant cell line SW620. We conclude that transfer of MDR1 retroviral vectors resulted in a significant increase in P-gp expression in most cases; however, aberrant splicing of MDR1 transcripts can result in reduced expression of vector-derived P-gp.