Visualization of spinal afferent innervation in the mouse colon by AAV8-mediated GFP expression.

BACKGROUND: Primary afferent neurons whose cell bodies reside in thoracolumbar and lumbosacral dorsal root ganglia (DRG) innervate colon and transmit sensory signals from colon to spinal cord under normal conditions and conditions of visceral hypersensitivity. Histologically, these extrinsic afferents cannot be differentiated from intrinsic fibers of enteric neurons because all known markers label neurons of both populations. Adeno-associated virus (AAV) vectors are capable of transducing DRG neurons after intrathecal administration. We hypothesized that AAV-driven overexpression of green fluorescent protein (GFP) in DRG would enable visualization of extrinsic spinal afferents in colon separately from enteric neurons. METHODS: Recombinant AAV serotype 8 (rAAV8) vector carrying the GFP gene was delivered via direct lumbar puncture. Green fluorescent protein labeling in DRG and colon was examined using immunohistochemistry. KEY RESULTS: Analysis of colon from rAAV8-GFP-treated mice demonstrated GFP-immunoreactivity (GFP-ir) within mesenteric nerves, smooth muscle layers, myenteric plexus, submucosa, and mucosa, but not in cell bodies of enteric neurons. Notably, GFP-ir colocalized with CGRP and TRPV1 in mucosa, myenteric plexus, and globular-like clusters surrounding nuclei within myenteric ganglia. In addition, GFP-positive fibers were observed in close association with blood vessels of mucosa and submucosa. Analysis of GFP-ir in thoracolumbar and lumbosacral DRG revealed that levels of expression in colon and L6 DRG appeared to be related. CONCLUSIONS & INFERENCES: These results demonstrate the feasibility of gene transfer to mouse colonic spinal sensory neurons using intrathecal delivery of AAV vectors and the utility of this approach for histological analysis of spinal afferent nerve fibers within colon.

Increased longevity and metabolic correction following syngeneic BMT in a murine model of mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive inherited disease caused by deficiency of the glycosidase α-L-iduronidase (IDUA). Deficiency of IDUA leads to lysosomal accumulation of glycosaminoglycans (GAG) heparan and dermatan sulfate and associated multi-systemic disease, the most severe form of which is known as Hurler syndrome. Since 1981, the treatment of Hurler patients has often included allogeneic BMT from a matched donor. However, mouse models of the disease were not developed until 1997. To further characterize the MPS-I mouse model and to study the effectiveness of BMT in these animals, we engrafted a cohort (n=33) of 4-8-week-old Idua(-/-) animals with high levels (88.4±10.3%) of wild-type donor marrow. Engrafted animals displayed an increased lifespan, preserved cardiac function, partially restored IDUA activity in peripheral organs and decreased GAG accumulation in both peripheral organs and in the brain. However, levels of GAG and GM3 ganglioside in the brain remained elevated in comparison to unaffected animals. As these results are similar to those observed in Hurler patients following BMT, this murine-transplantation model can be used to evaluate the effects of novel, more effective methods of delivering IDUA to the brain as an adjunct to BMT.

In vivo selection of human embryonic stem cell-derived cells expressing methotrexate-resistant dihydrofolate reductase.

Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug-resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase the engraftment of gene-modified, hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR-GFP-expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rgammac(null) (NSG) mice after injection of Tyr22-DHFR-hESC-derived cells significantly increases human CD34(+) and CD45(+) cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR cells in vivo, and provides a novel approach for combined human cell and gene therapy.

High-titer lentiviral vectors stimulate fetal calf serum-specific human CD4 T-cell responses: implications in human gene therapy.

Human immunodeficiency virus-1-derived lentiviral vectors have been increasingly used for gene delivery in both pre-clinical and clinical models. Numerous studies have shown that dendritic cells (DC) transduced with concentrated lentiviral vectors can induce primary T-cell responses to viral and tumor antigens. In this study, we attempted to generate influenza hemagglutinin-specific CD4 T cells using lentiviral vectors containing the signal sequence and human lysosome-associated membrane protein to target hemagglutinin to the major histocompatibility complex class II processing pathway. Autologous dendritic cells were generated in serum-free medium and transduced with concentrated, high-titer lentiviruses to stimulate autologous T cells. Unexpectedly, we failed to generate influenza hemagglutinin-specific CD4 T cells rather than T cells specific for fetal calf serum (FCS). By limiting dilution, we established several FCS-specific CD4 T-cell clones restricted by human leukocyte antigen-DR1 and human leukocyte antigen-DR4. Lentiviruses produced in human serum-adapted 293 cells or in serum-free medium were unable to sensitize dendritic cells for recognition by FCS-specific CD4 T-cell clones. Our results indicate that residual FCS in concentrated lentiviral pellets is, in part, responsible for its immunogenicity. These FCS-specific CD4 T cells may be useful in testing clinical grade lentiviral vectors for the presence of contaminating FCS.

Methotrexate accumulates to similar levels in animals transplanted with normal versus drug-resistant transgenic marrow.

Gene transfer and expression of methotrexate (MTX)-resistant variants of dihydrofolate reductase (DHFR) in normal hematopoietic cells is a potential strategy to permit administration of larger doses of MTX by alleviating drug toxicity in normal cells and tissues that are drug sensitive. We have previously demonstrated that transplantation of marrow from transgenic mice expressing drug-resistant DHFRs conferred upon normal recipient animals resistance to MTX at levels that are usually toxic for hematopoietic and gastrointestinal (GI) tissues. One explanation for the observed protection from GI toxicity by drug-resistant marrow is that MTX could be cleared more rapidly in animals maintaining a more healthy hematopoietic system. To evaluate this possibility, we carried out MTX pharmacokinetic studies in mice that received transplanted transgenic marrow expressing either of two different DHFR variants, administering increasing doses of MTX up to 4 mg/kg/day. Animals received i.p. injection precisely every 24 h. Every 4 days, three animals from each group were sacrificed, and their plasma and intestines were assayed for MTX. Animals transplanted with transgenic Arg-22 DHFR drug-resistant marrow maintained hematocrit levels that were about 4-fold higher at 3 weeks after transplant than those of untreated animals or animals that received normal marrow cells. Animals that received normal marrow did not survive beyond 25 days and did not accumulate higher levels of MTX than animals that received a transgenic marrow transplant. Untreated animals exhibited a higher rate of survival (36 days) but again did not accumulate higher levels of MTX than the transgenic marrow recipients. When the experiment was repeated using transgenic Tyr-22 DHFR marrow, the levels of MTX in the plasma or GI tissues did not differ significantly between groups. Intestinal concentrations of MTX in both experiments were about 4-5-fold higher than those in the plasma. These results indicate that protection from MTX toxicity conferred by expression of drug-resistant DHFR activity in the marrow is not the result of a higher rate of MTX clearance from the circulation in comparison with control animals but a true resistance of hematopoietic and GI tissues to MTX. The maintenance of antifolate levels in animals protected from MTX toxicity implies that this procedure should not compromise the antitumor efficacy of MTX.

Retroviral vector design studies toward hematopoietic stem cell gene therapy for mucopolysaccharidosis type I.

To optimize a gene transfer system for hematopoietic stem cell gene therapy of patients with mucopolysaccharidosis (MPS) type I, 10 retroviral vectors were constructed to express the human alpha-L-iduronidase (IDUA) cDNA. These vectors were designed to evaluate the potential effects of specific promoters, the addition of selectable markers, and the use of multiple promoters versus an internal ribosome entry site for expression of IDUA and selectable maker genes. The effect of vector design was investigated in primary patient fibroblasts (F(MPS)) or murine fibroblast cell lines; while overall comparison of transgene expression was determined in patients' peripheral blood lymphocytes (PBL(MPS)) and CD34+ progenitors (PBPC(MPS)). We observed that the human PGK promoter introduced the highest IDUA activity per 1% relative transgene frequency in F(MPS). Use of the same promoter to separately regulate both the therapeutic gene and a drug-resistance gene resulted in decreased expression of the unselected gene. Co-selection using bicistronic vectors not only increased the number of transductants, but also elevated transgene expression under selective pressure in transgene-positive progenitors. Bicistronic vector LP1CD overcame down-regulation and practically introduced the highest IDUA level in unselected PBL(MPS) and an intermediate level in PBPC(MPS). These studies provide a better understanding of factors contributing to efficient gene expression in hematopoietic cells.

In vivo transduction of cerebellar Purkinje cells using adeno-associated virus vectors.

We investigated whether adenovirus or adeno-associated virus vectors can transduce cerebellar Purkinje cells (PCs) in vivo. Mice were injected in the deep cerebellar nuclei (DCN) with lacZ-transducing adenovirus (Ad.RSV-betagal) or a recombinant AAV serotype 2 (rAAV2) vector (vTR-CMVbeta) mixed with wild-type adenovirus type 5 (Ad5). One week later, Ad.RSV-betagal transduced cells were found throughout the cerebellar white matter in a dose-dependent manner, but few transduced PCs were evident. In contrast, vTR-CMVbeta with Ad5 transduced several hundred PCs throughout the injected hemisphere. Using an rAAV2 vector transducing a CMV-regulated green fluorescent protein gene, we again found PC transduction, but only with Ad5 coinjection. To assess the effect of injection site and to determine whether the apparent requirement for Ad5 coinfection is observed with other promoters, a beta-actin-regulated vector was injected with or without Ad5 to DCN or cerebellar cortical sites. Thousands of transduced PCs were observed under each condition. Cortical injection yielded greater numbers of transduced cells. Injection of rAAV2 without Ad5 led to greater specificity for PC transduction. We conclude that injection of rAAV2 vectors into the cerebellum is an effective means for transferring genes into substantial numbers of Purkinje cells in vivo.

Mild preconditioning and low-level engraftment confer methotrexate resistance in mice transplanted with marrow expressing drug-resistant dihydrofolate reductase activity.

Effective engraftment of hematopoietic cells targeted for gene transfer is facilitated by cytoreductive preconditioning such as high-dose total body irradiation (TBI). To minimize the adverse side effects associated with TBI, experiments were conducted to determine whether sublethal doses of TBI would allow sufficient engraftment of MTX-resistant hematopoietic cells to confer survival on recipient mice administered MTX. FVB/N animals were administered 1, 2, or 4 Gy TBI (lethal dose, 8.5 Gy), transplanted with 10(7) FVB/N transgenic marrow cells expressing an MTX-resistant dihydrofolate reductase (DHFR) transgene, and then administered MTX daily for 60 days. Control mice administered 1 Gy with or without subsequent transplantation of normal marrow cells succumbed to MTX toxicity by day 45. In contrast, nearly all animals transplanted with transgenic marrow survived MTX administration, regardless of the TBI dose used for preconditioning. The donor DHFR transgenic marrow engraftment level was proportional to the preconditioning dose of TBI but was surprisingly reduced in animals given 2 or 4 Gy TBI and subsequently administered MTX when compared with control animals administered phosphate-buffered saline. Animals preconditioned with 1 Gy were also protected from MTX toxicity when transplanted with reduced amounts (5 x 10(6) and 1 x 10(6) cells) of DHFR transgenic donor marrow, resulting in low-level (approximately 1%) engraftment. In conclusion, very mild preconditioning allows sufficient low-level engraftment of genetically modified stem cells for in vivo manifestation of the modified phenotype, suggesting the usefulness of mild preconditioning regimens in human gene therapy trials targeting hematopoietic stem cells. (Blood. 2000;96:1334-1341)

"Supercharged Cells" for delivery of recombinant human iduronate-2-sulfatase.

Expression of iduronate-2-sulfatase (IDS) from three different promoters in four retroviral vectors was studied in peripheral blood lymphocytes from patients with Hunter syndrome (PBL(MPS)), i.e., the LTR in vectors L2SN and L2, avian beta-actin promoter in LB2, and the CMV early promoter in LNC2. PBL(MPS) were exposed to packaging cell supernatant resulting in transduction frequencies ranging 10-fold from 5 to 49%. Surprisingly, IDS activities were equally high in all transduced lymphocyte populations: 515 U/mg/h in PBL(MPS)-L2SN, 734 in PBL(MPS)-LB2, 352 in PBL(MPS)-L2, and 389 in PBL(MPS)-LNC2 compared to controls (<10 in PBL(MPS)-LXSN or PBL(MPS)). The half-life of endocytosed IDS in PBL(MPS) was 1.9 days. However, the level of lymphocyte IDS activity from proviral expression was found to be only a fraction of the total, a large portion being derived from reuptake of enzyme from murine packaging cells, i.e., a "second source" of enzyme. Therefore, measurement of transgene lysosomal enzyme soon after exposure of target cells to vector supernatant may yield a gross overestimate of long-term transgene expression by transduced cells. Nevertheless, patient fibroblasts cocultured with transduced PBL(MPS) had reduced (35)SO(4)-GAG accumulation, levels similar to those of normal fibroblasts. These studies revealed a broadly applicable phenomenon: cells can be charged with a lysosomal enzyme to levels much higher than those found in nature. By "supercharging" cells with a lysosomal protein (or other molecule bearing the mannose-6-phosphate ligand), such cells may be exploited as vehicles for systemic delivery of therapeutic or diagnostic agents.

Maintenance of differential methotrexate toxicity between cells expressing drug-resistant and wild-type dihydrofolate reductase activities in the presence of nucleosides through nucleoside transport inhibition.

Methotrexate (MTX), a potent inhibitor of dihydrofolate reductase (DHFR), has been used widely as a chemotherapeutic agent and as a selective agent for cells expressing drug-resistant DHFR activity. MTX deprives rapidly dividing cells of reduced folates that are necessary for thymidylate synthesis and de novo purine nucleotide synthesis. However, MTX toxicity can be circumvented by salvaging thymidine (TdR) and purine nucleosides. Here we have investigated conditions under which nucleoside transport inhibition can be used to maintain differential MTX toxicity between unmodified cells and cells expressing drug-resistant DHFR activity in the presence of exogenous nucleosides. PA317 cells (a 3T3 derivative cell line) were rescued from the toxicity of 0.1 microM MTX by 1.0 microM TdR in the presence of 100 microM inosine. The nucleoside transport inhibitor dipyridamole (DP) resensitized these cells to MTX, even in the presence of exogenous nucleosides. Furthermore, PA317 cells transduced with any of three retroviruses encoding drug-resistant DHFRs remained resistant to MTX over all concentrations tested (up to 10.0 microM) in the presence of DP. Similar results were obtained in transduced HuH7 and K562 cell lines, a human hepatoma and a human leukemia cell line, respectively. We conclude that nucleoside transport inhibition increases the toxicity and selectivity of MTX in cultured cells, and therefore is an effective way to maintain differential MTX toxicity between unmodified and DHFR-modified cells. Our results support the use of nucleoside transport inhibition in in vivo selection protocols involving the liver and hematopoietic systems.

Gene therapy of genetic diseases and cancer.

The scope of gene transfer applications in human therapy has expanded enormously over the last 15 years to include not only several types of genetic diseases but also a variety of genetic approaches to the treatment of cancer. Hematopoietic stem cells have been considered excellent targets for therapeutic gene transfer because of their capacity for self-renewal and for differentiation into multiple cellular lineages. Retrovirus-mediated gene transfer has been tested for treatment of diseases that specifically affect the hematopoietic system, such as adenosine deaminase deficiency and chronic granulomatous disease. Storage disorders such as Gaucher disease, Hurler syndrome and Hunter syndrome, genetic deficiencies that affect a broad range of tissue types, may also be amenable to treatment by gene transfer into hematopoietic cells, owing to the release of enzyme expressed in transduced cells with subsequent uptake by untransduced cells ("metabolic cross-correction"). Hematopoietic stem cells may also be targeted for introduction of drug-resistance genes for the purpose of protecting normal tissues from the toxic side-effects of cancer chemotherapeutic agents, thus allowing more effective antitumor chemotherapy. The danger of introducing drug-resistance function into tumor cells may be dealt with by including sequences specifically designed to reduce expression of oncogenes or to restore expression of tumor suppressor genes. Current limitations on the efficiency of gene transfer into hematopoietic stem cells may be alleviated by the development of new vector systems such as adeno-associated virus or lentivirus vectors, or by advances in cell processing that render hematopoietic cells more susceptible to transduction. Drug-resistance genes may also be applied for in vivo selection to expand the representation of a small proportion of transduced hematopoietic cells. These approaches toward increasing the frequency of hematopoietic cell transduction contribute to the anticipated feasibility of gene therapy for genetic diseases and cancer.

Enzymatic correction and cross-correction of mucopolysaccharidosis type I fibroblasts by adeno-associated virus-mediated transduction of the alpha-L-iduronidase gene.

Mucopolysaccharidosis type I (MPS I), a deficiency in the lysosomal enzyme alpha-L-iduronidase (IDUA), is characterized by skeletal abnormalities, hepatosplenomegaly and neurological dysfunction. To evaluate the potential for treatment of the disease using a gene delivery approach, recombinant adeno-associated virus (rAAV) vectors were constructed and evaluated for expression of the human IDUA cDNA in transduced cells. 293 cells transduced with these AAV vectors contained IDUA activity at 0.5 to 1.4 micromol/mg x hr, 50- to 140-fold above background (control-transduced) levels. In time course studies of transduced 293 cells, IDUA activity levels peaked 1 week after transduction and persisted at 50% of the peak level for at least 6 weeks. Transduced MPS I fibroblasts also expressed high levels of IDUA activity (114-290 nmol/mg x hr), which persisted for at least 3 weeks in the absence of selection. In addition, transduced MPS I fibroblasts were capable of clearing intracellular radiolabeled glycosaminoglycan (GAG). As a test of the ability of these vectors to mediate metabolic cross-correction, transduced HuH7 human hepatoma cells were demonstrated to release enzyme that was subsequently taken up by nontransduced MPS I fibroblasts. These results illustrate the effectiveness of AAV vectors for delivery and expression of human IDUA gene sequences and for potential treatment of MPS I.

Gene therapy for chronic myelogenous leukemia.

Chronic myelogenous leukemia (CML) is characterized by a balanced translocation that leads to the formation of the the BCR-ABL fusion gene. Although autografts can prolong the life of CML patients, patients relapse owing to malignant cells that persist in the graft and the host. This review discusses various experimental strategies that target the BCR-ABL gene or gene products that are downstream of it. Various strategies have been adopted to block BCR-ABL at the gene, mRNA and protein level. One promising strategy involves the cotransduction of a patient's hematopoietic stem cells (HSCs) with anti-BCR-ABL antisense sequences and a drug resistance gene. This might allow for the elimination of any residual disease in the graft or host by chemotherapy while rendering any drug-resistant, malignant CML HSCs functionally normal.

Methotrexate-resistant form of dihydrofolate reductase protects transgenic murine embryos from teratogenic effects of methotrexate.

Methotrexate, a potent inhibitor of the ubiquitously expressed enzyme dihydrofolate reductase, induces limb and facial anomalies that resemble vascular disruptions in their evolution and final outcome. Previous studies suggest that inhibition of dihydrofolate reductase is responsible for methotrexate-induced embryopathy, although specific sites of methotrexate activity have not been well defined. In this report, we show that constitutive expression of a methotrexate-resistant form of dihydrofolate reductase in transgenic embryos and their placentas ameliorates methotrexate teratogenicity. However, expression of the transgene in maternal tissues had no significant protective effect. The results confirm the role of dihydrofolate reductase inhibition in the pathogenesis of methotrexate-induced birth defects and provide a foundation for future studies of targeted transgene expression in select embryonic or placental cell populations.

Transgenic mice expressing the tyr22 variant of murine DHFR: protection of transgenic marrow transplant recipients from lethal doses of methotrexate.

Expression of the arg22, drug-resistant variant of dihydrofolate reductase (DHFR) in hematopoietic cells has been demonstrated to confer resistance to methotrexate (MTX) in mice, even though this variant suffers from low catalytic activity. The recently reported tyr22 variant has the advantage of higher catalytic activity combined with significant resistance to MTX. To evaluate the resistance conferred by tyr22-DHFR in vivo, we generated several transgenic mouse lines carrying a tyr22-DHFR minigene regulated by its natural promoter. The transgene copy number in 11 lines ranged from 6 to 68 copies and ribonuclease protection analysis demonstrated that 4 of these lines expressed significant transgenic DHFR mRNA at 20 to 68% of the endogenous DHFR mRNA level. Marrow from 4 of the 11 lines conferred significant increases in MTX-resistance in comparison with normal marrow when transplanted into lethally irradiated recipients. The ability of the tyr22-DHFR transgenic marrow to confer MTX-resistance to bone marrow transplant (BMT) recipients did not correlate with the level of mRNA expression or the number of transgene copies. However, two lines (lines 11 and 15) that were most effective in maintaining normal hematocrit levels in BMT recipients receiving 1 mg/kg/day MTX exhibited the greatest ability to form MTX-resistant hematopoietic progenitor colonies in vitro. Furthermore, MTX dose escalation studies demonstrated that line 11 marrow conferred resistance in BMT recipients receiving up to 6 mg/kg/day MTX. Southern blot analysis of the BMT recipients 7 months posttransplantation showed a preponderance of transgenic donor-derived cells in bone marrow and spleen, as well as a surprisingly high level in the small intestine. These results indicate that tyr22-DHFR is likely to be superior to arg22-DHFR in conferring MTX-resistance in BMT recipients, illustrating its usefulness for chemoprotection during MTX chemotherapy and also potentially for in vivo selection of transduced cells in gene therapy trials.

Retrovirally mediated gene transfer of Arg22 and Tyr22 forms of dihydrofolate reductase into the hematopoietic cell line K562: a comparison of methotrexate resistance.

Mutations in the enzyme dihydrofolate reductase (DHFR) can confer resistance to the inhibitory effects of folate analogs such as methotrexate (Mtx) and trimetrexate (Ttx). Retroviral vectors expressing the DHFR-Arg22 mutants and the newly described DHFR-Tyr22 mutant were used to transduce the hematopoietic cell line K562. In vitro selection of vector-containing cells was documented via polymerase chain reaction and Southern analysis. When proliferation of selected vector-containing cells was evaluated over a range of Mtx concentrations (0.01 to 10 micromol/L), both Arg22 and Tyr22 provided protection from Mtx, but Tyr22 proved superior to Arg22 in conferring Mtx resistance at low concentrations. Ttx proved to be 10- to 100-fold more potent than Mtx in inhibiting proliferation of nontransduced K562, but the relative effectiveness of individual mutants in conferring drug resistance was similar to that of Mtx. Decreasing the amount of folate in the culture medium to more physiological concentrations increased the potency of administered Mtx and Ttx. Drug resistance in retrovirally transduced K562 cells is consistent with the enzymatic characteristics of the individual mutants. Our findings suggest that the new Tyr22 form of DHFR may prove better in conferring drug resistance than the previously reported Arg22 mutant.

Gene therapy for chronic myelogenous leukemia (CML): a retroviral vector that renders hematopoietic progenitors methotrexate-resistant and CML progenitors functionally normal and nontumorigenic in vivo.

Chronic myelogenous leukemia (CML) is a malignant disease of the human hematopoietic stem cell caused by the BCR/ABL gene rearrangement. The only curative therapy is allogeneic transplantation. Although autologous transplants may prolong survival, most patients relapse because of disease persisting in the host and in the graft. Continued administration of chemotherapy after transplant could reduce the incidence of relapse provided that the autograft can be protected by transfer of a drug-resistance gene. However, CML autografts will almost certainly contain malignant stem cells that will also be rendered drug-resistant. The presence of the BCR/ABL oncoprotein is necessary and sufficient for malignant transformation seen in CML. We thus hypothesized that transfer of a vector that combines a drug-resistance gene with anti-BCR/ABL antisense (AS) sequences may allow for posttransplant chemotherapy to decrease persistent disease while rendering inadvertently transduced CML stem and progenitor cells functionally normal. We constructed a retroviral vector, LasBD, that combines the methotrexate (MTX)-resistant tyrosine-22 dihydrofolate-reductase (tyr22-DHFR) gene and AS sequences directed at the b3a2 BCR/ABL breakpoint. b3a2 BCR/ABL containing 32D and MO7e cells were transduced with LasBD and selected in MTX for 14 days. Expression of the AS sequences reduced BCR/ABL mRNA and p210(BCR/ABL) protein levels by 6- to 10-fold in most cells. This subsequently led to the restoration of normal function of BCR/ABL cDNA+ cells: they grew significantly slower in the presence of interleukin-3 (IL-3); they underwent apoptotic cell death when cultured without IL-3; and they had restored expression and function of adhesion receptors. These effects were specific, because LasBD-containing AS sequences directed at the b3a2 BCR/ABL breakpoint did not affect p190(BCR/ABL)-containing cells. LasBD also rendered 20% to 30% of primary Ph- and Ph+ CD34(+) cells MTX-resistant and decreased BCR/ABL mRNA levels in MTX resistant Ph+ CD34(+) cells by 10-fold. Expression of the MTX-resistant DHFR gene and the AS sequences has been stable for at least 1 year in vitro and for more than 70 days in vivo. Finally, LasBD decreased tumorigenicity of 32DBCR/ABL cells in vivo by 3 to 4 logs. In conclusion, the tyr22-DHFR gene in the LasBD vector can protect normal hematopoietic cells from MTX-mediated toxicity, whereas the AS sequences in LasBD can suppress expression of the BCR/ABL gene and restore normal function of BCR/ABL cDNA-containing cells. The LasBD vector may therefore prove to be an extremely useful adjunct in autologous transplantation for CML.

Drug-resistant dihydrofolate reductases: generation, expression and therapeutic application.

Although methotrexate (MTX) and other antifolates are effective antitumor chemotherapeutic agents, their utility is limited by toxicity for normal hematopoietic and gastrointestinal tissues. Gene transfer and expression of variant dihydrofolate reductase (DHFR) which confers resistance to MTX could protect these tissues from MTX toxicity. Drug-resistant DHFR variants have been cloned from a number of sources and have also been generated by in vitro mutagenesis. A procedure is described which we used for saturation mutagenesis of the murine DHFR coding sequence at codon positions 22 and 31, resulting in a number of mutant DHFR genes encoding enzyme exhibiting a favorable combination of drug resistance with retention of catalytic activity. To evaluate the in vivo effectiveness of variant DHFR expression, we established several lines of FVB/N transgenic mice which expressed drug-resistant DHFR activity and which exhibited varying degrees of increased resistance to MTX administration. Transplantation of bone marrow from drug-resistant DHFR transgenic animals into normal, irradiated recipients conferred resistance to MTX at surprisingly high levels, indicating that drug-resistant DHFR expression in hematopoietic cells also protects gastrointestinal tissues from MTX toxicity. These studies have thus provided encouraging results with respect to the potential of drug-resistant DHFR gene transfer for improved use of MTX as an antitumor agent and for its use as an in vivo selective agent.

Methotrexate dose-escalation studies in transgenic mice and marrow transplant recipients expressing drug-resistant dihydrofolate reductase activity.

Methotrexate (MTX) dose-escalation studies were conducted in two inbred lines of FVB/N transgenic mice expressing distinct drug-resistant dihydrofolate reductases (DHFRs) and in animals transplanted with transgenic marrow. Survival of animals expressing a tryptophan-31 variant DHFR transgene was only slightly improved over that of normal animals, and survival of tryptophan-31 variant DHFR marrow transplant recipients was indistinguishable from that of normal animals (at a MTX dose of 4 mg/kg i.p. daily). In contrast, extended survival was observed for animals expressing an arginine-22 variant (Arg22) DHFR transgene, with the last three of eight animals in this group succumbing at a final MTX dose of 14 mg/kg i.p. daily. Survival was slightly reduced for normal animals transplanted with Arg22 marrow. Interestingly, demise of animals in both Arg22 groups was not associated with the profound drop in hematocrit levels usually observed in MTX-treated animals. These animals were instead characterized by severe atrophy of the gastrointestinal tract, whereas hematocrit levels and marrow histology were relatively normal. Kidney pathology (mesangiocapillary glomerulopathy) was also observed in Arg22 marrow recipients but not in Arg22 transgenics, consistent with expression of the drug-resistance gene in kidney tissues of the transgenics, as demonstrated by ribonuclease protection analysis. Immediate dose-response studies in Arg22 marrow transplant recipients defined a maximum tolerated dose of 4 mg/kg/day MTX, 2 to 3 times that of animals transplanted with normal marrow or of normal untransplanted animals. These results define the extent of chemoprotection afforded by drug-resistant DHFR expression and serve to identify alternate sites of toxicity in animals administered the higher levels of MTX afforded by drug-resistant DHFR expression in the marrow.

Methotrexate resistance of mouse dihydrofolate reductase: effect of substitution of phenylalanine-31 by serine or tryptophan.

Steady state and preliminary pre steady state studies of the mouse DHFR indicate that the wild-type enzyme used for our mutagenic studies follows a significantly different in vitro kinetic pathway than previously reported. In particular, turnover does not appear to be governed by H4F release from the E.NADPH complex. The discrepancies in catalysis and binding behavior of the mouse DHFRs may be due to the isomeric nature of the DHFRs studied. The enhanced ability of the two mutations at position 31 to confer resistance to MTX, as expected, decreased the affinity of the enzyme for the inhibitor. A correlation between the increased size of the side chain at position 31 and decreased inhibitor affinity was observed. This findings is consistent with previous mutagenesis studies of mouse DHFR but is at odds with conclusions drawn from an analysis of the role of the position in inhibitor binding to human DHFR. It is generally agreed that a highly efficient enzyme is desired for most cellular metabolic functions; however, because substitution of position 31 with tryptophan impairs catalytic efficiency, it appears that there exists a high physiological tolerance for significantly impaired DHFR. Indeed, mice who have received transplants of bone marrow expressing the Trp-31 mutant or the severely impaired Arg-22 mutant are capable of surviving lethal doses of MTX. Nevertheless, the consequences in vivo of a reduction in the observed in vitro catalytic effectiveness of DHFR remain to be determined. Additional mutagenic studies attempting to select catalytically silent mutations that reduce inhibitor binding may further enhance the therapeutic potential of drug-resistant DHFR genes for improved folate antagonist mediated antitumor activity.