Loss of Ubr1 promotes aneuploidy and accelerates B-cell lymphomagenesis in TLX1/HOX11-transgenic mice.

The TLX1/HOX11 homeobox gene was originally identified at the recurrent t(10;14)(q24;q11) translocation breakpoint, a chromosomal abnormality observed in 5-7% of T-cell acute lymphoblastic leukemias (T-ALLs). Proviral insertional mutagenesis studies performed on transgenic mice ectopically expressing TLX1/HOX11 in B lymphocytes (IgHmu-HOX11(Tg) mice) revealed the Ubr1 gene locus as a frequent site of proviral insertion, concomitant with accelerated development of diffuse large B-cell lymphoma. Insertion into this genomic region was confirmed by Southern blotting and by the ability to generate a polymerase chain reaction (PCR) amplicon across the viral-genome junction. Western immunoblot and semiquantitative reverse transcriptase-PCR analysis revealed downregulated expression of the Ubr1 gene product subsequent to viral integration. Loss or reduced levels of Ubr1 expression was associated with 5/14 spontaneous B-cell lymphomas in IgHmu-HOX11(Tg) mice and one of nine primary human T-ALLs. To gain mechanistic insight into the cooperativity between TLX1/HOX11 and Ubr1, IgHmu-HOX11(Tg)/Ubr1(-/-) mice were generated. IgHmu-HOX11(Tg)/Ubr1(-/-) mice exhibited a modest but statistically significant acceleration of disease onset relative to IgHmu-HOX11(Tg)/Ubr1(+/-) mice. Moreover, micronucleus assays to detect for chromosome missegregation were conducted and revealed increased presence of micronuclei in IgHmu-HOX11(Tg)/Ubr1(-/-) primary B lymphocyte cultures, and in both TLX1/HOX11-overexpressing T cell lines and fibroblast cultures following transfection with short interfering RNAs (siRNAs) targeting Ubr1. Karyotyping of primary B lymphocyte cultures revealed increased incidences of hypodiploid karyotypes. Finally, mitotic figures analysed from Ubr1 siRNA-transfected fibroblast cultures revealed no defects in chromosome congression to the metaphase plate, but increased incidences of atypical anaphase figures, including the development of anaphase bridges and lagging chromosomes. Based on these findings, we identify a synergistic role between TLX1/HOX11 overexpression and Ubr1 inactivation in promoting chromosome missegregation, permitting the accrual of additional chromosome losses and cytogenic abnormalities en route to malignancy.

Dysregulated expression of mitotic regulators is associated with B-cell lymphomagenesis in HOX11-transgenic mice.

Dysregulated expression of the homeobox gene, HOX11 is a frequent etiologic event in T-cell acute lymphoblastic leukemias. HOX11-transgenic mice (IgHmu-HOX11Tg)-expressing HOX11 in the B-cell compartment develop B-cell lymphomas with extended latency. The latency suggests that additional genetic events are required prior to the onset of malignant lymphoma. We report the identification of 17 HOX11 collaborating genes, revealed through their propensity to be targeted in a proviral insertional mutagenesis screen. Seven integrations disrupted genes in mitotic spindle checkpoint control, suggesting that cells with elevated HOX11 expression are especially sensitive to dysregulation of chromosome segregation during mitosis. IgHmu-HOX11Tg primary B-lymphocyte cultures exposed to the aneugenic agents, colchicine and colcemid, exhibited increased incidences of chromosome missegregation as assessed by cytokinesis-block micronucleus assays. Additionally, IgHmu-HOX11Tg cultures were shown to exhibit aberrant bypass of spindle checkpoint arrest, as assessed by the increased presence of cycling cells determined by assessment of DNA content and by BrdU immunolabelling. Western immunoblotting revealed elevated expression of the mitotic effector molecules, cyclin A, cyclin B1 and cdc20 in IgHmu-HOX11Tg cultures. Moreover, spontaneously arising lymphoid neoplasms in IgHmu-HOX11Tg mice frequently exhibit aberrant expression of mitotic regulators, concomitant with increased development of micronuclei, abnormal mitotic checkpoint control and increased incidences of abnormal karyotypes when expanded in culture. Collectively, these findings indicate that abnormal regulation of spindle checkpoint control as a result of HOX11 overexpression leads to a heightened predisposition for development of aneuploidy, contributing to oncogenesis.

Induction of tolerance to immunogenic tumor antigens associated with lymphomagenesis in HOX11 transgenic mice.

Transgenic mice expressing human HOX11 in B lymphocytes die prematurely from lymphomas that initiate in the spleen and frequently disseminate to distant sites. Preneoplastic hematopoiesis in these mice is unperturbed. We now report that expression of the HOX11 transgene does not affect the ability of dendritic cells (DCs) to process and present foreign peptides and activate antigen-specific T cell responses. We also show that nontransgenic DCs presenting peptides derived from the human HOX11 protein are highly efficient stimulators of autologous T cells, whereas transgenic T cells are nonresponsive to peptides derived from the HOX11 transgene and the murine Meis1 protein. HOX11 transgenic mice thus show normal development of tolerance to immunogenic antigens expressed throughout B cell maturation. DCs pulsed with cell lysates prepared from lymphomas, obtained from HOX11 transgenic mice with terminal lymphoma, activate T cells from nontransgenic and premalignant transgenic mice, whereas T cells isolated from lymphomatous transgenic mice are nonresponsive to autologous tumor cell antigens. These data indicate that HOX11 lymphoma cells express tumor-rejection antigens that are recognized as foreign in healthy transgenic mice and that lymphomagenesis is associated with the induction of anergy to tumor antigen-specific T cells. These findings are highly relevant for the development of immunotherapeutic protocols for the treatment of lymphoma.

p53 gene mutations in sequential oral epithelial dysplasias and squamous cell carcinomas.

Previous studies of oral cancer have suggested that alterations of the p53 tumour suppressor gene occur early in the precancerous stage of development. However, these observations have been based on cross-sectional assessment of abnormal p53 protein staining by immunohistochemistry and may not necessarily reflect gene changes. The purpose of this longitudinal study was to examine the changes in the p53 gene in progressive, sequential epithelial dysplasias and carcinomas from the oral cavity. The study analysed 24 formalin-fixed, paraffin-embedded tissue biopsies from ten patients with two or more temporally distinct lesions from the same site in the oral cavity with the diagnosis of hyperkeratosis, epithelial dysplasia, carcinoma in situ or squamous cell carcinoma. Exons 5-8 of the p53 gene were amplified from genomic DNA using intronic primers and directly sequenced using fluorescent-labelled primers. Standard immunohistochemistry with the DO7 monoclonal antibody was used to detect mutant and wild-type p53 protein. Mutations of the p53 gene were identified in 9 of 24 samples. Eight were missense mutations and one occurred at a splice site. In six patients, mutations of the p53 gene occurred late after the transformation of epithelial dysplasia to carcinoma. In two patients with progressive dysplasia, but who had yet to develop invasive carcinoma, p53 missense mutations occurred at the carcinoma in situ stage in one case and in a moderate dysplasia in the other. There was an inconsistent relationship between gene mutations and the level of p53 protein staining by immunohistochemistry. It is concluded that during oral carcinogenesis, p53 gene mutations seem to occur relatively late and are associated with transformation to the invasive phenotype.

Engraftment of gene-marked hematopoietic progenitors in myeloma patients after transplant of autologous long-term marrow cultures.

We conducted a phase I hematopoietic stem cell (HSC) gene-marking trial in patients undergoing autologous blood or marrow stem cell transplant for the treatment of multiple myeloma. Between 500 and 1000 ml of bone marrow was harvested from each of 14 myeloma patients and 1 syngeneic donor. A mean of 3.3x10(9) cells per patient were plated in 20 to 50 long-term marrow culture (LTMC) flasks and maintained for 3 weeks. LTMCs were exposed on days 8 and 15 to clinical-grade neo(r)-containing retrovirus supernatant (G1Na). A mean of 8.23x10(8) day-21 LTMC cells containing 5.2x10(4) gene-marked granulocyte-macrophage progenitor cells (CFU-GM) were infused along with an unmanipulated peripheral blood stem cell graft into each patient after myeloablative therapy. Proviral DNA was detected in 71% of 68 tested blood and bone marrow samples and 150 of 2936 (5.1%) CFU-GM derived from patient bone marrow samples after transplant. The proportion of proviral DNA-positive CFU-GM declined from a mean of 9.8% at 3 months to a mean of 2.3% at 24 months postinfusion. Southern blots of 26 marrow and blood samples were negative. Semiquantitative PCR analysis indicated that gene transfer was achieved in 0.01-1% of total bone marrow and blood mononuclear cells (MNCs). Proviral DNA was also observed in EBV-transformed B lymphocytes, in CD34+ -enriched bone marrow cells, and in CFUs derived from the latter progenitors. Gene-modified cells were detected by PCR in peripheral blood and bone marrow for 24 months after infusion of LTMC cells. Sensitivity and specificity of the PCR assays were independently validated in four laboratories. Our data confirm that HSCs may be successfully transduced in stromal based culture systems. The major obstacle to therapeutic application of this approach remains the overall low level of genetically modified cells among the total hematopoietic cell pool in vivo.

Gene therapy for canine alpha-L-iduronidase deficiency: in utero adoptive transfer of genetically corrected hematopoietic progenitors results in engraftment but not amelioration of disease.

Canine alpha-L-iduronidase (iduronidase) deficiency is a model of the human lysosomal storage disorder mucopolysaccharidosis type I (MPS I). We used this canine model to evaluate the therapeutic potential of hematopoietic stem cell (HSC) gene therapy for enzyme deficiencies. In previous studies, iduronidase-deficient dogs infused with autologous marrow cells genetically modified to express iduronidase had long-term engraftment with provirally marked cells, but there was no evidence of proviral iduronidase expression or clinical improvement. The presence of humoral and cellular immune responses against iduronidase apparently abrogated the therapeutic potential of HSC gene therapy in these experiments. To evaluate HSC gene therapy for canine MPS I in the absence of a confounding immune response, we have now performed in utero adoptive transfer of iduronidase-transduced MPS I marrow cells into preimmune fetal pups. In three separate experiments, 17 midgestation fetal pups were injected with 0.5-1.5 x 10(7) normal or MPS I allogeneic long-term marrow culture (LTMC) cells transduced with neo(r)- or iduronidase-containing retroviral vectors. Nine normal and three MPS I pups survived the neonatal period and demonstrated engraftment of provirally marked progenitors at levels of up to 12% for up to 12 months. However, the proportion of provirally marked circulating leukocytes was approximately 1%. Neither iduronidase enzyme nor proviral-specific transcripts were detected in blood or marrow leukocytes of any MPS I dog. Humoral immune responses to iduronidase were not detected in neonates, even after "boosting" with autologous iduronidase-transduced LTMC cells. All MPS I dogs died at 8-11 months of age from complications of MPS I disease with no evidence of amelioration of MPS I disease. Our results suggest that iduronidase-transduced primitive hematopoietic progenitors can engraft in fetal recipients, contribute to hematopoiesis, and induce immunologic nonresponsiveness to iduronidase in MPS I dogs. However, the therapeutic potential of HSC gene transfer in this model of iduronidase deficiency appears to be limited by poor maintenance of proviral iduronidase gene expression and relatively low levels of genetically corrected circulating leukocytes.

Genetically corrected autologous stem cells engraft, but host immune responses limit their utility in canine alpha-L-iduronidase deficiency.

Canine alpha-L-iduronidase (alpha-ID) deficiency, a model of the human storage disorder mucopolysaccharidosis type I (MPS I), is an ideal system in which to evaluate the clinical benefit of genetically corrected hematopoietic stem cells. We performed adoptive transfer of genetically corrected autologous hematopoietic cells in dogs with alpha-ID deficiency. Large volume marrow collections were performed on five alpha-ID-deficient dogs. Marrow mononuclear cells in long-term marrow cultures (LTMCs) were exposed on three occasions during 3 weeks of culture to retroviral vectors bearing the normal canine alpha-ID cDNA. Transduced LTMC cells from deficient dogs expressed enzymatically active alpha-ID at 10 to 200 times the levels seen in normal dogs. An average of 32% of LTMC-derived clonogenic hematopoietic cells were provirus positive by polymerase chain reaction and about half of these expressed alpha-ID. Approximately 10(7) autologous gene-modified LTMC cells/kg were infused into nonmyeloablated recipients. Proviral DNA was detected in up to 10% of individual marrow-derived hematopoietic colonies and in 0.01% to 1% of blood and marrow leukocytes at up to 2 to 3 years postinfusion. Despite good evidence for engraftment of provirally marked cells, neither alpha-ID enzyme nor alpha-ID transcripts were detected in any dog. We evaluated immune responses against alpha-ID and transduced cells. Humoral responses to alpha-ID and serum components of the culture media (fetal bovine and horse sera and bovine serum albumin) were identified by enzyme-linked immunosorbent assay. Cellular immune responses to autologous alpha-ID but not neo(r) transduced cells were demonstrated by lymphocyte proliferation assays. To abrogate potential immune phenomena, four affected dogs received posttransplant cyclosporine A. Whereas immune responses were dampened in these dogs, alpha-ID activity remained undetectable. In none of the dogs engrafted with genetically corrected cells was there evidence for clinical improvement. Our data suggest that, whereas the alpha-ID cDNA may be transferred and maintained in approximately 5% of hematopoietic progenitors, the potential of this approach appears limited by the levels of provirally derived enzyme that are expressed in vivo and by the host's response to cultured and transduced hematopoietic cells expressing foreign proteins.

Deregulation of NPM and PLZF in a variant t(5;17) case of acute promyelocytic leukemia.

Greater than 95% of acute promyelocytic leukemia (APL) cases are associated with the expression of PML-RARalpha. This chimeric protein has been strongly implicated in APL pathogenesis because of its interactions with growth suppressors (PML), retinoid signaling molecules (RXRalpha), and nuclear hormone transcriptional co-repressors (N-CoR and SMRT). A small number of variant APL translocations have also been shown to involve rearrangements that fuse RARalpha to partner genes other than PML, namely PLZF, NPM, and NuMA. We describe the molecular characterization of a t(5;17)(q35;q21) variant translocation involving the NPM gene, identified in a pediatric case of APL. RT-PCR, cloning, and sequence studies identified NPM as the RARalpha partner on chromosome 5, and both NPM-RARalpha and RARalpha-NPM fusion mRNAs were expressed in this patient. We further explored the effects of the NPM-RARalpha chimeric protein on the subcellular localization of PML, RXRalpha, NPM, and PLZF using immunofluorescent confocal microscopy. While PML remained localized to its normal 10-20 nuclear bodies, NPM nucleolar localization was disrupted and PLZF expression was upregulated in a microspeckled pattern in patient leukemic bone marrow cells. We also observed nuclear co-localization of NPM, RXRalpha, and NPM-RARalpha in these cells. Our data support the hypothesis that while deregulation of both the retinoid signaling pathway and RARalpha partner proteins are molecular consequences of APL translocations, APL pathogenesis is not dependent on disruption of PML nuclear bodies.

Adoptive transfer of genetically modified human hematopoietic stem cells into preimmune canine fetuses.

To develop a surrogate model system for assaying gene transfer into human hematopoietic stem cells (HSCs) with in vivo repopulating potential, we injected human marrow cells transduced with a reporter retroviral vector in long-term marrow cultures (LTMCs), into the yolk sacs of preimmune canine fetuses. Of eight mid-gestation fetuses injected through the exteriorized uterine wall and under ultrasound guidance, seven were born alive. One puppy died in the neonatal period accidentally. The remaining six puppies are all healthy at 31 months of age. There was no evidence for graft-versus-host disease or any untoward effects of in utero adoptive transfer of transduced human LTMC cells. All puppies were chimeras. Human cells, detected by fluorescence in situ hybridization, were present in blood, declining from 38% to 0.05% between 10 and 44 weeks after birth. Corresponding numbers for marrow were from 20% to 0.05%. Human cells were also detected in assays of hematopoietic cell progenitors and in stimulated blood cultures. All six puppies were positive for the presence of proviral DNA at various time-points after birth. In three dogs, provirus was detected up to 41 weeks after birth in blood or marrow, and in one dog up to 49 weeks in blood. These data support the further development of this large-animal model system for studies of human hematopoiesis.

Retroviral-mediated transfer and expression of the multidrug resistance protein 1 gene (MRP1) protect human hematopoietic cells from antineoplastic drugs.

Multidrug resistance protein (MRP1) is a member of the ATP-binding cassette (ABC) transmembrane transporter superfamily that confers multidrug resistance. The transfer and expression of the MRP1 gene in human hematopoietic stem cells may be a useful alternative to multidrug resistance (MDR1) gene transfer for protection from the myelosuppressive effects of chemotherapy in cancer patients. We constructed a gibbon ape leukemia virus packaging cell line (PG13) using the human MRP1 cDNA in a Moloney murine leukemia virus (MoMuLV) backbone containing a modified LTR. This PG13-based cell line, designated MRP1-PG13, produces retroviral vectors bearing the MRP1 gene at a titer of 1.7x10(5) viral particles/ml. Transduction of the human leukemic cell line K562 showed that viral MRP1-PG13 supernatants routinely transfer the MRP1 gene to approximately 35% of target K562 cells, of which at least one third are capable of proliferating in the presence of otherwise toxic concentrations of etoposide. Southern blot analyses indicated that most clones had only one proviral integration. Northern blot analysis of expanded K562 clones showed the presence of a major full-length approximately 8-kb MRP1 transcript as well as a minor approximately 6-kb transcript in all clones. Flow cytometric analysis of the producer cells and clones of transduced K562 cells demonstrated significantly increased MRP1 expression in these cells (approximately 30-fold increase). Human bone marrow mononuclear cells and CD34+ cells were also transduced with MRP1-PG13 supernatants on fibronectin-coated culture flasks in the presence of SCF, IL-3, and IL-6. PCR analysis of individual hematopoietic colonies in methylcellulose cultures demonstrated proviral DNA in approximately 10% of unselected human hematopoietic progenitor cells cultured from nonsorted mononuclear cell samples and in up to approximately 75% of progenitors when CD34-enriched cell populations were targeted. To assess functional MRP1 gene expression, normal human hematopoietic progenitors and K562 cells were cultured in methylcellulose assays containing vincristine or etoposide. All transduced samples gave rise to approximately 10% drug-resistant colonies, which were shown to be provirus-positive by PCR. Our studies document the development of an amphotropic MRP1 retroviral vector producer cell line and pave the way for large animal and preclinical studies of chemoprotection by MRP1 gene transfer.

Fractionation of Gene Modified Hematopoietic Autografts into Multiple Weekly Infusions Does Not Improve Engraftment in Unconditioned Canine Recipients.

Hematopoietic stem cell (HSC) gene therapy will require efficient transfer of genes to HSCs and long term engraftment and proliferation of genetically modified HSCs following adoptive transfer. We evaluated whether fractionation of grafts into 4-5 weekly infusions to non-myeloablated, autologous canine recipients would improve engraftment of genetically modified HSCs. Experimental animals and controls receiving a single infusion had similar levels of engraftment with ∼3-10% of marrow derived progenitors carrying transgene sequences for up to 29 months. There appears to be no improvement of engraftment of genetically modified HSCs in non-myeloablated large animal recipients by dose fractionation.

A model for spontaneous B-lineage lymphomas in IgHmu-HOX11 transgenic mice.

HOX11, a divergent homeodomain-containing transcription factor, was isolated from the breakpoint of the nonrandom t(10;14)(q24;q11) chromosome translocation found in human T cell acute lymphoblastic leukemias. The translocation places the HOX11 coding sequence under the transcriptional control of TCR alpha/delta regulatory elements, resulting in ectopic expression of a normal HOX11 protein in thymocytes. To investigate the oncogenic potential of HOX11, we targeted its expression in lymphocytes of transgenic mice by placing the human cellular DNA under the transcriptional control of Ig heavy chain or LCK regulatory sequences. Only IgHmu-HOX11 mice expressing low levels of HOX11 were viable. During their second year of life, all HOX11 transgenic mice became terminally ill with more than 75% developing large cell lymphomas in the spleen, which frequently disseminated to thymus, lymph nodes, and other nonhematopoietic tissues. Lymphoma cells were predominantly clonal IgM+IgD+ mature B cells. Repopulation of severe combined immunodeficient mice with cells from hyperplastic spleens indicated that the HOX11 tumor phenotype was transplantable. Before tumor development, expression of the transgene did not result in perturbations in lymphopoiesis; however, lymphoid hyperplasia involving the splenic marginal zones was present in 20% of spleens. Our studies provide direct evidence that expression of HOX11 in lymphocytes leads to malignant transformation. These mice are a useful model system to study mechanisms involved in transformation from B-lineage hyperplasia to malignant lymphoma and for testing novel approaches to therapy. They represent a novel animal model for non-Hodgkin's lymphoma of peripheral mature B cell origin.

Retrovirus-mediated gene transfer into human hematopoietic stem cells.

Human hematopoietic stem cells genetically modified by retroviral-mediated gene transfer may offer new treatment options for patients with genetic disease. The potential of gene-modified hematopoietic stem cells as vehicles for gene delivery was first illustrated by the demonstration that hematopoietic systems of lethally irradiated mice can be reconstituted with retroviral vector transduced syngeneic bone marrow, and that these cells can in turn provide genetically marked progeny which persist in blood and marrow over extended time periods. In contrast, hematopoietic stem cells from large animals prove difficult to transduce with retroviral vectors and are consequently less likely to function as vehicles for long-term gene therapy. Indeed, clinically relevant levels of gene transfer into large animal and human hematopoietic stem cells has not been widely achieved. The need for improved retroviral vector systems and for understanding the biology of hematopoietic stem cell gene transfer continue to fuel intense research activity. Preliminary results from human stem cell gene marking and gene therapy trials currently underway are encouraging. This contribution reviews the underlying concepts relevant to retroviral-mediated gene transfer into hematopoietic stem cells. We survey the evolution of approaches for gene transfer into hematopoietic stem cells, from murine and large animal models to the first human clinical trials. Finally, we discuss new strategies which are currently being pursued.

In vitro maintenance and retroviral transduction of human myeloma cells in long-term marrow cultures.

One objective of clinical gene marking trials in multiple myeloma (MM) is to determine the extent to which relapse after stem cell transplant is attributable to contamination of the autograft with myeloma cells. A requirement in these studies is ex vivo genetic marking of malignant cells present in autografts which are derived from patients exposed to significant prior chemotherapy. We evaluated gene marking of cloonogenic myeloma cells in marrow aspirates from 14 patients with MM. To effect gene transfer we utilized a long-term marrow culture (LTMC) system previously shown to facilitate gene transfer into a spectrum of hematopoietic progenitor and stem cells. Transduction of cells in LTMC was performed by multiple supernatant exposure. At LTMC initiation and after 21 days of culture malignant cells were assessed by morphology, flow cytometry, and polymerase chain reaction (PCR). The mean number of day 21 LTMC adherent layer-derived granulocyte/macrophage progenitors as a percentage of the original inoculum was within the normal range for this technique. The efficiency of transduction of normal hematopoietic progenitors as determined by the number of colonies positive for proviral DNA by PCR, G418 resistance, and X-gal staining was also within the expected range; 65%, 44% and 23%, respectively. Thus, there was no evidence that prior chemotherapy exposure or malignant cell contamination compromised cell survival or gene transfer efficiency in LTMC. All patients retained plasma cells in LTMCs for the duration of the 21-day culture period. Molecular analysis confirmed the persistence of clonal IgVH gene rearrangements in day 21 LTMC-derived DNA from 6 of 12 informative patients (50%). PCR using allele-specific primers when available confirmed the specificity of IgVH rearrangements for the myeloma clone. In 2 of the 14 patients, expansion of clonogenic cells was demonstrated in LTMC. In both cases there was strong evidence for transfer of reporter genes (neo and LacZ) into the myeloma clone: morphologically abnormal G418-resistant colonies demonstrated intense staining for beta-galactosidase, and cytospin preparations showed 100% plasma cells with monoclonal heavy and light chain restriction. In one patient, individual colonies positive for beta-galactosidase bore a cytogenetic abnormality characteristic of the patient's myeloma clone. PCR of DNA from pooled plasma cell colonies using tumor-specific CDR3 primers was positive. Our results demonstrate the maintenance of myeloma cells in vitro for up to 21 days in LTMC. They further illustrate that these cells can be genetically marked using transduction protocols currently being tested in clinical trials of hematopoietic cell gene transfer.

Preclinical assessment of human hematopoietic progenitor cell transduction in long-term marrow cultures.

Long-term marrow cultures (LTMCs) were established from 27 human marrows. Hematopoietic cells were subjected to multiple rounds of exposure to retroviral vectors during 3 weeks of culture. Seven different retroviral vectors were evaluated. LTMCs were assessed for viability, replication-competent retrovirus, progenitors capable of proliferating in immune-deficient mice, and gene transfer. The average number of adherent cells and committed granulocyte-macrophage progenitors (CFU-GM) recovered from LTMCs was 28% and 11% of the input totals, respectively. There was no evidence by marker rescue assay or polymerase chain reaction (PCR) of replication-competent virus production during LTMC. No toxicity to cellular proliferation due to the transduction procedure was observed. The adherent layers of LTMCs exposed to retroviral vectors were positive for proviral DNA by PCR and by Southern blot analysis. Fifty-three percent of 1,427 individual CFU-GM from transduced LTMC adherent layers were positive for vector-derived DNA. For neocontaining vectors, the average G418 resistance was 28% of 1,393 LTMC-derived CFU-GM. Forty percent of 187 tissues from 30 immune-deficient mice injected with human LTMC cells were positive for human DNA 4-5 weeks after adoptive transfer. These studies indicate that multiple exposures of human LTMCs to retroviral vectors result in consistent and reproducible LTMC viability and gene transfer into committed progenitors. Our results further support the use of transduced LTMC cells in clinical trials of hematopoietic stem cell gene transfer.

Endogenous murine leukemia virus DNA sequences in murine cell lines: implications for gene therapy safety testing by PCR.

Safety testing for replication-competent retrovirus (RCR) is an important requirement in gene transfer clinical trials using retroviral vectors. A sensitive polymerase chain reaction (PCR) method is one approach to RCR detection. Only in the presence of RCR will the pol-env encoding sequences, necessary for viral replication and packaging, be amplified from proviral DNA in infected indicator cells. To avoid false-positive results in this assay it is crucial that indicator cell lines are free of endogenous retroviral sequences that could also be amplified with pol-env PCR primers. We screened candidate murine indicator cell lines and determined that while Mus dunni is free of detectable pol-env sequences, endogenous retroviral sequences do indeed exist in several cell lines and lead to false-positive results in the PCR assay for RCR. Furthermore, these endogenous retroviral sequences are expressed as RNA transcripts in NIH 3T3 and SC-1 cell lines, as determined by PCR amplification of cDNA but, nevertheless, do not give rise to replication-competent particles. We recognize the potential for murine cell lines to undergo spontaneous rearrangements of endogenous viral sequences in culture and give rise to recombinants containing newly acquired contiguous pol-env sequences. Indicator cell lines should thus be carefully selected and monitored on an ongoing basis when used in safety testing using PCR approaches for the detection of RCR.

Gene therapy: here to stay.

Advances in biotechnology have brought gene therapy to the forefront of medical research. The feasibility of gene transfer was first demonstrated in experiments using tumour viruses. This led to the development of a variety of viral and nonviral methods for the genetic modification of somatic cells. Two main approaches emerged: in-vivo modification, in which gene transfer vehicles are delivered directly into patients, and ex-vivo manipulation, in which cells from the patient are grown in culture, genetically modified and then returned to the patient. In 1990, shortly after the safety of retrovirus-mediated gene transfer was demonstrated in patients with malignant melanoma, the first clinical trial of gene therapy was initiated for adenosine deaminase deficiency. Since then, the number of clinical protocols initiated worldwide has increased exponentially. Although some clinical trials now in progress are concerned with relatively rare inborn errors of metabolism, most are concerned with more commonly encountered cancers and infectious diseases. Preliminary results suggest that by the turn of the century the dream of treating diseases by replacing or supplementing the products of defective genes or introducing novel therapeutic genes will become a reality.