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C Baum

Publications and source records attributed to C Baum.

At least 19 recordsLinked to original sources
PubMed2006

Overcoming promoter competition in packaging cells improves production of self-inactivating retroviral vectors.

Retroviral vectors with self-inactivating (SIN) long-terminal repeats not only increase the autonomy of the internal promoter but may also reduce the risk of insertional upregulation of neighboring alleles. However, gammaretroviral as opposed to lentiviral packaging systems produce suboptimal SIN vector titers, a major limitation for their clinical use. Northern blot data revealed that low SIN titers were associated with abundant transcription of internal rather than full-length transcripts in transfected packaging cells. When using the promoter of Rous sarcoma virus or a tetracycline-inducible promoter to generate full-length transcripts, we obtained a strong enhancement in titer (up to 4 x 10(7) transducing units per ml of unconcentrated supernatant). Dual fluorescence vectors and Northern blots revealed that promoter competition is a rate-limiting step of SIN vector production. SIN vector stocks pseudotyped with RD114 envelope protein had high transduction efficiency in human and non-human primate cells. This study introduces a new generation of efficient gammaretroviral SIN vectors as a platform for further optimizations of retroviral vector performance.

Animals↗
PubMed2006

Towards hematopoietic stem cell-mediated protection against infection with human immunodeficiency virus.

The failure of pharmacological approaches to cure infection with the human immunodeficiency virus (HIV) has renewed the interest in gene-based therapies. Among the various strategies that are currently explored, the blockade of HIV entry into susceptible T cells and macrophages promises to be the most powerful intervention. For long-term protection of both of these lineages, genetic modification of hematopoietic stem cells (HSCs) would be required. Here, we tested whether HSCs and their progeny can be modified to express therapeutic levels of M87o, a gammaretroviral vector encoding an artificial transmembrane molecule that blocks fusion-mediated uptake of HIV. In serial murine bone marrow transplantations, efficient and multilineage expression of M87o was observed for more than 1 year (range 37-75% of mononuclear cells), without signs of toxicity related to the transmembrane molecule. To allow enrichment of M87o-modified HSCs after transplant, we constructed vectors coexpressing the P140K mutant of O(6)-methylguanine-DNA-methyltransferase (MGMT-P140K). This clinically relevant selection marker mediates a survival advantage in HSCs if exposed to combinations of methylguanine-methyltransferase (MGMT) inhibitors and alkylating agents. A bicistronic vector mediated sufficient expression of both M87o and MGMT to confer a selective survival advantage in the presence of HIV and alkylating agents, respectively. These data encourage further investigations in large animal models and clinical trials.

AIDS Vaccines↗
PubMed2006

Woodchuck hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression.

Introduction of the post-transcriptional regulatory element (PRE) of woodchuck hepatitis virus (WHV) into the 3' untranslated region of retroviral and lentiviral gene transfer vectors enhances both titer and transgene expression. Optimal use of the PRE is often necessary to obtain vectors with sufficient performance for therapeutic applications. The enhancing activity of the PRE depends on the precise configuration of its sequence and the context of the vector and cell into which it is introduced. However, data obtained in the context of WHV-associated hepatocellular carcinomas suggests that the PRE might potentially contribute to tumorigenesis, especially if encoding a truncated version of the WHV X protein. Oncogenic side effects of lentiviral vectors containing the PRE have reinforced these safety concerns, although a causal role of the PRE remained unproven. Here, we demonstrate that PRE mutants can be generated that are devoid of X protein open reading frames (ORFs) as well as other ORFs exceeding 25 amino acids, without significant loss of RNA enhancement activity. Furthermore, the X protein promoter could be deleted without compromising the enhancement of vector titers and transgene expression. Such a modified PRE sequence appears useful for future vector design.

Gene Deletion↗
PubMed2004

Treatment simulation approaches for the estimation of the distributions of treatment quality parameters generated by geometrical uncertainties.

Geometric uncertainties arise during treatment planning and treatment and mean that dose-dependent parameters such as EUD are random variables with a patient specific probability distribution. Treatment planning with highly conformal treatment techniques such as intensity modulated radiation therapy requires new evaluation tools which allow us to estimate this influence of geometrical uncertainties on the probable treatment dose for a planned dose distribution. Monte Carlo simulations of treatment courses with recalculation of the dose according to the daily geometric errors are a gold standard for such an evaluation. Distribution histograms which show the relative frequency of a treatment quality parameter in the treatment simulations can be used to evaluate the potential risks and chances of a planned dose distribution. As treatment simulations with dose recalculation are very time consuming for sufficient statistical accuracy, it is proposed to do treatment simulations in the dose parameter space where the result is mainly determined by the systematic and random component of the geometrical uncertainties. Comparison of the parameter space simulation method with the gold standard for prostate cases and a head and neck case shows good agreement as long as the number of fractions is high enough and the influence of tissue inhomogeneities and surface curvature on the dose is small.

Body Burden↗
PubMed2004

Efficient expansion and gene transduction of mouse neural stem/progenitor cells on recombinant fibronectin.

Neural stem/progenitor cells (NSCs) are commonly grown as floating neurospheres in medium containing basic fibroblast growth factor and epidermal growth factor. Under these conditions, about 1% of the cells retain multipotentiality. We developed a protocol based on culture of NSCs in adherence on recombinant fibronectin (rFN) to transduce up to 90% NSCs at a multiplicity of infection of 2 with no need for viral concentration or production of serum-free retroviral supernatants. NSCs grew faster on rFN than as neurospheres on tissue culture plastic and did not lose their stem cell nature or multipotentiality. Furthermore, retroviral-mediated transgene expression was sustained with time in culture and upon differentiation into neurons and astrocytes. These experimental conditions may be utilized to study the function of various genes in NSCs, and to manipulate NSCs for gene and cell therapy of several neurological diseases.

Animals↗
PubMed2004

B-cell-specific transgene expression using a self-inactivating retroviral vector with human CD19 promoter and viral post-transcriptional regulatory element.

Retroviral gene transfer resulting in transgene expression selectively restricted to specific cell lineages would be desirable for many gene therapeutic applications. Such transcriptional targeting of retroviruses can be accomplished by employing eukaryotic control elements in self-inactivating (SIN) retroviral vectors, but use of these vectors is complicated by an accompanying reduction in viral titers. To overcome this restriction and address the influence of the post-transcriptional regulatory element of the Woodchuck hepatitis virus (WPRE) on viral titers and transgene expression, we developed SIN-vectors with and without WPRE. Using the enhancer-promoter of the Spleen Focus Forming virus (SFFV) to direct eGFP expression to multiple hematopoietic lineages, we show that WPRE significantly (>10 x) increased viral titers (>10(6) per ml of unconcentrated supernatant) and transgene expression in NIH3T3 cells in vitro. Gene expression in vivo was significantly lowered in lymphoid cells, but not in myeloid cells when WPRE was present. Furthermore, the use of WPRE in combination with the B-cell lineage-specific CD19 promoter significantly increased viral titers and allowed targeting of transgene expression by SIN-vectors specifically to B cells throughout their development in primary and secondary lymphoid organs.

Animals↗
PubMed2004

Self-inactivating retroviral vectors with improved RNA processing.

Three RNA features have been identified that elevate retroviral transgene expression: an intron in the 5' untranslated region (5'UTR), the absence of aberrant translational start codons and the presence of the post-transcriptional regulatory element (PRE) of the woodchuck hepatitis virus in the 3'UTR. To include such elements into self-inactivating (SIN) vectors with potentially improved safety, we excised the strong retroviral promoter from the U3 region of the 3' long terminal repeat (LTR) and inserted it either downstream or upstream of the retroviral RNA packaging signal (Psi). The latter concept is new and allows the use of an intron in the 5'UTR, taking advantage of retroviral splice sites surrounding Psi. Three LTR and four SIN vectors were compared to address the impact of RNA elements on titer, splice regulation and transgene expression. Although titers of SIN vectors were about 20-fold lower than those of their LTR counterparts, inclusion of the PRE allowed production of more than 10(6) infectious units per ml without further vector optimizations. In comparison with state-of-the-art LTR vectors, the intron-containing SIN vectors showed greatly improved splicing. With regard to transgene expression, the intron-containing SIN vectors largely matched or even exceeded the LTR counterparts in all cell types investigated (embryonic carcinoma cells, fibroblasts, primary T cells and hematopoietic progenitor cells).

Animals↗
PubMed2004

Stem cell clonality and genotoxicity in hematopoietic cells: gene activation side effects should be avoidable.

Two serious adverse events involving activation of the LMO2 oncogene through retrovirus vector insertion in the otherwise extremely successful first gene therapy trial for X-linked severe combined immunodeficieny type 1 (SCID-X1) had initially caused widespread concern in the patient and research communities. Careful consideration 1 year after diagnosis of the second case still finds 12 of the treated patients clearly benefiting from gene therapy (freedom from treatment failure, 80%; survival 100%), a situation that should not portend the end of gene therapy for this disease, and is, in fact encouraging. While current approaches are justified to treat patients with otherwise life-threatening disorders, a broad consensus has developed that systematic basic research is required to further understand the pathophysiology of these serious adverse events and to provide new insights, enabling safer and more effective gene therapy strategies. With the continued success of SCID-X1 gene therapy in the majority of patients treated, it is of even greater importance to understand exactly which vector element or combination of elements predispose to toxicity. An in-depth study of the mechanisms behind the activation of the LMO2 and gammac genes will be highly instructive for the development of safer procedures and vectors. We summarize the central observations, ongoing experimental approaches, new concepts, and developments relevant to understanding, interpreting, and eventually overcoming the real and perceived obstacles posed by insertional mutagenesis due to gene transfer vectors.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Retroviral transfer of MRP1 and gamma-glutamyl cysteine synthetase modulates cell sensitivity to L-buthionine-S,R-sulphoximine (BSO): new rationale for the use of BSO in cancer therapy.

MRP1 (multidrug resistance protein 1) co-exports glutathione (GSH) and drug(s) and exports GSH, glucuronide, and sulphate-conjugated drugs. Human Fly-eco fibrosarcoma cells producing the MRP1-expressing retrovirus SF91MRP (Fly-eco MRP1), as well as 3T3 cells transduced with SF91MRP (3T3/MRP1), presented a decrease in intracellular GSH levels, as measured by two different methods. The enhanced export of GSH caused by the overexpression of MRP1 was partially counterbalanced by an increased rate of GSH synthesis. Fly-eco MRP1 and 3T3/MRP1 were hypersensitive to the GSH-depleting and cytotoxic activities of L-buthionine-S,R-sulphoximine (BSO), compared with their parental counterparts. In addition, the potentiation by BSO of the cytotoxic activity of chlorambucil and doxorubicin in Fly-eco MRP1 cells was greater than in parental Fly-eco cells. Although the turnover time of GSH, i.e. the theoretical time in which the entire GSH pool is resynthesised, was approximately 50% faster in Fly-eco MRP1 cells than in parental cells, this was not sufficient to fully restore the intracellular GSH level. In addition, mrp1 (-/-) mice were resistant to the GSH-depleting activity of intraperitoneally (i.p.) injected BSO, compared with mrp1 (+/+) mice. Co-transfer of the cDNAs for MRP1 and the heavy subunit of gamma-glutamyl cysteine synthetase (GCS) resulted in increased intracellular GSH levels and in high-level resistance to the GSH-depleting and cytotoxic activities of BSO. These data, and in particular the elevated single-agent cytotoxicity of BSO, provide a new rationale for the use of BSO in the treatment of MRP1-overexpressing tumours.

3T3 Cells↗
PubMed2003

Impact of splice-site mutations of the human MDR1 cDNA on its stability and expression following retroviral gene transfer.

The multidrug resistance 1 (MDR1) gene transfer to hematopoietic cells for protection against cytotoxic drugs has received considerable attention in gene therapy. However, ectopic expression of MDR1 from retroviral vectors has been hampered by its genetic instability resulting from cryptic splice sites within the cDNA. We have evaluated the efficiency of retroviral MDR1 vectors with introduced mutations of the MDR1 cryptic splice donor (cSD) located at nucleotide +339 and of the cryptic splice acceptor (cSA) at nucleotide +2319 of the cDNA. Sequence alterations of the cSD reduced the expression of MDR1 P-glycoprotein (P-gp), even when generated as silent mutations. A silent mutation of the cSA reduced the splicing activity shifting the splice acceptor site one base downstream; however, it significantly improved the expression of P-gp. The incidence of wild-type MDR1 pregenome splicing was markedly reduced when vectors were produced in human 293 packaging cells as opposed to murine PG13 and GP+envAm12. We conclude that complete splice correction of MDR1 in retroviral vectors may only be achieved with extensive alterations of the cDNA or neighboring vector sequences and that the splicing is significantly influenced by the choice of the packaging cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

[Multiple superficial basal cell carcinomas following chronic ingestion of sodium hydrogen carbonate containing arsenic].

Basal cell carcinomas (BCC) are caused by various factors such as ultraviolet light, ionizing radiation, possibly human papilloma viruses and chemical agents. Among the latter inorganic trivalent arsenic has long been recognized as an important cause of BCC. A 70-year-old woman developed multiple basal cell carcinomas mainly on the trunk after taking sodium hydrogen carbonate contaminated with arsenic for many years for chronic gastritis.

Aged↗
PubMed2002

High expression of transgenes mediated by hybrid retroviral vectors in hepatocytes: comparison of promoters from murine retroviruses in vitro and in vivo.

To achieve high transgene expression in the liver, we have compared the reporter gene expression among various murine retroviral long terminal repeats (LTRs) or leader sequences in vitro. Transient reporter gene expression assays revealed the highest gene expression by the polycythemic strain of spleen focus-forming virus (SFFVp) LTR in differentiated hepatocellular carcinoma cell lines, HuH-7 and PLC/PRF/5. However, remarkable difference was not observed among LTRs in other types of human liver tumor cell lines. Essentially the same results were obtained by infecting these cells with a series of retroviral vectors. Repression of transgene expression was observed by the leader sequences from Moloney murine leukemia virus (MoMLV), but not from mouse embryonic stem cell virus (MESV). Strengths of the promoters were further compared in murine hepatocytes in vivo. Although the proportions of genomic integration were almost the same, higher gene expression was observed by the FMEV-type vector, which contained the SFFVp LTR and the MESV leader, in comparison with that by the MoMLV-based vector. Thus, FMEV-type vectors may represent a novel type of vectors for human gene therapy with hepatocytes.

Animals↗
PubMed2002

A novel 'sort-suicide' fusion gene vector for T cell manipulation.

Retroviral suicide gene vectors have successfully been used in clinical studies to improve the safety of adoptive immunotherapy with allogeneic T lymphocytes in the treatment of malignant and viral diseases. At the same time these studies have revealed several problems that are yet to be resolved including impaired T cell function due to long ex vivo culture. Here we present new retroviral vectors co-expressing truncated CD34, a gene transfer marker which ensures rapid enrichment of transduced cells using commercially available GMP-approved devices, and a splice-corrected variant of Herpes simplex virus thymidine kinase (scHSVtk) which confers high sensitivity to the prodrug ganciclovir. We show that a retroviral hybrid vector, MP71, based on the myeloproliferative sarcoma virus (MPSV) and the murine embryonic stem cell virus (MESV), encoding a tCD34/scHSVtk fusion protein mediates high expression of the 'sort-suicide' selection marker, thereby allowing for highly efficient purification and selective elimination of transduced cells.

Antigens, CD34↗
PubMed2002

Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell.

Many applications of hematopoietic gene therapy require selection for clones with active transgene expression. However, it was unclear whether the clonal progeny of a retrovirally transduced hematopoietic stem cell would be capable of maintaining transgene expression through serial repopulation and multilineage differentiation. Such investigations require simultaneous analyses of clonality, multilineage activity and transgene copy numbers. Using a mouse model, the present study demonstrates that a single hematopoietic stem cell expressing a marker gene from one or two insertions of a simple retroviral vector actively maintains multilineage transgene expression in the vast majority (80-99%) of bone marrow and peripheral blood cells. Gene expression persisted through serial transplantations for at least 97 weeks post gene transfer and was observed in the lymphoid (B, T and NK cells), myeloid (CD11b(+), Gr-1(+)), erythroid (Ter119(+), mature red blood cells) and megakaryocytic (as indicated by platelets) progeny. Therefore, a single immunoselection for hematopoietic stem cells expressing the transgene in vivo was sufficient to establish a completely chimeric hematopoiesis. These observations imply that the retroviral vectors used in this study contain cis-elements that mediate expression through massive clonal expansion and multilineage differentiation, provided the insertion occurred in genetic loci permissive for expression in hematopoietic stem cells.

Animals↗
PubMed2002

A phase II trial of farnesyl protein transferase inhibitor SCH 66336, given by twice-daily oral administration, in patients with metastatic colorectal cancer refractory to 5-fluorouracil and irinotecan.

BACKGROUND: ras genes encode Ras proteins that are important for signal transduction in cancer cells. Farnesyl protein transferase (FPTase) is an enzyme that is responsible for a critical post-translational modification of Ras. PATIENTS AND METHODS: We report the results of a phase II trial of SCH 66336, an FPTase inhibitor, in patients with metastatic colorectal cancer. This is the first reported experience of an FPTase inhibitor in this disease. All patients were considered refractory to first- and second-line therapy. A total of 21 evaluable patients were treated with a starting dose of 200 mg b.i.d. given continuously. RESULTS: The major side-effects were fatigue (grade 1 in 42%, grade 2 in 42% and grade 3 in 14%), diarrhea (grade 1 in 23% and grade 3 in 42%) and nausea (grade 2 in 16%). Elevations in serum creatinine (grade 2 or 3) were observed in 19% of patients and appeared to be related to dehydration induced by diarrhea. Significant hematological toxicity was not observed (only grade 1 thrombocytopenia in 19% and grade 2 or 3 anemia in 28%). Pharmacological studies revealed adequate mean pre-dose plasma concentrations in this group of patients on day 15 of therapy. No objective responses were observed, although stable disease was seen in three patients for several months. Administration of SCH 66336 was accompanied by gastrointestinal toxicity. CONCLUSIONS: Future development of this compound cannot be recommended as monotherapy in this disease.

Administration, Oral↗
PubMed2002

Gene therapy for sarcoma.

Soft tissue sarcomas are mesenchymal tumors which respond poorly to systemic therapy. Recent studies suggest a higher response rate with an increased doxorubicin dosage. However, this was parallel with a profound hematotoxicity in 75% of patients. Transfer of the human multidrug resistance 1 (MDR1) gene to normal hematopoietic stem cells and transplantation may significantly reduce the hematotoxicity of anthracyclin-based chemotherapy. To test this concept of supportive gene therapy in advance of a clinical study, we transduced mobilized peripheral blood progenitor cells (PBPC) with the retroviral vector SF91m3 containing the human MDR1 gene, transplanted these cells to immune-deficient mice, allowed 6 weeks for engraftment to occur and treated the animals with MDR1-based chemotherapy. In the MDR1-transduced group the human leukocytes were significantly protected from the toxicity of chemotherapy (p < 0.05). While the gene transfer rate was in the range of 10% and thus comparable to recent clinical trials, the gene expression was 59% of transduced cells and thus significantly higher than previously reported for less-advanced vectors. On the other hand, ifosfamide, a drug which has been used successfully for stem cell mobilization, is active in soft tissue sarcoma. Due to these favorable characteristics sarcoma is an attractive target to test the efficacy of MDR1 gene therapy in a clinical setting. Gene therapeutic strategies may also be used to directly target sarcoma cells, e.g. by transfer of suicide genes. We found that adenoassociated virus 2 (AAV-2) vectors efficiently transduce human HS-1 and HT1080 sarcoma cells (>90%) while other tumor cell lines and primary human PBPC were less susceptible. The thymidine kinase (TK) suicide gene was cloned into an AAV-2 vector and a complete kill of TK-transduced HS-1 and HT1080 cells was observed following exposure to aciclovir or ganciclovir (GCV), while >90% of mock-transduced HS-1 cells survived at these dosages. Transplantation of those sarcoma cells to nonobese diabetic (NOD)/LtSz-severe-combined immunodeficient (scid)/scid (NOD/SCID) mice resulted in a survival of >5 months in the AAV-TK-transduced/GCV-treated group, while the mice in the mock-transduced/GCV-treated group had died after 3 weeks. These data show that soft tissue sarcomas are a particularly suitable model system for the development and clinical testing of new gene therapeutic concepts.

Animals↗