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Biomedical subjects

D von Laer

Publications and source records attributed to D von Laer.

At least 19 recordsLinked to original sources
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↗
PubMed2005

Suppression of HIV-1 infection in primary CD4 T cells transduced with a self-inactivating lentiviral vector encoding a membrane expressed gp41-derived fusion inhibitor.

Peptidomimetics of HIV-1 gp41 sequences required for membrane fusion are potent inhibitors of HIV-1 entry. We hypothesize that expression of a membrane-bound gp41-derived fusion inhibitor will confer HIV-1 resistance to primary CD4 T cells. Efficient gene delivery and stable expression of a membrane-bound gp41-derived fusion inhibitor to primary CD4 T cells was accomplished using a self-inactivating lentiviral vector. A potent antiviral effect was observed when transduced CD4 T cells were challenged with a highly virulent CXCR4-tropic strain of HIV-1. Production of soluble p24 in the supernatant was inhibited 100-fold, and cytopathic effects were evident early in non-transduced cells and absent in transduced cells. Expression of the gp41 sequences was not detrimental to CD4 cells as transduced CD4 T cells exhibited a population doubling time that was equivalent to T cells transduced with a control vector. Results from this study support the rationale to use this lentiviral vector targeted at HIV entry as a potential gene therapy for HIV infection.

Anti-HIV Agents↗
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↗
PubMed2001

Low-level expression of functional foamy virus receptor on hematopoietic progenitor cells.

Foamy viruses have several qualities favorable for vector development: they are not known to cause disease; they can transduce stationary cells; and the foamy virus receptor is expressed on a wide variety of cells. Here, we analyzed the level of virus receptor expression on hematopoietic progenitor cells. Foamy virus binding was measured by a flow cytometric assay and was found to be considerably reduced in hematopoietic progenitors cell lines as well as in primary CD34(+) cells when compared to fibroblasts. Retroviral vectors based on murine leukemia virus (MLV) pseudotyped with a foamy virus envelope transduced hematopoietic cell lines with a more than 10-fold lower efficiency than fibroblasts. Moreover, less than 1% of primary CD34(+) hematopoietic progenitor cells were transduced with the foamy virus pseudotypes, while gene transfer efficiencies of 8-40% were achieved using pseudotypes with amphotropic envelope or the G protein of vesicular stomatitis virus. In conclusion, the expression of functional foamy virus receptors on hematopoietic progenitors cells was found to be insufficient to achieve high levels of gene transfer into CD34(+) hematopoietic progenitor cells with cell-free vector supernatants using current transduction protocols.

Animals↗
PubMed2001

Lack of superinfection interference in retroviral vector producer cells.

Vesicular stomatitis virus G protein (VSV-G)-pseudotyped retroviral vectors have become more feasible for clinical gene transfer protocols since stable tetracycline (tet)-regulated packaging cell lines have become available. Here, we analyzed superinfection interference in VSV-G-pseudotyped and classic amphotropic packaging cell lines. No superinfection interference was observed in VSV-G-pseudotyped packaging cell lines. Thus, integrated retroviral vector genomes accumulated during culture. Similar results were obtained with the amphotropic packaging cells, but to a lesser degree. In addition, VSV-G packaging cells were susceptible to infection with vector particles devoid of envelope proteins, which are produced by these cells in high titers when VSV-G expression is suppressed by tetracycline. For both packaging systems, superinfection could be blocked by azidothymidine (AZT). With regard to safety, this study suggests that in clinical protocols amphotropic producer clones should be tested for superinfection interference and VSV-G packaging cells should always be cultured in the presence of AZT.

Cells, Cultured↗
PubMed2001

Influence of multiplicity of infection and protein stability on retroviral vector-mediated gene expression in hematopoietic cells.

Using retroviral vectors encoding enhanced green fluorescent protein (EGFP), we addressed to what extent expression of retroviral transgenes in hematopoietic cells depends on the multiplicity of infection (MOI) and on the half-life of the encoded protein. We show that an elevation of the MOI not only elevates the frequency of transduced cells, but also increases transgene expression levels and reduces interanimal variability in vivo (hematopoietic cells of C57BL/6J mice analyzed 13 weeks after transplantation). This suggests that the MOI has to be carefully controlled and should be adapted as desired for clinical studies when evaluating vector performance in preclinical models. The impact of protein stability is demonstrated by comparing vectors expressing EGFP or a destabilized variant with a C-terminal PEST-sequence, d2EGFP. The loss of expression with d2EGFP was more pronounced in terminally differentiated cells of the peripheral blood (>30 fold) than in progenitor cells (five- to 10-fold), indicating a stronger transcription of the retroviral promoter in progenitor cells and a predominant role of protein inheritance over de novo synthesis of transgenic protein in mature blood cells. This analysis reveals an important and differentiation-dependent contribution of protein half-life to the expression of retroviral vectors in hematopoietic cells, establishes d2EGFP as a more accurate reporter for determination of vector transcription, and also suggests that preclinical data obtained under conditions of high transduction rates or with vectors expressing stable reporter proteins require careful interpretation.

Animals↗
PubMed2001

Recombinant expression of lymphocytic choriomeningitis virus strain WE glycoproteins: a single amino acid makes the difference.

Cytoplasmic vector systems are generally used for expression of lymphocytic choriomeningitis virus (LCMV) proteins. However, we achieved high levels of cell surface glycoproteins using a standard nuclear expression plasmid. Expression was independent of other LCMV proteins but was blocked by a missense mutation within the original LCMV(WE) glycoprotein cDNA.

Amino Acid Substitution↗
PubMed2001

Membrane-anchored peptide inhibits human immunodeficiency virus entry.

Peptides derived from the heptad repeats of human immunodeficiency virus (HIV) gp41 envelope glycoprotein, such as T20, can efficiently inhibit HIV type 1 (HIV-1) entry. In this study, replication of HIV-1 was inhibited more than 100-fold in a T-helper cell line transduced with a retrovirus vector expressing membrane-anchored T20 on the cell surface. Inhibition was independent of coreceptor usage.

Amino Acid Sequence↗
PubMed2000

Amphotropic and VSV-G-pseudotyped retroviral vectors transduce human hematopoietic progenitor cells with similar efficiency.

One restriction of retroviral gene transfer into hematopoietic stem cells is the low level of amphotropic virus receptor. In the present study, we examined whether retroviral vectors pseudotyped with the G-protein of vesicular stomatitis virus (VSV) can overcome this restriction. Human progenitor cells purified by magnetic beads and cell sorting were transduced with an amphotropic or VSV-G-pseudotyped retroviral vector containing the truncated human nerve growth factor receptor as a marker gene. Cells were prestimulated with flt-3 ligand, stem cell factor, and interleukin-3 and transduced on fibronectin. Marker gene expression was analyzed by flow cytometry. Transduction efficiencies of amphotropic and VSV-G-pseudotyped virus for CD34+ cells did not differ significantly. Gene transfer into CD34+CD38- cells, which are enriched in more immature progenitors, was not restricted and transfer efficiencies for this subset were also similar for both pseudotypes. The addition of fibronectin improved gene transfer with the amphotropic vector considerably (5- to 19.3-fold, mean 12.6), while the effect on the VSV-G-pseudotype was far less pronounced (1- to 3.9-fold, mean 2.1, P = 0.04). In conclusion, high levels of gene transfer to human hematopoietic progenitors were achieved with an optimized transduction protocol, and transduction efficiencies could not be improved further by the use of VSV-G-pseudotypes.

ADP-ribosyl Cyclase↗
PubMed1999

Downregulation of c-kit (stem cell factor receptor) in transformed hematopoietic precursor cells by stroma cells.

We show a dramatic downregulation of the stem cell factor (SCF) receptor in different hematopoietic cell lines by murine stroma. Growth of the human erythroid/macrophage progenitor cell line TF-1 is dependent on granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3). However, TF-1 cells clone and proliferate equally well on stroma. Independent stroma-dependent TF-1 clones (TF-1S) were generated on MS-5 stroma. Growth of TF-1S and TF-1 cells on stroma still requires interaction between c-kit (SCF receptor) and its ligand SCF, because antibodies against c-kit inhibit growth to less than 2%. Surprisingly, c-kit receptor expression (RNA and protein) was downregulated by 2 to 3 orders of magnitude in TF-1S and TF-1 cells grown on stroma. This stroma-dependent regulation of the kit receptor in TF-1 was also observed on exposure to kit ligand-negative stroma, thus indicating the need for heterologous receptor ligand interaction. Removal of stroma induced upregulation by 2 to 4 orders of magnitude. Downregulation and upregulation of c-kit expression could also be shown for the megakaryocytic progenitor cell line M-07e and was comparable to that of TF-1, indicating that stroma-dependent regulation of c-kit is a general mechanism. Downregulation may be an economic way to compensate for the increased sensitivity of the c-kit/ligand interaction on stroma. The stroma-dependent c-kit regulation most likely occurs at the transcriptional level, because mechanisms, such as splicing, attenuation, differential promoter usage, or mRNA stability, could be excluded.

Animals↗
PubMed1999

Retroviral vectors pseudotyped with lymphocytic choriomeningitis virus.

Pseudotyping can improve retroviral vector stability and transduction efficiency. Here, we describe a novel pseudotype of murine leukemia virus packaged with lymphocytic choriomeningitis virus (LCMV). This pseudotype was stable during ultracentrifugation and infected several cell lines from different species. Moreover, LCMV glycoproteins were not cell toxic.

Animals↗
PubMed1998

CD4+ T cells migrate into inflamed skin only if they express ligands for E- and P-selectin.

Previous data suggested a role of endothelial selectins in skin homing of lymphocytes. In the current study, we have analyzed the expression and functional role of E-and P-selectin ligands on CD4+ T cells induced in vivo upon skin sensitization, using soluble selectin-Ig chimera and blocking Abs. Only low numbers of CD4+ cells expressing significant levels of E- or P-selectin ligands were present in s.c. lymph nodes of untreated mice (0.5-1.5% and 2-4%, respectively). Induction of a delayed-type hypersensitivity reaction increased the percentage of E-selectin-binding CD4+ cells in the draining lymph nodes up to 6 to 9% and that of P-selectin-binding cells up to 14%. The majority of E- and P-selectin-binding cells displayed an activated phenotype as judged by the increase in IL-2R, CD71, or cell size. The populations of E- and P-selectin-binding cells were largely overlapping; all E-selectin-binding cells also bound to P-selectin, whereas only a subfraction of P-selectin-binding cells reacted with E-selectin. Both E- and P-selectin-binding CD4+ cells, isolated by FACS, efficiently migrated into inflamed, but not normal skin, whereas P- or E-selectin ligand-negative CD4+ T cells did not. Abs against one of the two endothelial selectins partially inhibited the entry of isolated, ligand-positive cells, whereas a combination of Abs against both selectins almost completely abrogated skin homing. These data indicate that the expression of functional ligands for E- and for P-selectin is essential for homing of CD4+ T cells into the inflamed skin.

Animals↗
PubMed1998

Intragenic variability of human cytomegalovirus glycoprotein B in clinical strains.

Human cytomegalovirus (HCMV) strains can be classified into four glycoprotein B (gB) genotypes, and there has been evidence of differences in viral virulence. In this study, intragenic variability of HCMV gB strains was analyzed. The gB gene was amplified by nested polymerase chain reaction using samples from immunosuppressed patients. The genotype of fragments corresponding to the cleavage site of gB was determined by restriction fragment analysis; fragments corresponding to the N- and C-termini (gBn and gBc) were sequenced and compared with published sequences. At the cleavage site, the four known genotypes were found. Typing revealed four major genotypes at the N-terminus and two at the C-terminus. In 22 of 44 strains, the gB type determined at the cleavage site was different from the gBn or gBc type (or either), indicating that intragenic variability within the gB gene occurs frequently.

Base Sequence↗
PubMed1998

Entry of amphotropic and 10A1 pseudotyped murine retroviruses is restricted in hematopoietic stem cell lines.

Although transduction with amphotropic murine leukemia virus (MLV) vectors has been optimized successfully for hematopoietic differentiated progenitors, gene transfer to early hematopoietic cells (stem cells) is still highly restricted. A similar restriction to gene transfer was observed in the mouse stem cell line FDC-Pmix compared with transfer in the more mature myeloid precursor cell line FDC-P1 and the human erythroleukemia cell line K562. Gene transfer was not improved when the vector was pseudotyped with gp70SU of the 10A1 strain of MLV, which uses the receptor of the gibbon ape leukemia virus (Pit1), in addition to the amphotropic receptor (Pit2). Although 10A1 and amphotropic gp70SU bound to FDC-P1, K562, and fibroblasts, no binding to FDC-Pmix cells was detected. This indicates that FDC-Pmix cells lack functional Pit2 and Pit1 receptors. Pseudotyping with the vesicular stomatitis virus G protein improved transduction efficiency in FDC-Pmix stem cells by 2 orders of magnitude, to fibroblast levels, confirming a block to retroviral infection at the receptor level.

Animals↗
PubMed1998

Lack of functional Pit-1 and Pit-2 expression on hematopoietic stem cell lines.

Hematopoietic stem cells (HSC) are an important target for retroviral gene transfer. However, transduction efficiency in these HSC is extremely low compared to fibroblasts or more mature hematopoietic cells. This infection block was analyzed in the HSC line FDC-Pmix. The infection frequency with the amphotropic murine leukemia virus (MLV-A) is more than 100-fold lower in FDC-Pmix cells as compared to fibroblasts. Pseudotyping with the env of the 10A1 strain (MLV-10A1), which uses both the amphotropic receptor (Pit-2) and the receptor for gibbon ape leukemia virus (Pit-1), did not improve the infection efficiency. Vectors pseudotyped with VSV G protein were found to overcome the infection block in FDC-Pmix, confirming that the block is at the level of virus binding and possibly penetration. Accordingly, we could not detect virus binding of MLV-A or MLV-10A1 to FDC-Pmix cell lines. Northern blot analysis was performed to detect whether the defect is at the level of transcription. Surprisingly, similar levels of Pit-2 receptor transcripts were detected in all cell types. The overexpression of rat Pit-2 DNA in CHO but not in FDC-Pmix cells improved amphotropic infection frequency after introducing rat Pit-2 DNA into the cells. Taken together these results show that the inefficient infection of FDC-Pmix is due to a lack of functional receptors. Either the receptor protein is incorrectly processed in these cells or a cofactor is missing in FDC-Pmix cells that is necessary for efficient binding and/or penetration.

Animals↗
PubMed1998

Glycoprotein B genotype correlates with cell tropism in vivo of human cytomegalovirus infection.

Human cytomegalovirus (HCMV) strains can be classified into four genotypes of the glycoprotein B (gB). In a previous study, the gB genotype 1 was found more frequently in bone marrow transplant recipients with nonfatal HCMV infection than in patients who died from HCMV disease [Fries et al. (1994): Journal of Infectious Diseases 169:769-774]. The distribution and cell tropism of different gB types in vivo were investigated. The gB type of HCMV was determined in blood or urine specimen from 76 organ and 47 bone marrow transplant recipients using PCR and restriction fragment length polymorphism (RFLP). The leukocyte populations (polymorphonuclear leukocytes, monocytes, T lymphocytes, non-T lymphocytes) of 20 viremic patients were purified by a fluorescence-activated cell sorter (FACS) and examined for HCMV infection by PCR. Sequence analysis of four randomly selected strains showed that gB types were similar to published sequences and no atypical gB types were found. Within the compartments blood and urine, the gB types were almost equally distributed, whereas the gB type 1, in contrast to gB types 2 and 3, did not infect T lymphocytes in vivo. These data show that the gB type correlates with viral tropism in vivo and thus provides further evidence that the gB variation may indeed influence the virulence of HCMV.

Bone Marrow Transplantation↗