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Biomedical subjects

W F Anderson

Publications and source records attributed to W F Anderson.

At least 127 records · Page 7Linked to original sources
PubMed1994

Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences.

A replication-competent retrovirus (RCR) was detected by S+/L- assays in three lots of retroviral vector G1Na that were harvested on consecutive days from a single culture of PA317/G1Na producer cells. Using a number of retrovirus-specific primer pairs, it was shown that this RCR was a novel recombinant created by exchanges between G1Na and helper sequence pPAM3 and was not an existing RCR introduced by cross-contamination. Sequencing of clones of DNA amplified in six independent PCR reactions confirmed that the 3' portion of this RCR was composed of retroviral envelope sequences unique to pPAM3 joined to a 3' long terminal repeat (LTR) unique to G1Na. Comparison of pPAM3 and G1Na sequences at the site corresponding to this junction revealed a short segment of patchy nucleotide identity (8 out of 10 bp), suggesting that these helper and vector sequences were joined by homologous recombination. Generation of RCR by exchanges between helper and vector sequences underscores the necessity of testing by efficient methods all retroviral vectors for the presence of RCR before their use. Production of 171 lots (855 liters) of various retroviral vectors that were free of RCR, including 42 lots of G1Na, however, indicates that the combination of exchanges required to generate an RCR are infrequent in this system.

Animals↗
PubMed1994

Uncoupled expression of Moloney murine leukemia virus envelope polypeptides SU and TM: a functional analysis of the role of TM domains in viral entry.

Moloney murine leukemia virus ecotropic envelope expression plasmids were used to demonstrate that the synthesis of the retroviral envelope SU and TM polypeptides can be uncoupled with retention of biologic activity. By substituting a glycosyl-phosphatidylinositol (GPI) membrane anchor for part or all of the retroviral envelope transmembrane protein and creating several deletion variants of the TM subunit, we have begun to dissect the role of the TM protein in envelope function. We show that a GPI-anchored envelope can be incorporated into virions and binds receptor. We found that the envelope cytoplasmic tail, while not required, influences the efficiency of retroviral transduction at some step after membrane fusion (possibly by interacting with core). The membrane-spanning domain of TM is involved in membrane fusion, and this function is distinct from its role as a membrane anchor. As few as eight amino acids of the putative membrane-spanning domain are sufficient to achieve membrane anchoring of envelope but not to mediate membrane fusion. In addition, though not required, the membrane-spanning domain may have some direct role in the incorporation of envelope into virions. Finally, the extracellular domain of TM, besides containing the putative fusion domain and interacting with SU, may influence the synthesis or stability and the glycosylation of envelope, possibly by affecting oligomerization of the complex and proper intracellular transit.

3T3 Cells↗
PubMed1994

pH-independent murine leukemia virus ecotropic envelope-mediated cell fusion: implications for the role of the R peptide and p12E TM in viral entry.

Murine leukemia virus ecotropic and amphotropic envelope expression vectors were genetically engineered to generate truncations of the p15E TM cytoplasmic tail. The ecotropic construct CEET has the entire cytoplasmic tail of TM deleted, while the CEETR construct has only the R peptide portion of the tail deleted, thereby producing a TM subunit (p12E) that is identical to the one found in mature virions. The analogous amphotropic constructs were called CAET and CAETR. These envelopes, as opposed to their p15E TM counterparts, mediate cell-to-cell fusion at neutral pH in both transformed and nontransformed cell lines. Though the TM cytoplasmic domain is not required, its presence appears to augment such cell-to-cell fusion. This envelope-dependent fusion requires the presence of the viral receptor on the cell surface. Ecotropic virions bearing the p12E TM contain wild-type levels of the envelope complex and have near-normal titers. In contrast, virions which lack the cytoplasmic domain of TM (e.g., CEET) have 10- to 100-fold-lower titers but contain normal amounts of envelope. Both of the corresponding amphotropic virions contain normal amounts of envelope but have 10- to 100-fold-lower titers. Using immunofluorescent detection of envelope to monitor the fate of receptor-bound virions, we found that ecotropic murine leukemia virus envelope disappears from the cell surface while amphotropic envelope persists on the cell surface after virus binding. This pattern of immunofluorescence is consistent with the proposed routes of cell entry for these viruses, i.e., by endocytosis and direct fusion, respectively. In this assay, ecotropic virions bearing the genetically engineered p12E TM also appear to be internalized despite the ability of their envelope to mediate fusion at neutral pH in the same target cells. Our results show that direct fusion at neutral pH is a natural consequence of the surface expression of the mature ecotropic envelope and its receptor. We propose that the processing of the R peptide from the envelope TM (p15E) to yield p12E, at the time of virus budding or within virions, renders the envelope competent to fuse.

Amino Acid Sequence↗
PubMed1993

Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients.

The contribution of infused bone marrow cells to long-term haemopoietic recovery in patients undergoing autologous bone marrow transplantation is unknown. Such information would help to clarify the role of this procedure in cancer therapy and would aid in the development of strategies to reduce the risk of subsequent aplasia. By transferring a neomycin resistance marker gene into the marrow cells of 20 patients before transplantation, we were able to trace the pattern of haemopoietic reconstitution postinfusion. The marker gene was present and expressed in all haemopoietic lineages in vivo in 15 of 18 evaluable patients at 1 month post-transplantation, in 8 of 9 patients at 6 months, and in 5 of 5 at 1 year. The marker has remained detectable for up to 18 months--the duration of our study. Our findings indicate that harvested bone marrow consistently contributes to long-term multilineage recovery of haemopoiesis after autologous marrow transplantation in cancer patients. These results provide a rationale for the continued exploration of more ablative preparative regimens with single or sequential autologous marrow transplants.

Adolescent↗
PubMed1993

Residues that mediate DNA binding of autoimmune antibodies.

Somatic mutations to arginine (R) are a common feature of a subset of J558 H chain genes that code for the majority of high-affinity, anti-dsDNA antibodies in autoimmune MRL/lpr mice. To examine the consequences of such amino acid substitutions on DNA binding, we reverted three somatic mutations of a prototypic anti-dsDNA H chain gene, VH3H9, and assayed the effect of those reversions by expression in a V lambda 1 L chain-only plasmacytoma line. Reversion of R53 eliminated virtually all dsDNA binding and sharply reduced ssDNA affinity. While the complete germ-line revertant of VH3H9 retained a low level of DNA binding, the substitution of R96, a product of N base addition in the third complementarity determining region (CDR3), with glycine (G) was sufficient to abolish measureable DNA specificity. Antibodies with higher affinity for DNA were generated by introducing arginines into VH3H9 at any one of four positions where somatic mutations to arginine had been identified by sequencing other anti-dsDNA J558 H chain genes. All four arginine mutants showed affinity increments consistent with their direct involvement in DNA binding, although one such mutant, K64R, required the simultaneous reversion of an adjacent aspartic acid (D) to the germ-line glycine. Two variants with three nongerm-line arginines showed further improvements in DNA affinity suggesting that their contributions to DNA binding may be additive. Molecular modeling of antibody and mutant F(ab) structures and calculations of their electrostatic potentials were used as an aid in interpreting the results and in predicting the location and size of possible combining sites.

Amino Acid Sequence↗
PubMed1993

Functional and molecular characterization of tumor-infiltrating lymphocytes transduced with tumor necrosis factor-alpha cDNA for the gene therapy of cancer in humans.

TNF is effective in causing the regression of selected murine tumors when administered at high concentrations. Therapeutic levels in humans cannot be obtained systemically, however, because of dose-limiting toxicity. The development of immunotherapy with IL-2 and tumor-infiltrating lymphocytes (TIL), which can accumulate at tumor sites in some patients, and of efficient retroviral techniques for gene transfer into eukaryotic cells has allowed new therapeutic approaches using TNF. We have retrovirally transduced human TIL with the gene for TNF in an attempt to deliver high concentrations of TNF to the tumor site without dose-limiting systemic toxicity. Successful gene insertion was confirmed by Southern hybridization in 16 of 16 transduced and selected TIL cultures from 15 different patients, with an estimated 28 to 93% transduced cells within each culture. Transduced selected TIL cultures produced greater amounts of TNF, compared with nontransduced controls, in 11 of 16 cultures evaluated. However, overall production of TNF was > 30-fold lower, compared with a transduced and highly selected tumor cell line control (MEL-TNF). In addition, steady state levels of vector-derived transcript in nine of 10 transduced selected TIL cultures were < 14% of the amount seen in the MEL-TNF control line. In an attempt to increase TNF production, TIL were transduced with a mutated form of TNF containing the IFN-gamma signal peptide in place of the transmembranous region, to enhance secretion into the endoplasmic reticulum. By using this vector, TNF production increased by an average of fivefold. These studies demonstrate that TIL can be genetically modified to express and secrete a protein for use in targeted cancer therapy but that partial expression blockades exist that prevent maximal cytokine production by introduced genes in TIL.

DNA↗
PubMed1993

Gene-marking to trace origin of relapse after autologous bone-marrow transplantation.

Bone marrow harvested for autologous bone-marrow transplantation may contain residual malignant cells even when it is judged to be in remission. Genetic marking and subsequent detection of these cells in recipients would give useful information about the origin of relapse after transplantation. We transferred the neomycin-resistance gene into bone-marrow cells harvested from children with acute myeloid leukaemia in remission. Two patients have relapsed since reinfusion of the marked cells. In both, the resurgent blast cells contained the neomycin-resistance gene marker; thus, remission marrow can contribute to disease recurrence. This method of tracking malignant cells should enable the development of better marrow purging strategies.

Bone Marrow Cells↗
PubMed1993

In vitro infection of human bone marrow by feline leukemia viruses.

Four subgroup feline leukemia viruses (FeLV) were tested for their ability to infect primary cultures of human bone marrow in vitro. Using three sequential exposures to both concentrated and unconcentrated virus, FeLV infection of human bone marrow was documented by indirect immunofluorescence, ELISA, and polymerase chain reaction (PCR) assays. Based on comparison to known standards, it was estimated that between 5 and 10% of the bulk human marrow culture could be infected by FeLV. Further evidence for FeLV reverse transcription, integration, and expression was obtained using specific PCR assays.

Bone Marrow↗
PubMed1993

Gene therapy for the treatment of brain tumors using intra-tumoral transduction with the thymidine kinase gene and intravenous ganciclovir.

Malignant brain tumors are responsible for significant morbidity and mortality in both pediatric and adult populations. These common tumors present an enormous therapeutic challenge due to their poor outcome despite radical surgery, high dose radiotherapy and chemotherapy. Survival of patients from the time of diagnosis is measured in months and recurrence after treatment is associated with a life expectancy of weeks. In an attempt to improve this grim prognosis of patients with malignant brain tumors (both primary tumors and secondary metastasis from systemic cancer such as melanoma, lung and breast cancer), we have developed a novel approach to the therapy of brain tumors. This approach makes use of recombinant DNA technology to transfer a sensitivity gene into a brain tumor. This is achieved by direct injection of the tumor with a cell line actively producing a retroviral vector carrying a gene conferring drug sensitivity to the tumor. A retroviral vector is a mouse retrovirus genetically engineered to replace its own genes with a new gene. Such vectors are capable of "infecting" mammalian cells and stably incorporate their new genetic material into the genome of the infected host. The producer cell is an NIH 3T3 cell that has been genetically engineered to continually produce retroviral vectors. The new gene is incorporated into the genome of the tumor cells and expresses the protein which is encoded by the new gene. This protein (the herpes simplex virus enzyme thymidine kinase, HS-tk) sensitizes the tumor cells to an antiviral drug (ganciclovir, GCV) which is a natural substrate for HS-tk. The enzymatic process induced by GCV leads to death of the cell expressing the herpes TK activity, i.e., death of the tumor cells. Since the HS-tk enzyme which is normally present in mammalian cells has very low affinity for GCV, systemic toxicity related to this mechanism is not observed. This type of in vivo gene transfer has several unique features. First, these retroviral-vectors will only integrate and express their genes in cells which are actively synthesizing DNA. Therefore, surrounding non-proliferating normal brain tissue should not acquire the HS-tk gene and will remain insensitive to GCV. Second, all of the transduced tumor cells (and retroviral vector producing cells) will be killed by the host immune response and/or GCV treatment eliminating potential concern about insertional mutagenesis giving rise to malignant cells. This is the first clinical attempt to treat malignant tumors in human beings by in-vivo genetic manipulation of the tumor's genome.

Adult↗