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Biomedical subjects

A D Miller

Publications and source records attributed to A D Miller.

At least 217 records · Page 12Linked to original sources
PubMed1988

Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans.

Purine nucleoside phosphorylase (PNP; purine-nucleoside orthophosphate ribosyltransferase, EC 2.4.2.1) deficiency is an inherited disorder associated with a severe immune defect that is fatal. Enzyme replacement therapy is an attractive approach to treatment of this disease. To this aim we constructed retroviral vectors containing a human PNP cDNA and a selectable gene encoding neomycin phosphotransferase. PNP expression was controlled by either the early promoter from simian virus 40, the immediate early promoter from human cytomegalovirus, or the retroviral promoter. Cultured skin fibroblasts from two unrelated PNP-deficient patients that were infected with these vectors expressed mean PNP activities of 0.03, 0.74, and 5.9 mumol/hr per mg of protein, respectively. The latter infectants had PNP activities eight times the level of 0.74 mumol/hr per mg of protein observed in normal skin fibroblasts, enabling rapid metabolism of exogenous deoxyguanosine, the cytotoxic metabolite that accumulates in the plasma of PNP-deficient patients. These experiments indicate that viral long terminal repeat was the strongest promoter for expression of PNP and suggest the potential of human skin fibroblasts as vehicles for therapeutic gene expression.

Cells, Cultured↗
PubMed1988

Identification of a signal in a murine retrovirus that is sufficient for packaging of nonretroviral RNA into virions.

A region near the 5' end of Moloney murine leukemia virus (MoMLV) is required for packaging of viral RNA into virions. Retroviral vectors based on MoMLV have been constructed that are also efficiently packaged into virions despite removal of most of the interior region of the parental virus. To further localize sequences which are sufficient for packaging, we inserted various fragments from an MoMLV-based retroviral vector into a nonretroviral transcription unit, transfected these constructs into retrovirus-packaging cells, and measured packaging of RNA transcribed from these constructs into virions. Transcripts from some of these constructs were packaged at least as well as those from the parental vector or MoMLV itself. Sequences extending into the gag region, but not the long terminal repeat or tRNA-binding sequences, were required for efficient RNA packaging. RNAs transcribed from constructs which did not contain an insert, or in which the orientation of the insert was reversed, were not packaged at detectable levels. These studies define sequences which are necessary and sufficient for encapsidation of murine leukemia virus RNA into virions.

Animals↗
PubMed1988

Design of retrovirus vectors for transfer and expression of the human beta-globin gene.

Regulated expression of the human beta-globin gene has been demonstrated in cultured murine erythroleukemia cells and in mice after retrovirus-mediated gene transfer. However, the low titer of recombinant viruses described to date results in relatively inefficient gene transfer, which limits their usefulness for animal studies and for potential gene therapy in humans for diseases involving defective beta-globin genes. We found regions that interfered with virus production within intron 2 of the beta-globin gene and on both sides of the gene. The flanking regions could be removed, but intron 2 was required for beta-globin expression. Inclusion of beta-globin introns necessitates an antisense orientation of the gene within the retrovirus vector. However, we found no effect of the antisense beta-globin transcription on virus production. A region downstream of the beta-globin gene that stimulates expression of the gene in transgenic mice was included in the viruses without detrimental effects on virus titer. Virus titers of over 10(6) CFU/ml were obtained with the final vector design, which retained the ability to direct regulated expression of human beta-globin in murine erythroleukemia cells. The vector also allowed transfer and expression of the human beta-globin gene in hematopoietic cells (CFU-S cells) in mice.

Animals↗
PubMed1988

Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells.

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.

Animals↗
PubMed1988

Canine model for gene therapy: inefficient gene expression in dogs reconstituted with autologous marrow infected with retroviral vectors.

Successful retroviral gene transfer into murine hematopoietic stem cells indicates the potential for somatic gene therapy in the treatment of certain human hereditary diseases. We developed a canine model to test the applicability of these techniques to a preclinical model of human marrow transplantation. Previously we reported that canine CFU-GM could be infected with retroviral vectors carrying either the gene for a mutant dihydrofolate reductase (DHFR) or neomycin phosphotransferase (NEO). This study reports six lethally irradiated dogs transplanted with autologous marrow cocultivated with retroviral vector-producing cells. This procedure conferred drug resistance to 3% to 13% of the CFU-GM. Three dogs infected with either the NEO or DHFR virus engrafted, but we detected no drug-resistant CFU-GM. Three dogs were given marrow infected with a DHFR virus and received methotrexate (MTX) as in vivo selection; all three had evidence of engraftment. In the surviving dog, we detected 0.03% to 0.1% MTX-resistant CFU-GM at 3 to 5 weeks posttransplant during in vivo selection. These results indicate that we can reconstitute lethally irradiated dogs with autologous marrow exposed to retroviral vectors and suggest that gene transfer into hematopoietic cells is feasible on a large scale. However, the low-level transient gene expression indicates that considerable obstacles remain before human gene therapy can be considered.

Animals↗
PubMed1987

Localization of motoneurons innervating individual abdominal muscles of the cat.

The motor pools of the individual abdominal muscles of the cat were localized in studies by using either intramuscular injections of horseradish peroxidase (HRP) to retrogradely label abdominal motoneurons or electrical microstimulation of the ventral horn at different segmental levels to produce localized twitches of the abdominal muscles. The segmental distribution of each motor pool was as follows: rectus abdominis, T4-L3; external oblique, T6-L3; transverse abdominis, T9-L3; and internal oblique, T13-L3. The differences in the rostral extents of the individual motor pools reflect the greater rostral extents of the different muscles (rectus abdominis greater than external oblique greater than transverse abdominis greater than internal oblique). Labeled motoneurons were also found at other segmental levels; however, it was concluded that this labeling occurred because of spread of HRP from the injected muscle since localized abdominal muscle twitches could not be produced by electrical stimulation in these regions. In addition, control experiments showed that HRP can spread from the injected muscle and identified the sources of some of this spurious labeling. Motoneurons labeled after injections into the four abdominal muscles overlapped extensively on transverse sections of the spinal cord; however, rectus abdominis motoneurons were located more medially than the others from about T11 to L3. Soma diameters ranged between 12 and 41 microns (average 24-26 microns per cat). In summary, this study has provided a systematic description of the innervation of the individual abdominal muscles of the cat.

Abdominal Muscles↗
PubMed1987

Epitope insertion into the human hypoxanthine phosphoribosyltransferase protein and detection of the mutant protein by an anti-peptide antibody.

The translational stop codon TAA of the human hypoxanthine phosphoribosyltransferase (HPRT) cDNA has been changed to GAA by site-specific mutagenesis. This modification extends the open reading frame to a downstream stop codon and results in the addition of a unique negatively charged hexapeptide to the C terminus of human HPRT protein. The mutated cDNA was transferred into HPRT-deficient rodent cells by retroviral vector infection, and the expressed enzyme was found to be fully active. An antibody against a synthetic octapeptide corresponding to the mutated HPRT C terminus precipitated the HPRT protein specifically from cells infected with the mutant virus and not infected with the wild-type HPRT virus. The technique of inserting a novel epitope into a protein by site-directed mutagenesis should be generally applicable in studies of the regulation of gene expression in vitro and in vivo.

Epitopes↗
PubMed1987

Efficient retrovirus-mediated transfer and expression of a human adenosine deaminase gene in diploid skin fibroblasts from an adenosine deaminase-deficient human.

Skin fibroblasts might be considered suitable recipients for therapeutic genes to cure several human genetic diseases; however, these cells are resistant to gene transfer by most methods. We have studied the ability of retroviral vectors to transfer genes into normal human diploid skin fibroblasts. Retroviruses carrying genes for neomycin or hygromycin B resistance conferred drug resistance to greater than 50% of the human fibroblasts after a single exposure to virus-containing medium. This represents at least a 500-fold increase in efficiency over other methods. Transfer was achieved in the absence of helper virus by using amphotropic retrovirus-packaging cells. A retrovirus vector containing a human adenosine deaminase (ADA) cDNA was constructed and used to infect ADA-fibroblasts from a patient with ADA deficiency. The infected cells produced 12-fold more ADA enzyme than fibroblasts from normal individuals and were able to rapidly metabolize exogenous deoxyadenosine and adenosine, metabolites that accumulate in plasma in ADA-deficient patients and are responsible for the severe combined immunodeficiency in these patients. These experiments indicate the potential of retrovirus-mediated gene transfer into human fibroblasts for gene therapy.

Adenosine↗
PubMed1987

Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region.

Replication-competent retroviruses can be modified to carry nonviral genes. Such gene transfer vectors help define regions of the retroviral genome that are required in cis for retroviral replication. Moloney murine leukemia virus has been used extensively in vector construction, and all of the internal protein-encoding regions can be removed and replaced with other genes while still allowing production of virions containing and transmitting the altered retroviral genome. However, inclusion of a portion of the gag region from Moloney murine leukemia virus markedly increases the titer of virus derived from these vectors. We determined that this effect was due to more efficient packaging of the vector RNA into particles and did not depend on protein synthesis from the gag region. We conclude that the retrovirus packaging signal extends into the gag region. We have found that retroviral vectors containing the complete packaging signal allow more efficient gene transfer into a variety of cell types. In addition, these results may help explain why many oncogenic retroviruses have retained gag sequences and often express transforming proteins that are gag-onc hybrids.

Gene Products, gag↗
PubMed1987

Human purine nucleoside phosphorylase and adenosine deaminase: gene transfer into cultured cells and murine hematopoietic stem cells by using recombinant amphotropic retroviruses.

Cell lines were established which produced high titers (approximately 10(6) infectious units per ml) of amphotropic, replication-defective recombinant retroviruses which transduced sequences encoding either human purine nucleoside phosphorylase (PNP) or adenosine deaminase (ADA). These viruses also contained a human hypoxanthine phosphoribosyltransferase gene as a selectable marker and a mouse metallothionein promoter (MMP) sequence just upstream from the PNP or ADA genes. Virus structure was maintained through the replication cycle if a short (216-base pair) MMP sequence was used. However, the use of a longer (1,834-base pair) MMP sequence resulted in the deletion of a significant portion of the recombinant virus genome, including the transcriptional regulatory elements of the MMP sequence. Northern analysis indicated a predominance of genome length transcripts in cells infected with deleted virus. The demonstration of substantial human PNP or ADA activity in virus-infected mouse fibroblasts by isozyme analysis suggested that active gene product was translated from either spliced or bicistronic message. The deleted ADA and PNP viruses were introduced into mouse hematopoietic stem cells by cocultivating freshly explanted bone marrow with virus producer cells. The infected marrow cells were injected into irradiated, syngeneic recipient mice, and the presence of integrated ADA or PNP proviral sequences was demonstrated in the DNA of spleen colonies by Southern analysis. Failure of these integrated proviral sequences to express active, human isozyme in spleen colony tissue indicated the existence of some regulatory constraint not active in cultured mouse cells.

Adenosine Deaminase↗
PubMed1987

Control of abdominal and expiratory intercostal muscle activity during vomiting: role of ventral respiratory group expiratory neurons.

The role of ventral respiratory group (VRG) expiratory (E) neurons in the control of abdominal and internal intercostal (expiratory) muscle activity during vomiting was examined in decerebrate cats by recording from these neurons during fictive vomiting in paralyzed animals and comparing abdominal muscle activity during vomiting before and after sectioning the axons of these descending neurons. Fictive vomiting was defined by a series of bursts of coactivation of abdominal and phrenic nerves elicited by either subdiaphragmatic vagus nerve stimulation or emetic drugs. Such coordinated activity would be expected to produce vomiting if the animals were not paralyzed. Data were recorded from 27 VRG E neurons that were antidromically activated from the lower thoracic (T13) or lumbar spinal cord. During fictive vomiting, almost two-thirds of these neurons (17/27) were mainly active in between periods of abdominal and phrenic nerve coactivation, when the internal intercostal motoneurons are known to be active. This group of neurons was termed INT neurons. INT neurons were subdivided according to whether they were active between every burst of phrenic and abdominal nerve coactivation (INTa neurons, n = 10) or only between some bursts (INTb neurons, n = 7). Another one-third of the VRG E neurons had normal or increased levels of activity when the abdominal nerves were active during fictive vomiting (ABD neurons). The one remaining neuron was mainly silent throughout fictive vomiting. ABD neurons were indistinguishable from INT neurons on the basis of their location in the VRG, type of firing pattern (ramp versus step ramp), conduction velocity, or extent of projection in the lumbar cord. However, INTa neurons had a significantly higher discharge rate during respiration than either ABD or INTb neurons. Abdominal muscle EMG and nerve activity were recorded from six unparalyzed cats before and after cutting the axons of VRG E neurons as they cross the midline between C1 and the obex. The lesions abolished or almost eliminated expiratory modulation of abdominal muscle activity. In contrast, the abdominal muscles were always active during vomiting; however, the amplitude of postlesion abdominal activity varied from approximately 70-100% of prelesion values in three cats to 60-70% of normal in a fourth animal to only approximately 20% of prelesion values in two other cats.(ABSTRACT TRUNCATED AT 400 WORDS)

Abdominal Muscles↗
PubMed1987

Possible role of brain stem respiratory neurons in mediating vomiting during space motion sickness.

Vomiting is produced primarily by the coordinated action of the respiratory muscles. The object of this study was to determine if brain stem expiratory neurons control abdominal muscle activity during vomiting. We recorded the activity of 27 ventral respiratory group (VRG) expiratory neurons, which are known to be of primary importance for control of abdominal muscle activity during respiration. During fictive vomiting produced by emetic stimulants in decerebrate, paralyzed cats, only one-third of these neurons had the appropriate firing pattern to contribute to abdominal muscle activity during vomiting. In a second series of experiments, we recorded abdominal muscle activity in five cats before and after cutting the axons of VRG expiratory neurons. Abdominal activity remained at 60-100% of prelesion values in three animals. Thus, abdominal muscle activity during vomiting must be controlled not only by some brain stem expiratory neurons but also by other input(s).

Abdominal Muscles↗
PubMed1987

Towards gene therapy for hemophilia B.

Hemophilia B is an X-chromosome-linked bleeding disorder resulting from lack of clotting factor IX activity and affects about 1 in 30,000 males. Current therapy involves injection of crude factor IX prepared from pooled human plasma. Treatment is complicated by viral contaminants in factor IX preparations, such as non A-non B hepatitis and the AIDS virus, and by the practical difficulties of chronic injections. An alternative therapy might include the insertion of a factor IX expression vector into the somatic cells of affected individuals to allow continued production of factor IX. Toward this end, we have constructed a retrovirus vector for transfer and expression of factor IX. Despite the fact that factor IX is normally synthesized in hepatocytes and requires extensive post-translational modification for activity, we have shown that fully active factor IX can be made by human skin-derived fibroblasts. These results open the way to testing the use of skin grafts for gene therapy of hemophilia B.

Biological Assay↗
PubMed1986

Innervation of periesophageal region of cat's diaphragm: implication for studies of control of vomiting.

The extent of the region of the diaphragm around the esophagus that displays greatly reduced activity during the expulsive phase of vomiting was determined from electromyographic studies in cats to be about 0.75-1.0 cm from the esophagus. Horseradish peroxidase injected into this region retrogradely labeled motoneurons throughout most of the rostral-caudal extent of the phrenic nucleus, with the exception of caudal C6 and rostral C7. This widespread intermingling of motoneurons that innervate the region of reduced activity with other phrenic motoneurons creates a difficulty for needed follow-up studies of diaphragmatic control during vomiting.

Abdominal Muscles↗
PubMed1986

Factors involved in production of helper virus-free retrovirus vectors.

Retrovirus vectors allow efficient transfer of genetic material into cells. We describe an improved method for making cell lines which secrete broad host range retrovirus vectors in the absence of helper virus. This method was used to make virus-producing cell lines from several retrovirus vector constructions that encode dominant selectable markers. Virus titers from such lines exceeded 10(6) colony-forming units per milliliter of medium exposed to the cells. Cell lines that secreted certain vectors remained free of helper virus, while cell lines made using other vectors always secreted helper virus. Secretion of helper virus apparently depended on recombination between vector and the retrovirus packaging system, and factors involved in this event were investigated.

Animals↗
PubMed1986

Retroviral transfer of genes into canine hemopoietic progenitor cells in culture: a model for human gene therapy.

Amphotropic retroviral vectors containing either a mutant dihydrofolate reductase gene (DHFR) or the bacterial neomycin phosphotransferase gene (neo) were used to infect canine hemopoietic cells. We report successful transfer and expression of the DHFR and neo genes in canine hemopoietic progenitor cells (colony-forming units, granulocyte/macrophage) as measured by the ability of the viruses to confer resistance to either methotrexate or the aminoglycoside G418, respectively. Transfer was achieved in the absence of helper virus by using retrovirus packaging cell lines. Successful transfer of these genes into canine hemopoietic progenitor cells in vitro indicates the feasibility of gene transfer into canine marrow for autologous reconstitution. Studies of transfer of new genetic information into a large, outbred animal such as the dog will provide a preclinical model for future gene therapy in humans.

Animals↗