Transfer and expression of the human multiple drug resistance gene as potential human gene therapy

The human multiple drug resistance (MDR) gene has been used as a model for human gene transfer which could lead to human gene therapy. MDR is a transmembrane protein which pumps a number of toxic substances out of cells including several drugs used in cancer chemotherapy. Normal bone marrow cells express low levels of MDR and are particularly sensitive to the toxic effects of these drugs. There are two general applications of MDR gene therapy: (1) to provide drug-resistance to the marrow of cancer patients receiving chemotherapy, and (2) as a selectable marker which when co-transferred with a non-selectable gene such as the human beta globin gene can be used to enrich the marrow for cells containing both genes. We demonstrate efficient transfer and expression of the human MDR gene in a retroviral vector into live mice and human marrow cells including CD34⁺ cells isolated from marrow and containing the bulk of human hematopoietic progenitors. MDR gene transduction corrects the sensitivity of CD34⁺ cells to taxol, an MDR drug substrate, and enriches the marrow for MDR-transduced cells. The MDR gene-containing retroviral supernatant used has been shown to be safe and free of replication-competent retrovirus. Because of the safety of the MDR retroviral supernatant, and efficient gene transfer into mouse and human marrow cells, a phase 1 clinical protocol for MDR gene transfer into cancer patients has been approved to evaluate MDR gene transfer and expression in human marrow.     

Localization of the human alpha-globin structural gene to chromosome 16 in somatic cell hybrids by molecular hybridization assay

We have used 16 human × mouse somatic cell hybrids containing a variable number of human chromosomes to demonstrate that the human α-globin gene is on chromosome 16. Globin gene sequences were detected by annealing purified human α-globin complementary DNA to DNA extracted from hybrid cells. Human and mouse chromosomes were distinguished by Hoechst fluorescent centromeric banding, and the individual human chromosomes were identified in the same spreads by Giemsa trypsin banding. Isozyme markers for 17 different human chromosomes were also tested in the 16 clones which have been characterized. The absence of chromosomal translocation in all hybrid clones strongly positive for the α-globin gene was established by differential staining of mouse and human chromosomes with Giemsa 11 staining. The presence of human chromosomes in hybrid cell clones which were devoid of human α-globin genes served to exclude all human chromosomes except 6, 9, 14 and 16. Among the clones negative for human α-globin sequences, one contained chromosome 2 (JFA 14a 5), three contained chromosome 4 (AHA 16E, AHA 3D and WAV R4D) and two contained chromosome 5 (AHA 16E and JFA14a 13 5) in >10% of metaphase spreads. These data excluded human chromosomes 2, 4 and 5 which had been suggested by other investigators to contain human globin genes. Only chromosome 16 was present in each one of the three hybrid cell clones found to be strongly positive for the human α-globin gene. Two clones (WAIV A and WAV) positive for the human α-globin gene and chromosome 16 were counter-selected in medium which kills cells retaining chromosome 16. In each case, the resulting hybrid populations lacked both human chromosome 16 and the α-globin gene. These studies establish the localization of the human α-globin gene to chromosome 16 and represent the first assignment of a nonexpressed unique gene by direct detection of its DNA sequences in somatic cell hybrids.     

The isolation,characterisation, and chromosomal assignment of the gene for human 3-hydroxy-3-methylglutaryl coenzyme A reductase, (HMG-CoA reductase)

Summary We have used a cDNA clone for Chinese hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase to isolate a genomic recombinant for human HMG-CoA reductase. The identity of the gene was confirmed by partial sequence analysis. Several unique fragments that will be useful for restriction fragment length polymorphism (RFLP) studies were identified. In situ hybridisation of a 2.6kb unique fragment of the gene to human metaphase chromosomes localised the human HMGCoA reductase gene to human chromosome 5q12.     

Intron 1 sequences are required for pancreatic expression of the human proglucagon gene

The mammalian proglucagon gene is expressed in pancreatic islet A-cells, intestinal L-cells, and select neurons of the brain, where posttranslational processing results in the liberation of a unique profile of peptides. Despite the importance of proglucagon-derived peptides in human biology, little is known about the regulation of the human gene, as the rat gene has been the preferred model for understanding the regulation of proglucagon gene expression. Previously, we have shown that although the immediate promoter region of the rat proglucagon gene is sufficient for expression in pancreatic islet cells, the homologous human proglucagon promoter sequences are not sufficient. We have now used a comparative genomic approach to identify noncoding sequences near the human proglucagon gene that are conserved among mammals, and thus potentially are regulatory sequences. Our alignments identified three evolutionarily conserved noncoding regions (ECR), one is the immediate promoter region (ECR1), the second is about 5 kb 5' to the mRNA start site (ECR2), and the third is near the 3' end of the first intron (ECR3). Our in vitro transient transfection assays with reporter gene constructs that include the human ECR3 support expression in rodent islet cell lines. Complementary studies with transgenic mice possessing a reporter gene regulated by a human proglucagon gene promoter-intron 1 (including ECR3) sequences express the reporter gene in the pancreas, as well as the intestine and selected neurons. These studies suggest that conserved sequences within intron 1 of the human proglucagon gene are important for expression in the pancreas.     

An evolution revolution provides further revelation

The extent of copy-number variation (CNV) in the human genome has been appreciated only recently. Nevertheless, for almost four decades, gene duplication has been a prevailing hypothesis for evolutionary change. Recently, gene CNV spanning 60 million years of human and primate evolution has been determined enabling lineage-specific gene CNV to be identified. Primate lineage-specific gene CNV studies reveal that almost one third of all human genes exhibit a copy-number change in one or more primate species. Intriguingly, human lineage-specific gene amplification can be correlated to the emergence of human-specific traits such as cognition and endurance running.     

Adeno-associated virus type 2-mediated transduction of murine hematopoietic cells with long-term repopulating ability and sustained expression of a human globin gene in vivo.

Adeno-associated virus type 2 (AAV), a nonpathogenic human parvovirus, is gaining attention as a vector for potential use in human gene therapy. We and others have described AAV-mediated beta-globin gene transfer and expression in established human and murine erythroleukemia cell lines in vitro. However, successful AAV-mediated globin gene transduction of hematopoietic stem cells and long-term expression in vivo in progeny cells have not been documented. We report here that infection of murine hematopoietic bone marrow cells ex vivo with a recombinant AAV vector containing the genomic copy of a normal human globin gene followed by transplantation of these cells into lethally irradiated congenic mice resulted in efficient gene transfer into hematopoietic cells with long-term repopulating ability as detected by the presence of the human globin gene sequences in bone marrow and spleen in primary recipient mice for at least 6 months. Long-term expression of the human globin gene was also detected in bone marrow, but not in spleen, in primary recipient mice. Furthermore, in secondary-transplant experiments, we were also able to document the presence as well as expression of the transduced human globin gene in mouse bone marrow for up to 3 months. These results provide further support for potential use of the AAV-based vector system in gene therapy of human hemoglobinopathies in general and sickle-cell anemia and beta-thalassemia in particular.     

Expression profile of human immune-responsive gene 1 and generation and characterization of polyclonal antiserum

Murine immune-responsive gene 1 (IRG1) plays significant roles in embryonic implantation and neurodegeneration. The expression pattern of the human IRG1 gene, however, has not yet been established, and the predicted gene sequence has been revised several times according to computed expressed sequence tags (ESTs). To determine the human IRG1 gene expression profile, human fetal tissue samples, peripheral blood mononuclear cells (PBMCs) from normal healthy subjects, and the human leukemia cell lines THP-1 and K-562 challenged with lipopolysaccharide (LPS) were subjected to RT-PCR using degenerate primers. The results indicated that the IRG1 gene is differentially expressed in human fetal PBMCs and LPS-stimulated adult PBMCs. The amplified gene fragment was cloned into the pET32a(+) vector and fusion-expressed with a His-tag in a prokaryotic system. After affinity chromatography, human IRG1h fusion proteins were isolated by SDS-PAGE and identified by mass spectrometric analysis for use as an immunogen to immunize rabbits. The titer and specificity of the purified rabbit antiserum were sufficient to measure human IRG1 gene expression in various tissues and cultures. This purified polyclonal antiserum will allow us to initiate studies to elucidate the biological roles of the human IRG1 gene.     

Sequence of the cDNA and gene for angiogenin, a human angiogenesis factor

Human cDNAs coding for angiogenin, a human tumor derived angiogenesis factor, were isolated from a cDNA library prepared from human liver poly(A) mRNA employing a synthetic oligonucleotide as a hybridization probe. The largest cDNA insert (697 base pairs) contained a short 5'-noncoding sequence followed by a sequence coding for a signal peptide of 24 (or 22) amino acids, 369 nucleotides coding for the mature protein of 123 amino acids, a stop codon, a 3'-noncoding sequence of 175 nucleotides, and a poly(A) tail. The gene coding for human angiogenin was then isolated from a genomic lambda Charon 4A bacteriophage library employing the cDNA as a probe. The nucleotide sequence of the gene and the adjacent 5'- and 3'-flanking regions (4688 base pairs) was then determined. The coding and 3'-noncoding regions of the gene for human angiogenin were found to be free of introns, and the DNA sequence for the gene agreed well with that of the cDNA. The gene contained a potential TATA box in the 5' end in addition to two Alu repetitive sequences immediately flanking the 5' and 3' ends of the gene. The third Alu sequence was also found about 500 nucleotides downstream from the Alu sequence at the 3' end of the gene. The amino acid sequence of human angiogenin as predicted from the gene sequence was in complete agreement with that determined by amino acid sequence analysis. It is about 35% homologous with human pancreatic ribonuclease, and the amino acid residues that are essential for the activity of ribonuclease are also conserved in angiogenin. This provocative finding is thought to have important physiological implications.     

A second human calcitonin/CGRP gene

The calcitonin (CT) gene is alternatively expressed in a tissue-specific fashion producing either the calcium regulatory hormone CT in the thyroid or the neuropeptide calcitonin gene related peptide (CGRP) in the brain. In medullary carcinoma of the thyroid both peptides are produced. We present here evidence for the existence in the human genome of a second CT gene, which is also expressed in human medullary thyroid carcinoma. This gene encodes a second human CGRP, differing from the known human CGRP in 3 of the 37 amino acids.     

Human intestinal mucin-like protein (MLP) is homologous with rat MLP in the C-terminal region, and is encoded by a gene on chromosome 11 p 15.5.

A cDNA specific for a human intestinal mucin (MLP) was amplified by PCR from cDNA of cultured human colonic adenocarcinoma cells, LS174T. The human cDNA shared high sequence homology with a corresponding rat intestinal mucin (MLP) cDNA in the 3' terminal region, and hybridized to the same mRNA (approximately 9.0 Kb) that was recognized by a probe for the MUC-2 human intestinal mucin gene. The gene encoding our human mucin peptide also mapped to chromosome 11 p 15.5, the known locus of MUC-2. Our findings suggest that human MLP and MUC-2 are encoded by the same gene and that rat and human intestinal mucin share a common C-terminal amino acid structure.     

Transient expression of human neutral alpha-glucosidase AB (glucosidase II) in enzyme-deficient mouse lymphoma cells

To define new methods for gene isolation exploiting mutant mammalian cells we transformed a mutant mouse cell line deficient in glucosidase II with total human genomic DNA and detected transient expression of the human glucosidase II gene. Maximum gene expression was detected 48 h after addition of DNA as a 2.5-fold increase in neutral alpha-glucosidase activity (2.47 +/- 0.15, n = 4). When mutant mouse DNA was used for transformation, no increase in enzyme activity was seen. The increased enzyme activity was due to expression of the human gene product. Thus, by rocket immunoelectrophoresis, cells transformed with human DNA yielded a "rocket" which reacted with antibody to human but not to mouse glucosidase II and which hydrolyzed substrate in situ. Specific DNA sequences were required for expression of the enzyme activity, since digestion of DNA with EcoRI and SstI rendered the DNA ineffective for eliciting expression of the enzyme, while digestion of DNA with BamHI and XhoI did not affect the increase. Transfection with intact phage from a human genomic DNA library also resulted in transient expression of the human gene. These results demonstrate the feasibility of detecting, by enzymatic assay, transient expression of a human gene for an intracellular enzyme following DNA-mediated transformation both with total human DNA and with intact phage from a human recombinant library. This system could be used as an assay for isolation of a gene from a genomic library by sibling selection.     

Detection of a unique human V kappa IV germline gene by a cloned cDNA probe.

We have cloned the cDNA encoding the KIV chain of a human antibody with specificity against the major carbohydrate antigen of Streptococcus A. The cDNA has been used as a genetic probe to estimate the number of germline VKIV genes in human DNA. The presence of unique hybridizing bands on digestion of human DNA with several restriction endonucleases and the equivalence of the DNA in a band to a single gene per haploid genome point to the conclusion that there is a unique human VKIV germline gene. The corollary of this conclusion is that the diversity of human VKIV chains must be exclusively due to somatic mutation. This is supported by examination of the sequences of human KIV chain genes and their KIV chain products. Fusion of the unique germline VKIV gene (1) with one of several JK segments, followed by somatic mutations in the V region of the rearranged KIV gene, can account for the known sequences. The restricted germline gene repertoire may account for the small proportion of human KIV chains in the human K chain sequence library (2).