It is well recognized that haemoglobin variants can be detected during the measurement of HbA1c by high-performance liquid chromatography (HPLC). A number of variants have been reported as compromising the quantification of HbA1c, a marker used in the assessment of glycaemic control in diabetes. We describe a case of haemoglobin Etobicoke, a rare alpha chain variant detected in an Irish diabetic during HbA1c analysis. Its identity was confirmed using a series of investigations. These included haemoglobin electrophoresis at alkaline and acid pH, isoelectric focusing and globin chain electrophoresis. Ultimately mass spectrometry isolated the mutation at position alpha 84 (F5). Haemoglobin Etobicoke, first described in Canada in 1969 has not previously been detected on HbA1c analysis. In the presence of this rare variant, HbA1c, a standard method using HPLC to assess glycaemic control in diabetes is unreliable and alternatives such as fructosamine need to be considered. HbA1c measured by automated HPLC will effectively screen populations where haemoglobin variants were not previously known. Precise identity of these variants when they are detected is crucial to the reliable interpretation of HbA1c analyses.

Major advances in the use of site-specific recombinases to facilitate sustained gene expression via chromosomal targeting have been made during the past year. New tools for genomic manipulations using this technology include the discovery of epitopes in recombinases that confer nuclear localization, crystal structures that show the precise topology of recombinase-DNA-substrate synaptic complexes, manipulations of the DNA recognition sequences that select for integration over excision of DNA, and manipulations that make changes in gene expression inducible by drug administration, in addition, endogenous eukaryotic and mammalian DNA sequences have been discovered that can support site-specific recombinase-mediated manipulations.

This chapter presents chloramphenicol acetyltransferase (CAT) as a gene expression tool, the system has been widely employed to characterize transfection techniques for tissues and cell lines, for characterizing DNA mutagenesis and repair systems, for genetic and biochemical analyses of transcription factors and transcription initiation complexes, for characterization of signaling pathways, for characterizing the tissue specificity of promoters in transgenic animals, and for evaluating the efficacy of delivery systems for gene therapy in intact animals. The chapter focuses on three types of CAT assay methods, a thin-layer chromatography (TLC) assay, a mixed phase assay, and a high-performance liquid chromatography (HPLC) assay. Parameters that can influence the sensitivity and reproducibility of the assays include the concentration and stability of the reagents, the temperature of the reaction, the reaction time, the extraction efficiency of reaction products in solvents, the presence of other acetylases in the reaction, the reaction volume, the type of cell transfected, the preparation of cell extracts, and the amount of extracts used in the reaction.

We report gene transfer to the normal and injured murine pulmonary circulation via systemic (intravascular) and airway (intratracheal) delivery of novel polycationic liposomes (imidazolium chloride, imidazolinium chloride-cholesterol, and ethyl phosphocholine). With use of the reporter genes chloramphenicol acetyltransferase (CAT) or human placental alkaline phosphatase (hpAP), intravascular injection of lipid-DNA complexes resulted in gene expression primarily in the lung, with lesser expression in the heart (11% of lung, P < 0.05) and spleen (8% of lung, P < 0.05). Histochemical staining for the hpAP reporter gene showed localized transgene expression in the microvascular endothelium. Monocrotaline (80 mg/kg body wt sc) treatment produced endovascular inflammation and reduced lung CAT activity (2 days postintravascular transfection) by 75 +/- 8 and 86 +/- 6% at 7 and 21 days, respectively, after monocrotaline (P < 0. 05). Despite the apparent decrease in functional CAT protein, Southern blot analysis suggested maintained plasmid delivery, whereas quantitative PCR (TaqMan) showed decreased CAT mRNA levels in monocrotaline mice. In contrast, intratracheal delivery of lipid-DNA complexes showed enhanced CAT expression in monocrotaline mice. Transfection in alternate pulmonary vascular disorders was studied by inducing hypoxic pulmonary hypertension (4 wk at barometric pressure of 410 mmHg). Efficiency and duration of gene transfer, assessed by CAT activity, were similar in pulmonary hypertensive and normal lungs. We conclude that imidazolinium-derived polycationic liposomes provide a means of relatively selective and efficient gene transfer to the normal and injured murine microvascular circulation, although translation of transgene mRNA may be reduced by preexisting endothelial injury.

As the sequencing of the human genome proceeds, the need for a new screen for in vivo function is becoming apparent. Many investigators are turning to various transgenic models as a means of studying function. However, these approaches are very time consuming, with a transgene-expressing mouse model often taking months to establish. We have developed an efficient system for delivering genes in vivo, which allows the gene product to be studied as early as 24 h after introduction into the mouse model. The delivery system employs a novel cationic lipid, 1-[2-(9-(Z)-octadecenoyloxy)ethyl]-2-(8-(Z)-heptadecenyl)-3- (hydroxyethyl)imidazolinium chloride (DOTIM), and a neutral lipid, cholesterol, complexed with an expression vector containing the reporter gene chloramphenicol acetyl transferase (CAT). After a single intravenous injection of these complexes, several tissues were seen to express the transgene. High, persistent expression in the vascular endothelial cells in the mouse lung was obtained. Delivery of DNA in vivo has been evaluated by quantitative polymerase chain reaction and protein expression by CAT activity assays. In vivo studies showed reproducible expression in more than 500 mice injected via the tail vein. An early peak of expression was followed by lower, but sustained, expression for > 50 days. Transgene expression of CAT could also be identified by immunohistochemistry staining in mouse lung and appeared to be located within the capillaries. The pattern of in vivo expression could be modulated and targeted to specific organs by altering the lipid-DNA formulation. New expression vectors with altered introns and polyadenylation sites further improved expression. The expression reported here may be sufficient in magnitude, duration, and flexibility to be an attractive alternative, in some cases, to establishing transgenic animals by stable gene transfer.

In vivo transfection of established tumors with immunostimulatory genes can elicit antitumor immunity. Therefore, we evaluated the safety and efficacy of intratumoral injections of a bacterial superantigen with a cytokine gene in dogs with malignant melanoma, a spontaneous and highly malignant canine tumor. 26 dogs with melanoma were treated with lipid-complexed plasmid DNA encoding staphylococcal enterotoxin B and either GM-CSF or IL-2. Dogs were evaluated for treatment-associated toxicity, tumor responses, immunologic responses, and survival times. The overall response rate (complete or partial remissions) for all 26 dogs was 46% (12 of 26), and was highest in patients with smaller tumors. Toxicity was minimal or absent in all dogs. Injected tumors developed marked infiltrates of CD4+ and CD8+ T cells and macrophages, and tumor regression was associated with development of high levels of antitumor cytotoxic T lymphocyte activity in peripheral blood lymphocytes. Survival times for animals with stage III melanomas treated by intratumoral gene therapy were prolonged significantly compared with animals treated with surgical tumor excision only. Thus, local tumor transfection with superantigen and cytokine genes was capable of inducing both local and systemic antitumor immunity in an outbred animal with a spontaneously developing malignant tumor.

Purpose. The potential use of aerosol delivery for non-viral gene therapy was tested by nebulization of lipid:DNA complexes to the lungs of rhesus monkeys.

Novel treatments for acquired diseases may be accomplished by the transfer of therapeutic genes into somatic cells. This report demonstrates gene transfer to the lung circulation with a novel polycationic liposome from an imadzolinium derivative with the formula CH3(CH2)7CH = CH(CH2)7 (lipid B).

We compared the efficacy of gene transfer in vitro and in vivo using various formulations of DNA–lipid complexes based on the novel cationic lipid EDMPC (1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine, chloride salt). In vitro studies analyzed delivery of marker genes to four established cell lines, including two of pulmonary origin. The in vivo analysis used intralobar delivery of marker genes and CFTR to mice and rats. We observed a lack of positive correlation between those DNA–EDMPC formulations that delivered DNA most efficiently in vitro and those that worked best in vivo. Intralobar DNA delivery to rodents mediated by EDMPC was efficient. The high level of gene delivery by DNA–EDMPC formulations demonstrates that efficient lipid-mediated gene transfer to the lung is possible.

Rat myoblast primary cultures were tested as a model for proinsulin synthesis and processing and unregulated insulin delivery for insulin-dependent diabetes mellitus (IDDM) gene therapy. Three human proinsulin cDNA constructs containing genetically engineered furin endoprotease cleavage sites between the B-chain and C-peptide (IFur) and between the C-peptide and A-chain (IIFur) and/or containing a histidine B10 to aspartic acid point mutation were subcloned into a mammalian expression vector (pCMV) containing the cytomegalovirus (CMV) promoter. The altered cleavage sites enable the insulin to be processed by the ubiquitous endoprotease furin. The histidine B10 to aspartic acid mutation creates a more stable form of insulin leading to an increase in insulin accumulation. Myoblasts transfected with a proinsulin cDNA construct mutated at all three sites (pCMV.IFur.IIFur.B10), a construct with only the furin sites (pCMV.IFur.IIFur), and a construct containing only the mutation at the B10 position (pCMV.B10) accumulated 852 +/- 16, 150 +/- 13, and 883 +/- 39 microU (pro)insulin/ml, respectively, in the culture medium during a 48-hr incubation. (Pro)insulin was detected in the culture medium within 2 hr post-transfection. Significant (pro)insulin release continued for 1 week and gradually diminished over a month. Approximately 50% of the proinsulin released from rat myoblasts transfected with pCMV.IFur.IIFur.B10 was completely processed into mature insulin based on densitometric analysis of autoradiographs of gels containing immunoprecipitated 35S-Cys-labeled (pro)insulin. However, only a trace of the proinsulin encoded by pCMV.B10 was processed. In an isolated rat adipocyte [14C]glucose oxidation assay, insulin released from myoblasts transfected with pCMV.IFur.IIFur.B10 was active biologically, displaying more biological activity than normal human insulin. Plasmid expression was studied by transfecting myoblasts with the beta-galactosidase (beta-Gal) gene in pCMV, allowing them to divide and fuse into multinucleated myotubes, followed by staining for beta-Gal. Approximately 80% of myotubes expressed beta-Gal. The results indicate that proinsulin encoded by genetically modified proinsulin cDNA is processed into mature insulin, which is secreted at high levels, making myoblasts a viable target cell for gene therapy of IDDM.

Growth of human breast cells is closely regulated by steroid hormone as well as peptide hormone receptors. Members of both receptor classes are important prognostic factors in human breast cancer. Clinical data indicate that overexpression of the HER-2 gene is associated with an estrogen receptor-negative phenotype. In this study, we demonstrate that introduction of a HER-2 cDNA, converting non-overexpressing breast cancer cells to those which overexpress this receptor, results in development of estrogen-independent growth which is insensitive to both estrogen and the antiestrogen, tamoxifen. Moreover, activation of the HER-2 receptor in breast cancer cells by the peptide growth factor, heregulin, leads to direct and rapid phosphorylation of ER on tyrosine residues. This is followed by interaction between ER and the estrogen-response elements in the nucleus and production of an estrogen-induced protein, progesterone receptor. In addition, overexpression of HER-2 receptor in estrogen-dependent tumor cells promotes ligand-independent down-regulation of ER and a delayed autoregulatory suppression of ER transcripts. These data demonstrate a direct link between these two receptor pathways and suggest one mechanism for development of endocrine resistance in human breast cancers.