Frontiers in Human Genetics

The following sections are included:

• LIST OF CORRESPONDING AUTHORS

• FOREWORD

• PREFACE

• CONTENTS

Routine cytogenetic techniques of karyotyping and banding chromosomes have been the usual conventional testing procedures for detection of chromosomal anomalies in prenatal diagnostics. With the advent of molecular techniques of fluorescence in situ hybridization, the field of cytogenetics has been revolutionalized. Detection of some genes on chromosomes, which previously could not be detected by cytogenetics can now be visualized. Also, samples from which chromosomes in the metaphase plate maybe difficult to obtain, can be probed by FISH in all stages of the cell cycle. Hence applications in preimplantation genetics are increasing rapidly. The rapidity and efficiency of the technique makes it very attractive. This article reviews the applications as well as drawbacks of the FISH technique in different areas, mainly in prenatal and preimplantation diagnosis.

Single-nucleotide polymorphisms (SNP) are an important class of DNA polymorphisms as they are common, have relatively low mutation rates, and can be genotyped using rapid high throughput methods. Currently efforts are underway to discover and map SNP in the human genome. We report here a novel in silico approach to predict the presence of novel SNP in a specific gene, and demonstrate its utility in a candidate gene for myopia, the tissue inhibitor of metalloproteinase-2 (TIMP-2) gene. TIMP-2 proteins are inhibitors of metalloproteinases that are responsible for the degeneration of connective tissue including the sclera tissue of ocular globe. The TIMP-2 gene is located on chromosome 17q25 and is encoded by five exons spanning 83 kb of genomic DNA. No intragenic or flanking DNA markers had previously been reported. Genomic, cDNA and EST (expressed sequence tags) sequences were obtained from DNA databases, and multiply aligned using sequence analysis software. Several putative sequence variants were found, and two were studied further by direct sequencing and PCR genotyping. New restriction sites were introduced by deliberate primer mismatches and PCR samples were genotyped by restriction digestion ("artificial RFLP"). The presence of one polymorphism in exon 3 was confirmed both by sequencing and by PCR genotyping of 187 unrelated individuals of Chinese descent. Allelic frequencies were 0.27 and 0.73, and observed heterozygosity was close to 0.40. This approach for in silico detection and ex silico confirmation of novel polymorphisms is extremely rapid and can be generalised to any gene, EST or genomic sequences. These polymorphisms may serve as useful markers for maps and for localising disease genes by linkage and association analysis.

We report the development of a homogenous assay for the genotyping of single-nucleotide polymorphisms (SNPs), utilizing a fluorescent dsDNA-binding dye. Termed T_M-shift genotyping, this method combines multiplex allele-specific PCR with sequence differentiation based on the melting temperatures of amplification products. Allele-specific primers differing in length were used with a common reverse primer in a single-tube assay. PCR amplification followed by melt curve analysis was performed with a fluorescent dsDNA-binding dye on a real-time capillary thermocycler. Genotyping was carried out in a single-tube homogeneous assay in 25 minutes. We compared the accuracy and efficiency of this T_M genotyping method with conventional restriction fragment genotyping of a novel single nucleotide polymorphism in the Jagged1 (JAG1) gene. The flexibility, economy and accuracy of this new method for genotyping polymorphisms could make it useful for a variety of research and diagnostic applications.

With the completion of the human genome project, scientists will face the daunting task of analysing the function of all human genes obtained from the project. Global analysis of gene expression and DNA polymorphisms and mutations, however, are facilitated with the development of tools such as microarrays and "Lab-on-a-Chip" systems. This chapter gives an overview of the BioMEMs/"Lab-on-a-Chip" systems, the components that constitute such systems and some commercial chip systems currently being developed or sold.

The advancements in molecular biology have made biotechnology a billion-dollar business over the last two decades. Recent developments in instrumentation, nano-technology and information technology have provided the biomedical research community with enormous amounts of diverse information governing biological systems. Consequently, there is an urgent need for information storage, curation, analysis and retrieval (ISCAR) using bioinformatics tools. Though the very definition of bioinformatics is debatable, there is a general agreement about the importance of certain fundamental concepts. Broadly, bioinformatics is the marriage between modern biology and information technology to glean new knowledge from redundant databases. Bioinformatics helps researchers gather, standardize, combine and manipulate data to tease out the knowledge they contain. In future, it will guide in performing in silico biotechnological experiments to aid biomedical research and application. Hence "SILICO BIOTECH" highlights the simple relationships between different disciplines that govern the complex drug discovery process and its relevance in health care.

The power of the bioinformatics integration system called KRIS, better known as Kleisli, is reviewed through a series of examples.

Secondary structure prediction is one of the most reliable of protein structure prediction methodologies, but the rapid developments require constant evaluation in order to select the best method. Key criteria such as selection of data for training sets, cross-validation, prediction accuracy, are reviewed and discussed in the context of judging secondary structure prediction methods. Where possible, a comparison with prediction methods is presented.

SNAREs participate in various transport events, in both the secretory and endocytotic pathways. Here we report the identification and characterisation of a new human SNARE, GS27. GS27 has two mRNA transcript isoforms as a result of alternative RNA splicing. GS27a has 6 exons and encodes a predicted protein of 212 amino acids, whereas GS27b has 7 exons and encodes a predicted protein of 213 amino acids. The gene spans 16 kb of genomic DNA and locates on the human chromosomal position 17q21. The antibodies raised against the cytoplasmic domain of GS27a protein showed specific staining in the Golgi apparatus. It could also inhibit transport in vitro from the endoplasmic reticulum (ER) to the trans-Golgi, suggesting a function in the ER-to-Golgi transport. It may be a coincidence but one of the loci implicated in human familial essential hypertension is lying near the GS27 gene locus making it a potential candidate for this disease.

Nine newborns and infants with sex ambiguity and congenital adrenal hyperplasia (CAH) were analysed for the presence of the testis-determining factor (TDF). They required an urgent genetic evidence for a sex assignment both for clinical management and parental request. The sex-determining region on Y chromosome (SRY gene) was used as a molecular marker in combination with the Fluorescence in situ hybridization (FISH) to identify the chromosome X and Y material on the interphase cells to complement the conventional karyotyping. The molecular analysis of SRY gene to define the association with the testicular-determining factor (TDF) supports the presence of the Y chromosome material. Our results showed that the sex genetics of the newborns were assigned then as male and female respectively and one newborn was reassigned as female. In the nine samples that we carried out the SRY analysis, seven showed the presence of SRY gene and in two samples it was absent. Sex assignment earlier not determined by the clinicians in three patients showed the presence of SRY gene in two patients while one was absent. All these sex reassignment and confirmation were supported by the molecular-cytogenetics and karyotyping studies. This approach provided a fast sex genetic information when the hormonal assay and karyotyping were not available and/or delayed. It is very useful to complement an immediate information for the clinicians to rule out cases of ambiguous genitalia due to CAH and subsequently clinical investigations that affects the long term management.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive enzyme disorder that affects an estimated 200-400 million people. More than four hundred biochemical variants have been identified reflecting the vast heterogeneity of this disorder. At the molecular level, less than a hundred mutations have been characterized indicating a need for a rapid and efficient approach for mutational screening of G6PD deficient individuals in different populations. A technique called multiplex PCR using multiple tandem forward primers and a common reverse primer (MPTP technique) was recently developed. It was initially tested in Singapore population where the incidence of this deficiency occurs at approximately 3% of newborn males. The MPTP technique allows quick and comprehensive screening of point mutations and can be adopted to detect common variants such as 1376G→T (G6PD Canton) and 1388G→A (G6PD Kaiping) in mutation hot spots for specific populations. This paper presents a brief review on G6PD deficiency and the application of the MPTP technique for screening this disorder.

G6PD is one of the enzymes needed by erythrocytes to regenerate ADP to ATP for recovering energy resource. Deficiency of this enzyme will lead to an increase of erythrocyte lysis. The incidence of this disease in Indonesia is considerably high possibly due to malarial selection. It is estimated that 2-6 % of the population are carriers. Routine sequencing of the 60 common mutations covering all 12 exons of G6PD gene is laborious, time consuming and expensive. Therefore the MPTP method was used to detect the most common mutations located in exons 6, 11 and 12.

Among 57 samples of normal newborn babies from Yogyakarta, Indonesia that were screened by the Formazan method, 5 were G6PD deficient whereas among 5 newborns with severe jaundice, 2 were G6PD deficient. Of these seven deficiency cases, one was shown to carry the Silent variant (1311 C→T mutation).

The MPTP method for detecting the mutation of G6PD gene was found to bed useful for screening G6PD cases from Yogyakarta, Indonesia.

Newborn screening for 21-hydroxylase deficiency, which is the most common type of congenital adrenal hyperplasia (CAH), was started in the Philippines in June 1996. CAH was screened using the Delfia 17-OHP time-resolved fluoroimmunoassay with a false positive rate of 0.02% and false negative rate of 0.002%. Radioimmunoassay was used as a confirmatory test. A total of 13 cases of 21-hydroxylase deficiency cases were detected out of 100,369 newborns screened. Females outnumbered the males (1.6:1). Mean age when screening was done was 13.8 days. The mean age when screening results were made available was 27.5 days. This was mainly attributed to the delay in sending samples to the screening laboratory which was on the average 11.9 days. The salt-losing type of 21-hydroxylase was the most common clinical form. All 13 cases are alive.

Duchenne and Becker muscular dystrophies are allelic X-linked disorders resulting from defects in the gene coding for the dystrophin muscle protein. The dystrophin gene is more than 2300kb in size and consists of 79 exons. This large size and complexity presents a challenge to direct identification of point mutations and small deletions that cannot be identified by multiplex deletion testing or Southern blotting. One approach to this problem is to analyse the expression of ectopic dystrophin mRNA transcripts. Although the dystrophin gene transcript is distributed only over approximately 0.1% of the genome, analysis of such ectopic lymphocyte dystrophin transcripts can shed light on the pathogenic events at the transcriptional level.

Duchenne muscular dystrophy (DMD) is an X-linked recessive lethal disorder affecting 3500 live born males. This disorder is caused by mutations in the gene that encodes dystrophin, a high molecular weight cytoskeletal protein. The large size and complexity of the gene pose limitations for detailed mutational analysis in patients, especially for non-deletion cases. In this report, we describe the characterization of a single nucleotide alteration in exon 37 from ectopic transcripts of immortalized lymphocytes from a DMD patient. This mutation is predicted to result in termination of translation of the dystrophin protein. In vitro translation of polypeptide using an amplified fragment of 2105 bases covering the nucleotide change was carried out from the extracted mRNA transcripts using the coupled reticulocyte lysate transcription/translation system. A truncated polypeptide of approximately 46 kDa was obtained, confirming premature chain termination had occurred. Demonstration of this mutational effect proves that the identified mutation is responsible for the pathogenic phenotype in the patient. This approach may be applied in future for direct identification of causative mutations resulting in truncated dystrophin in other DMD patients.

Maple syrup urine disease (MSUD) is an autosomal recessive disorder in the oxidative decarboxylation of branched chain alpha-ketoacids (BCKAs) derived from leucine, isoleucine, and valine. Impaired activity of the branched chain ketoacid dehydrogenase complex causes MSUD's heterogenous clinical phenotype. Severity of the disease ranges from classical to mild variant types, thus the diagnosis is often overlooked and considered least likely to occur in clinical practice. In the Philippines, the local incidence has not been reported as yet. Using two-dimensional thin layer paper chromatography, we describe here 26 cases of MSUD diagnosed in different hospitals in the country from 1992 to 1998. A review of their demographic data, symptomatology, diagnosis, management and outcome is presented. Our results depict heretofore the importance of early detection of MSUD to ensure reduced morbidity, mortality, and length of hospitalization for these patients.

Southeast (SE) Asia has high frequencies of thalassemias. The most common abnormal genes are α-thalassemia, β-thalassemia, hemoglobin (Hb) E and Hb Constant Spring (CS). The severe form of α-thalassemia, α-thalassemia 1, is most common in this region and HbE is the hallmark of SE Asia. β-thalassemia is very heterogeneous at the molecular level. They are grouped into β⁰-thalassemia which is associated with absence of β-globin chain and β⁺-thalassemia with partial production of the β-globin chain.

The two types of α-thalassemia genes (α-thalassemia 1 and α-thalassemia 2), β-thalassemia, HbE, HbCS and some other mutations, in different combinations, lead to more than 60 thalassemia syndromes. HbBart's hydrops fetalis (homozygous α-thalassemia 1) associated with total lethality is the most severe thalassemic disease. HbH disease results from interaction between α-thalassemia 1 and α-thalassemia 2 or between α-thalassemia 1 and HbCS. Compound heterozygosity between β-thalassemia and HbE leads to β-thalassemia/HbE disease.

α-Thalassemias are most often due to gene deletions. While HbCS occurs from the mutation at the termination codon of the α2-globin gene. α-thalassemia results from a variety of molecular mechanisms, most of which are single base substitutions, deletions or insertions. Hemoglobin E occurs from a Glu→Lys substitution at position 26 of the β-globin chain. The abnormal gene also results in reduced amounts of β^E-mRNA. Therefore, HbE has a mild β⁺-thalassemia phenotype. Despite the same genotype, a remarkable variability in severity of β-thalassemia is observed. Genetic factors which influence the severity of anemia include: nature of the mutation, coinheritance of α-thalassemia gene and association with increased HbF.

Mesomelic dysplasia Kantaputra type (MDK) (MIM *156232) is a new autosomal dominant skeletal dysplasia characterized by bilateral symmetrical marked shortening of the ulnae and shortening and bowing of the radii. The proximal fibula is usually short and synostoses are present between the tibia and fibula and the small malformed calcaneus and talus. The prominent calcanei on the ventral surfaces of the distal fibulae are a characteristic feature of this new syndrome. Carpal and tarsal synostoses are present in some affected people. We studied this Thai family in which 15 members in 3 generations were affected. With reference to the breakpoints of a balanced translocation [t(2;8)(q31;p21)] in patients from a previously reported Italian family with a skeletal dysplasia that appears similar to MDK, a linkage analysis was performed using 50 CA-repeat markers mapped to nearby regions (2q22-q34 and 8p24-p21) of the translocation breakpoints. The results clearly ruled out a linkage of MDK to marker loci at the 8p24-p21 region, whereas all nine affected members available for the study shared a haplotype at four loci (D2S2284, D2S326, D2S2188, and D2S2314) spanning about 22.7cM in the 2q24-q32 region. The computer-assisted two-point linkage analysis revealed maximum logarithm of odds (lod) scores of 4.82, 4.21, 4.82, and 4.21 (θ = 0) at these loci, respectively. These data indicated that the MDK locus is in the vicinity of D2S2284 and D2S2188 loci that are the most likely mapped to 2q24-q32.

The role of genetic influences on the course of mycobacterial infections, particularly in tuberculosis is presented in this review. Studies have shown racial differences in susceptibility to tuberculosis, familial clustering of cases and a higher concordance rate of susceptibility among monozygotic twins compared to dizygotic twins. Animal models of infection have been used as tools in the genetic analysis of host susceptibility or resistance to tuberculosis. More recently, an autosomal dominant gene called Bcg has been identified which is believed to be responsible for a non-specific macrophage activation for bactericidal function. Nramp1 or natural resistance associated macrophage protein 1 is the candidate gene for Bcg. It is suggested that Nramp1 has a macrophage-specific membrane transport function. This review is presented to emphasize the important role of identifying genes regulating host innate and antigen-specific responses for better prevention and control of infections such as tuberculosis.

Loss of heterozygosity (LOH) of the chromosomal region 11q22-q24 is common in several types of human solid neoplasms, including carcinomas of the breast, lung, colon, nasopharynx, cervix, ovary, uterus and malignant melanoma, suggesting the presence of one or several tumour suppressor genes in this region. To determine if LOH occurs in this chromosomal region in tumours of the central nervous system, we screened for LOH in matched normal and tumour DNA samples from 32 patients with primary tumours of the CNS, of which 20 were benign and 12 were malignant. Nine polymorphic microsatellite loci from the 11q22-q23 region, between D11S940 and D11S934 were analyzed by the polymerase chain reaction (PCR). Six of the 32 cases (18.8%) demonstrated LOH at one or more of the nine loci studied and no significant increase in the frequency of LOH was observed at any particular locus. Of these six cases with LOH, two were benign and four were malignant tumours. Since LOH was detected in both benign and malignant cases, we conclude that LOH at the chromosomal region 11q22-q23 in tumors of the CNS is a random event and may not play an important role in the pathogenesis of these tumours.

Retinoblastoma is an embryonic neoplasm of retinal origin. Tumour development in retinoblastoma is initiated by the somatic inactivation of both alleles of the RB1 gene. It is predicted that 40% of all cases of retinoblastoma are hereditary with all bilateral and 10-15% of unilateral cases being constitutional in origin. In this study, the incidence of somatic loss of heterozygosity (LOH) as a mutational event in retinoblastoma was studied using four intragenic DNA markers. These include a microsatellite polymorphism, RB1.20 and four restriction fragment length polymorphisms (RFLPs) namely, intron 1/BamHI, intron 17/XbaI, intron 24/Tth1 III and intron 25/DraI. LOH was found in 17 out of 27 (63%) tumours analysed. A higher frequency of maternal loss compared to paternal loss was observed in bilateral cases (75%) as compared to unilateral cases (54%). Direct exon-by-exon sequencing revealed mutations in 15 out of 17 (88%) tumours with LOH. In three out of ten tumours without LOH, one mutation was detected and two mutations were found in five tumours without LOH. Seven of the mutations were found to be constitutional (germline) in origin with three of these mutations occurring in unilateral cases of retinoblastoma. The presence of constitutional mutations did not correlate with early age of tumour development. Our results show that there was no association between the types of mutation and constitutional origin of the mutation.

Familial adenomatous polyposis (FAP) is a familial form of colon cancer caused by mutation of the adenomatous polyposis coli (APC) gene. We investigated the APC mutation and phenotypic spectrum in 172 members of 36 Singapore FAP families. The protein truncation test (PTT) and DNA sequencing were used to screen the entire APC coding region for germline mutations. APC mutations were found in 28 families (78 %). 65 patients tested positive while 63 non-affected members tested negative. The correlation of PTT to clinical diagnosis is therefore 100%, suggesting that PTT is a highly reliable presymptomatic test for FAP. Twenty different APC mutations were identified, eleven of those were novel. All mutations, except one, resulted in the classical colonic phenotype. Interestingly, mutation at codon 332 resulted in attenuated FAP with left-sided predominance of polyps rather than the right. For the eight families without APC mutations, we screened for β-catenin mutation which was shown to be able to substitute for APC mutation in sporadic colorectal cancer. No germline β-catenin mutation was found. Further analysis reveals atypical clinical features such as the co-existence of adenomatous and hyperplastic polyps and other non-FAP associated cancers in these patients. Our results suggest the involvement of other genes and possibly new variants for the polyposis syndrome.

In the National University of Singapore Genetics of Hypertension (NUSIGHT) Study, we compared allele and genotype frequencies of three dopamine receptor D2 (DRD2) gene markers in Chinese hypertensive sibling-pairs and unrelated Chinese servicemen to test the hypothesis that the markers are associated with essential hypertension. Essential hypertension is a complex multifactorial disease caused by the interaction of genetic and behavioural factors. Several candidate predisposing genes have been proposed, including those in the dopaminergic neurotransmission pathway. We selected 62 ethnic Chinese non-obese, non-diabetic sibling-pairs on blood pressure > 150/90 mmHg or 24-hour ambulatory blood pressure > 140/90 mmHg, and disease onset < 60 years. We enrolled 176 unrelated ethnic Chinese men as population controls. Two polymorphisms in the coding region (Ser311Cys and NcoI) and one in the 3'UTR (TaqIA) of DRD2 were genotyped. Sib-pair linkage analysis of alleles identical by state and case-control linkage dysequilibrium analysis of allele, genotype and haplotype frequencies were performed. Significantly increased sharing of the marker TaqIA (pZ < 0.004), but not NcoI and Ser311Cys, was observed. However, no association between the three polymorphisms and hypertension was detected. The results suggest that linkage exists between DRD2 and essential hypertension in Singaporean Chinese, hence justifying further and larger genetic studies of this locus.

After identification of the dystrophin gene in 1987, there were confident claims that a cure for Duchenne muscular dystrophy would soon be available. It was thought that introducing a new or functional dystrophin gene into dystrophic tissue would compensate for the defective copy. Sadly, this approach has not turned out to be as simple as was anticipated.

The introduction of a functional dystrophin gene through cell therapy was encouraging in mice but failed dismally in human trials. A range of viral vectors have been evaluated for their potential to introduce a replacement dystrophin gene into muscle but the immune response and long-term transgene expression is an on-going problem.

Some alternative therapies have been considered, with intervention at one of the basic steps in the gene expression: (1) Transcription: re-activation of homologous genes. (2) Splicing: specific removal of an exon carrying a nonsense mutation. (3) Translation: suppression of nonsense mutations.

Gene therapy is a very young science. To date, it has promised much but delivered little. Nevertheless, there is still great optimism that therapies for some disorders, either gene replacement or genetically based, will prove effective in the near future.

We have developed HVJ (hemagglutinating virus of Japan; Sendai virus)-liposomes that are efficient in vitro and in vivo gene delivery vehicles using the fusion-mediated gene delivery. The HVJ-liposome was highly efficient for the introduction of oligonucleotides into cells in vivo as well as the transfer of genes less than 100 kbp without damaging cells. By coupling the Epstein-Barr (EB) virus replicon apparatus with HJV-liposomes, transgene expression was sustained in vitro and in vivo. Most animal organs were found to be suitable targets for the fusigenic-viral liposomes, and numerous gene therapy strategies using this system were successful in animals. Here we demonstrate cancer gene therapy using HVJ-liposomes.

• INTRODUCTION

• DEVELOPMENT OF NOVEL LIPOSOMAL VECTORS

• CLINICAL APPLICATIONS OF GENE THERAPY
  • Gene Therapy for Cystic Fibrosis (CF)
  • Cancer Gene Therapy

• PERSPECTIVE

• References

The development of DNA vaccines and plasmid vectors for gene therapy in recent years is based on the capacity of mammalian cells and tissues to take up exogenous DNA spontaneously. Naked DNA uptake is a universal property of mammalian cells, having been demonstrated in several different mammalian cell lines, primary cells in culture, solid tumours as well as in normal tissues (e.g. skeletal muscle and liver) in vivo. While mechanisms of uptake of exogenous DNA by bacteria are understood in some detail, almost nothing is known about this process in mammalian cells. It is unlikely that the extremely poor efficiency of naked plasmid entry into mammalian cells and tissues can be overcome until eukaryotic mechanisms are elucidated. We review the current state of knowledge of naked plasmid entry into mammalian cells, and present preliminary evidence for saturable plasmid binding sites on the plasma membrane of C2C12 myoblasts followed by time-dependent translocation of internalized plasmid into the nuclear compartment. Myoblasts can be engineered for insulin secretion via plasmid-mediated gene transfer. Thus, muscle and myoblast implants may be a suitable platform for developing genetic treatment of systemic metabolic disorders.

Mutations in the dystrophin gene are responsible for the severe X-linked recessive Duchenne muscular dystrophy. Affected boys show signs of muscle weakness between the ages of 3 and 5 years and will be restricted to a wheelchair by 12 years. Becker muscular dystrophy also arises from mutations in the dystrophin gene but in these cases, the typical in-frame deletion allows a semi-functional dystrophin protein to be produced.

The mdx mouse has a nonsense mutation in exon 23 of the dystrophin gene. We wished to evaluate intervention at the stage of dystrophin pre-mRNA processing (splicing) where exon 23 could be deleted from the mature transcript which could then be translated into a slightly shorter but functional dystrophin.

Antisense 2'-O-methyl oligonucleotides directed to the splice sites flanking exon 23 induced 100% skipping of that exon in primary cultured mdx myotubes. Induced exon skipping was not as efficient in immortalized mouse myoblast cell lines.

The deletion of exon 23 does not disrupt the reading frame of the dystrophin transcript and should allow the synthesis of a shorter but presumably functional protein. Such an approach has the potential to reduce the severity of a Duchenne mutation to the milder Becker phenotype.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease and so far, the treatment of DMD has not yet been established. Here, gene therapy is proposed as a novel treatment for DMD whereby the correction of the translational reading frame transforms severe DMD into the milder Becker muscular dystrophy. Based on the molecular analysis of dystrophin Kobe where the presence of an intra-exon deletion caused exon skipping during splicing, a part of the exon 19 sequence of the dystrophin gene was found to function as a splicing enhancer sequence, a sequence necessary for proper splicing. When oligonucleotides complementary to this sequence were added to the culture medium of lymphoblastoid cells, exon 19 skipping was specifically induced. This raises the possibility of a new therapeutic approach for DMD where dystrophin pre-mRNA splicing can be modulated by an oligonucleotide against a splicing enhancer sequence to produce an in-frame transcript that is able to produce truncated dystrophin. One natural example of this transformation is described.

Liver cirrhosis and liver cancer are major cause of morbidity and mortality worldwide with no effective therapy. Our aim is to establish a novel approach for liver cirrhosis and liver cancer utilizing in vivo gene therapy. To achieve effective gene expression in vivo, we used well established transfection method, hemagglutinating virus of Japan (HVJ)-liposome. Liver cirrhosis induced by dimethylnitorosamine (DMN) in rat is characterized by parenchymal collapse followed by hyper-accumulation of fiber tissue. All cirrhotic rats died of liver dysfunction by 7 weeks after the initial injection of DMN. Muscle directed gene transfer of hepatocyte growth factor (HGF) was performed. After repetitive gene therapy, the massive fibrosis was in nearly total remission and all cirrhotic rats were rescued from fatal cirrhosis. Alpha-fetoprotein (AFP) is highly expressed in most patients with liver cancer. To achieve specific gene expression in AFP-producing tumor, we used AFP promoter. AFP producing HUH7 tumors were established in the liver of SCID mice, vector consisting of HSV-TK gene with AFP promoter (AFP-TK) was introduced to these tumor bearing mice via splenic/portal injection. Repetitive in vivo transfection of AFP-TK followed by ganciclovir treatment can achieve abrogation of tumors in the liver. Our present results indicate that the gene therapy may hold promise for the treatment of patients with liver cirrhosis/ liver cancer.

We have defined the androgen responsiveness and status of prostate specific antigen (PSA) secretion of prostate cancer cells as well as non-prostatic cells transduced with PSA promoter reporter constructs. Androgen-independent (AI) human prostate cancer remains a lethal phenotype for which there is no effective therapy. AI prostate cancer produces and secretes large amounts of PSA at both primary and metastatic sites. The aim of this investigation is to explore the use of the PSA promoter as a mean of prostate cell specific expression of a toxic gene thymidine kinase (TK) to an AI PSA-producing human prostate cancer cell line. An adenovirus vector carrying human herpes simplex thymidine kinase (TK) gene under control of the PSA promoter (Ad-PSA-TK) was generated to target PSA-producing AI prostate cancer cells. Upon the administration of acyclovir, Ad-PSA-TK efficiently killed the AI PSA-producing human prostate cancer cells. This paper discusses the importance of tissue specific promoter system for using gene therapy of prostate cancer, and summarizes recent gene therapy strategies developed to target this process. This strategy can potentially be used in combination with current therapeutic modalities to achieve more effective tumor cell-kill with much reduced toxicity.

The following sections are included:

• ABSTRACTS