Prospects For Gene Therapy Research Articles

Imported RNA in mammalian mitochondria

The ‘division of genetic labour’ between nuclear and mitochondrial DNA (mtDNA) in mitochondrial protein synthesis in animal cells is controversial. As far as is known, all mitochondrial tRNAs, plus the two major rRNAs, are encoded by mtDNA, whereas all of the proteins involved in mitochondrial gene expression are nuclear encoded. In plants, fungi and protists, there are many examples of imported (nuclear-encoded) tRNAs, but compelling evidence for RNAimport into mammalian mitochondria has been lacking – until now.One recently documented example is the 5S rRNA, a proportion of which is found inside mitochondria 1xEvidence for the presence of 5S rRNA in mammalian mitochondria. Magalhaes, P.J et al. Mol. Biol. Cell. 1998; 9: 2375Crossref | PubMedSee all References1, although its function there is unknown. Other putatively imported RNAs have long been suggested to have roles in mitochondrial RNA processing. A particularly curious example concerns RNase P, the ribozyme involved in tRNA 5′ end maturation, which is apparently conserved from bacteria to man. No distinct mitochondrial RNase P has been characterized, and no gene for the catalytically essential RNA component of the enzyme can be found in mammalian mtDNA, although it is clearly present in yeast mtDNA. This leaves us with two alternatives: either mitochondrial pre-tRNA 5′ processing uses the same enzyme as in the nucleus, or a distinct enzyme inside mitochondria is entirely protein based. Preliminary reports in the literature unfortunately seemed to favour both options!The controversy has probably been laid to rest by Puranam and Attardi 2xThe RNase P associated with HeLa cell mitochondria contains an essential RNA component identical in sequence to that of the nuclear RNase P. Puranam, R.S and Attardi, G. Mol. Cell. Biol. 2001; 21: 548–561Crossref | PubMed | Scopus (82)See all References2, who report the characterization of an RNaseP in highly purified mitochondria that appears to be identical to the nuclear enzyme. Crucially, the enzyme is able in vitro to process correctly a mitochondrial pre-tRNA substrate, for Ser-tRNAUCN. The RNA component of the nuclear and mitochondrial enzymes is the same 340-nt H1 RNA species, encoded by a nuclear gene. Only a fraction of a percent of the total cellular H1 RNA content is recovered with the mitochondria, but it can be removed only by treatments that destroy organelle integrity. These findings raise the obvious questions of how H1 RNA (or intact RNase P holoenzyme, in a 17S particle also containing protein) enters mitochondria. The same authors also found tiny amounts of other small nuclear RNAs inside mitochondria, notably the RNA component of MRP, a ribozyme involved in nuclear pre-rRNA processing that was also originally proposed as a mitochondrial endoribonuclease.Clearly, if the biochemical pathways of mitochondrial RNA import in mammalian cells can be elucidated, a novel route will be established, whereby mitochondrial RNA can be manipulated, even if mtDNA transformation itself remains technically unfeasible. This could well offer a long-term prospect of gene therapy for correcting mitochondrial disorders involving tRNA gene mutations. At the very least, it would allow us to investigate more directly the molecular and physiological consequences of the presence inside mitochondria of mutant tRNAs.

Prospects for gene therapy using HIV-based vectors.

Recombinant vectors derived from murine leukemia virus (MLV) have been widely used to introduce genes in human gene therapy clinical trials and have shown the potential for medical applications and the promise of significantly improving medical therapies. Yet, the demonstrated limitations of these vectors support the need for continued development of improved vectors. The intrinsic properties associated with the MLV genome and its life cycle do not favor the successful application of this vector system in certain human gene transfer applications. Since MLV integrates randomly into the host genome, transgene expression is frequently affected by the flanking host chromatin. MLV insertions can often result in silencing or position effect variation of gene expression either immediately after insertion or following cell expansion in culture or in vivo. Migration of the MLV pre-integration complex from the cytoplasm into the nucleus of infected cells requires mitosis for nuclear membrane breakdown. Since a majority of human cells exist in a quiescent state in vivo, it is unlikely that direct in vivo gene delivery into target tissues can be achieved with the MLV vector system. Finally, insertion of tissue-specific cis-regulatory sequences to direct transgene expression frequently results in either the rearrangement of the vector sequence or disruption of the cis-regulatory sequence functions. The long terminal repeat (LTR) of MLV, which contains a ubiquitously active enhancer/promoter element, may partially account for this problem. Together, these problems pose a major obstacle for the use of MLV vectors in the treatment of human diseases. This Chapter discusses some of the potential targets to which HIV vectors might be applied in clinical settings and some of the issues surrounding use of HIV vectors in gene transfer clinical trials.

Cystic fibrosis: psychological issues.

To most parents the diagnosis of cystic fibrosis (CF) in their child represents a severe blow because they are confronted with an unwanted and unexpected disease that completely changes their whole life. Common reactions such as shock, denial, sadness and anger have to be mastered before a gradual adaptation to reality will be possible. The provision of support by a multidisciplinary team, including a psychologist, that offers its services from diagnosis and puts an emphasis on preventive care may help to achieve, maintain and improve physical and mental health, and social functioning in both patients and parents. CF not only affects the individual but the whole family, and the presence of biopsychosocial stressors may add to the burden caused by the disease. CF does not necessarily cause long-term serious family dysfunction, but it changes family structures and often taxes the family system beyond its strength. Even if there is only partial adherence to the demanding and complex treatment regimen, health professionals need to acknowledge the tremendous underlying effort on the part of the families. As a consequence of their continuous endeavours, many individuals with CF do lead remarkably normal lives with the prospect of gene therapy and lung transplantation, maintaining hope in case of severe deterioration.

Gene therapy for the hemoglobin disorders: past, present, and future.

With the development in the early 1980s of technology to transfer genes to murine hematopoietic stem cells by using recombinant murine oncoretroviral vectors (1), the possibility of genetic therapy for a number of human disorders of the lympho-hematopoietic system seemed an attainable goal. Hemoglobin disorders were among the first diseases to be considered for gene therapy. Both sickle cell disease and the thalassemias are common monogenic disorders worldwide that cause serious morbidity and mortality. The pathophysiology of these disorders is amenable to correction by the addition of a functional globin gene to stem cells with subsequent high level expression in maturing erythroid cells (Fig. 1). Encouraging reports of successful transfer of a β-globin gene into the hematopoietic stem cells of mice using an oncoretroviral vector appeared in 1988 (2, 3). Erythroid lineage-specific expression of the globin gene was observed, but the levels of expression were much too low (<1% of mouse β-globin) for a potential therapeutic effect. Recent studies using murine models of both sickle cell disease and β-thalassemia and γ-globin expressing transgenic mice substantiate the clinical impression that sustained expression of a transferred globin gene in the range of 10–20% of the level of endogenous globin in a majority of developing erythroblasts will be required for a therapeutic benefit (4).† Figure 1 Pathophysiology of hemoglobin disorders: Prospects for gene therapy. Normal human red cells contain 30 pg of hemoglobin, which in the adult is composed of two α and two β-globin chains (HbA = α2β2). The two globin chains are synthesized in nearly equal amounts in maturing erythroblasts. Before birth, γ-globin rather than β-globin is present in the hemoglobin tetramer. During the perinatal period, there is a switch from γ- to β-globin synthesis, resulting in the replacement of HbF with HbA in neonatal red blood …

Current and Future Gene Therapy for Malignant Gliomas.

Malignant gliomas are the most common neoplasm in the central nervous system. When treated with conventional treatments including surgery, irradiation, and chemotherapy, the average life expectancy of the most malignant type, glioblastoma multiforme is usually less than 1 year. Therefore, gene therapy is expected to be an effective and possibly curative treatment. Many gene therapeutic approaches have demonstrated efficacy in experimental animal models. However, the current clinical trials are disappointing. This review focuses on current therapeutic genes/vectors/delivery systems/targeting strategies in order to introduce updated trends and hopefully indicate prospective gene therapy for malignant gliomas.

The Prospects for Gene Therapy

Gene therapy will probably not be inordinately complex. Although extremely hightech in its design and manufacture, it is likely to be easy to administer in most practice settings. It will be widely used for classic genetic and common diseases alike.

How realistic is cutaneous gene therapy?

Recent progress with innovative, experimental gene therapy approaches in animals, and recent improvements in our understanding and manipulation of stem cells, gene expression and gene delivery systems, have raised plenty of hopes in essentially all branches of clinical medicine that hitherto untreatable or poorly manageable diseases will soon become amenable to treatment. Few other organ systems have received such enthusiastic reviews in recent years as to the chances and prospects of gene therapy as the skin, with its excellent accessibility and its pools of--seemingly--readily manipulated epithelial stem cells (cf. Cotsarelis et al., Exp Dermatol 1999: 8: 80-88). However, as in other sectors of clinical medicine, the actual implementation of general gene therapy strategies in clinical practice has been faced with a range of serious difficulties (cf. Smith, Lancet 1999: 354 (suppl 1): 1-4; Lattime & Gerson (eds.), Gene Therapy of Cancer, Academic Press, San Diego, 1999). Thus, it is critically important to carefully distinguish unfounded hype from justified hope in this embryonal area of dermatologic therapy, to discuss in detail what can be realistically expected from cutaneous gene therapy approaches in the next few years, and importantly, what kind of promises should not be made to our patients at this time.

Transient cyclophosphamide treatment before intraportal readministration of an adenoviral vector can induce re-expression of the original gene construct in rat liver.

Although adenovirus is an attractive vehicle for transferring therapeutic genes in vivo, animal studies have indicated that the clinical usefulness of adenoviruses may be limited by their immunogenicity. Although immunosuppressive strategies around the time of initial exposure of adenoviruses have been shown to prevent the formation of neutralizing antibodies and permit the successful readministration of adenoviruses in animals, the practicality of the approaches remains questionable. Because the majority of prospective gene therapy patients have already been infected with wild-type adenoviruses, initial treatment with adenoviruses in humans may correspond to readministration of adenoviruses into animals. It is shown here that although intraportal infusion of adenoviruses carrying a reporter lacZ gene resulted in transient high levels of transgene expression in the rat liver, intraportal readministration of adenoviruses failed to induce detectable levels of transgene expression. Conversely, when animals were treated transiently with cyclophosphamide before the intraportal readministration of adenoviruses, development of neutralizing antibodies and antigen-specific T cell proliferation in response to adenoviral readministration was significantly suppressed and successful re-expression of the transgene was achievable. These results may have important implications for efficacy considerations when adenoviral vectors are employed in clinical settings.

Prospects for gene therapy in cystic fibrosis.

Since the discovery of the gene encoding cystic fibrosis (CF), there has been much excitement about the possibility of gene therapy, which has now reached the stage of phase I clinical trials in adults. The baby born with CF has normal lungs at birth, but evidence of inflammation and lung changes are present as early as 4 weeks of age. Ideally, future novel treatments such as gene therapy should begin before the onset of airway damage. We review the pathophysiology of CF, including current molecular understanding, and set out the issues involved in gene therapy for children with CF. CF is the commonest lethal inherited disease in white people in the UK, with an incidence of ∼ 1/2000 live births. The median estimated life expectancy of children born in the 1990s is 40 years, which represents a doubling in the past 20 years. It is estimated that in the next millennium half of all patients will be adults. In 1989, the gene responsible for CF was localised to the long arm of chromosome 7. This gene encodes a protein called the CF transmembrane conductance regulator (CFTR), which is situated in the apical membrane of epithelial cells and functions as a chloride channel regulated by a cAMP dependent protein kinase. It also modulates the activity of other ion channels including the downregulation of the amiloride sensitive sodium channel. More than 800 mutations of the gene have been identified and they are categorised into five classes on the basis of CFTR protein alterations. Class I mutations result in either unstable mRNA or an abnormal protein that is rapidly degraded. Class II mutations result in faulty processing of the protein and failure of the protein to traffic to the apical membrane. This class includes the ΔF508 mutation, which is a codon deletion resulting in …

Current status and future prospects of gene therapy

Current status and future prospects of gene therapy

Gene Therapy for Hypertension

Somatic gene delivery approaches have received wide attention as a new technique for studying gene expression and as a potential therapeutic tool in treating both inherited and acquired diseases. Recent studies using nonviral and viral vectors have shown great promise for gene therapy in hypertensive diseases. Potential targets for prospective gene therapy in hypertension include vasopressor renin-angiotensin system components and a number of vasodilator polypeptides such as tissue kallikrein-kinin, atrial natriuretic peptide, adrenomedullin and nitric oxide synthase. Antisense inhibition with oligonucleotides or cDNAs encoding renin, angiotensinogen, angiotensin-converting enzyme and angiotensin receptors has been shown to cause a prolonged blood pressure reduction in spontaneously hypertensive rats. To evaluate the therapeutic potential of vasodilator proteins or peptides in high blood pressure, we delivered the genes encoding human tissue kallikrein, atrial natriuretic peptide, nitric oxide synthase, and adrenomedullin into hypertensive rat models and showed that a single injection resulted in a significant and sustained reduction of blood pressure for several weeks. The potency and duration of blood pressure reduction depends on the dose and the promoter of the gene administered, age and sex of the hypertensive animals as well as the vehicle used for gene delivery. Somatic gene transfer of human tissue kallikrein or atrial natriuretic peptide not only attenuated hypertension but also exerted a protective effect against salt-induced renal damage and cardiac hypertrophy in Dahl salt-sensitive rats after high salt loading. These results suggest that the application of antisense inhibition of vasopressors, or gene delivery of vasodepressors for gene therapy, may have potential in treating human hypertension, and cardiovascular and renal disorders.

Prospects of gene therapy in treatment of coronary heart disease

Gene therapy describes the transfer of genetic material into a cell for therapeutic purposes. This opens new therapeutic perspectives for cardiovascular medicine. It includes the inhibition of restenosis following angioplasty, e.g., by transfer of suicide genes such as CMV-thymidine kinase or by inhibition of the cell cycle of cells within the vessel wall. On the other hand, there is promising data concerning the induction of therapeutic angiogenesis using the transfer of angiogenic genes such as the one for vascular endothelial growth factor VEGF. During the past three years significant progress was made by a number of preclinical studies. On the other hand, the therapeutic success of gene therapy in humans is still missing, and this is true for all different strategies tested so far. Important and basic issues of gene transfer and the resulting cellular response need to be solved before a therapeutic use might become a routine procedure. In the meanwhile, an important focus of the experimental work lies in the identification and characterization of molecular targets for therapeutic interventions, another in the improvement of gene transfer systems. Such work will provide new information about the biology of cellular and viral structures including their functional interrelation; in addition a better insight into the pathogenesis of the various disease processes should be obtained.

Problems and prospects in gene therapy for hemophilia.

The aim of gene therapy for hemophilia is the stable introduction and expression of a gene encoding functional blood coagulation factor VIII or IX. Although there are as yet no published studies demonstrating long-term expression of therapeutic levels in large animal models of the disease, there have been several reports over the past year of sustained expression of therapeutic levels of clotting factors in small animals, and some of these strategies are currently being applied to hemophilic dogs. Recent advances include optimized retroviral gene transfer, improved adenoviral vectors for high levels of sustained expression of factor VIII in mice, stable therapeutic levels of factor IX expression in mice after transduction of muscle or liver with adenoassociated virus vector, as well as new nonviral gene delivery strategies. Finally, several important mouse and dog models of hemophilia have been characterized during the past year.

Extracellular matrix dynamics in heart failure: A prospect for gene therapy

In chronic congestive heart failure, an illness affecting more than 4 million Americans, there is impairment of myocardial extracellular matrix (ECM) remodeling. Failing human ventricular myocardium contains activated matrix metalloproteinases (MMPs), which are involved in adverse ECM remodeling. Our studies support the concept that impaired ECM remodeling and MMP activation are, in part, responsible for the cardiac structural deformation and heart failure. There is no known program that has declared its aim the investigation of the role of ECM gene therapy in heart failure. The development of transgenic technology, and emerging techniques for in vivo gene transfer, suggest a strategy for improving cardiac function by overexpressing or downregulation of the ECM components such as MMPs, tissue inhibitor of metalloproteinases (TIMPs), transforming growth factor-β1 (TGF-β), decorin, and collagen in cardiomyopathy and heart failure. J. Cell. Biochem. 68:403–410, 1998. © 1998 Wiley-Liss, Inc.

Extracellular matrix dynamics in heart failure: A prospect for gene therapy

In chronic congestive heart failure, an illness affecting more than 4 million Americans, there is impairment of myocardial extracellular matrix (ECM) remodeling. Failing human ventricular myocardium contains activated matrix metalloproteinases (MMPs), which are involved in adverse ECM remodeling. Our studies support the concept that impaired ECM remodeling and MMP activation are, in part, responsible for the cardiac structural deformation and heart failure. There is no known program that has declared its aim the investigation of the role of ECM gene therapy in heart failure. The development of transgenic technology, and emerging techniques for in vivo gene transfer, suggest a strategy for improving cardiac function by overexpressing or downregulation of the ECM components such as MMPs, tissue inhibitor of metalloproteinases (TIMPs), transforming growth factor-beta1 (TGF-beta), decorin, and collagen in cardiomyopathy and heart failure.

Inhibition of pancreatic tumor cell growth in culture by p21WAF1 recombinant adenovirus.

New innovations are needed for the treatment of pancreatic cancer, as current treatments do not offer significant improvements in overall survival. p21WAF1--a tumor suppressor gene--acts as a downstream effector of p53 function and mediates G1 cell cycle arrest by inhibiting cyclin-dependent kinases, which promote cell growth. p21 expression has also been shown to increase more than 20-fold in senescent cells in culture. The replication-defective recombinant adenoviral system (rAd), a major innovation in gene transfer technology, has recently been used in gene therapy applications for various malignancies but not for pancreas cancer. In this study we used rAd-p21 in cell growth inhibition studies of pancreatic tumor cell lines in vitro to explore its potential as a prospective gene therapy for pancreatic adenocarcinoma. We studied two pancreatic cell lines in culture, HPAC and Hs766T. HPAC revealed higher endogenous levels of p21 gene expression at the protein and RNA levels compared to Hs766T. p21 induction was tested using different doses of rAd-p21 to establish an optimum dose for significant induction of p21 gene expression. Tumor cell growth in culture following rAd-p21 infection was also analyzed in both cell lines. HPAC and Hs766T cell lines showed a significant dose-dependent increase in p21 protein expression when infected with rAd-p21. Both cell lines showed significant growth arrest, but Hs766T showed less cell growth inhibition than HPAC cells. Flow cytometric cell cycle analysis of rAd-p21-infected cells showed a statistically significant increase in the number of cells in G0/G1 in HPAC cells. Similar results were also obtained in Hs766T cells, however, the data were not statistically significant. In conclusion, pancreatic tumor cell growth can be inhibited by rAd-p21 in vitro, with significant numbers of tumor cells reverting from S to G0/G1. Thus rAd-p21 may be effective as a candidate gene therapy for pancreatic cancer and should be further evaluated with in vivo studies.

High density lipoproteins and coronary heart disease. Future prospects in gene therapy

Atheroselorosis cardiovascular disease is the leading cause of death in industrial societies. For coronary heart disease, hypercholeterolemia and dyslipoproteinemia are the major risk factors. Low serum levels of cholesterol in the HDL fraction is the most common abnormality found in patients with confirmed coronary artery disease. A therapeutical strategy consists in increasing the serum HDL, cholesterol concentration in order to improve the ‘reverse cholesterol transport’. Studies in transgenic mice and rabbits for human apo A-I or human lecithin cholesterol acyl-transferase showed that overexpression of these proteins increases serum HDL cholesterol concentration and reduces diet induced atherogenesis. Furthermore, adenovirus-mediated transfer of human apo A-I and LCAT genes in mice also increases circulating apo A-I and LCAT. Apo A-I and LCAT are two potential targets for gene therapy of patients with atherosclerosis associated with a low HDL cholesterol level.

ADULT LIVER TRANSPLANTATION FOR METABOLIC LIVER DISEASE

Liver replacement provides an effective method of replacing a failing liver, and corrects the underlying defect in many metabolic conditions. Results of liver transplantation for metabolic diseases have been encouraging, with the exception of hereditary hemochromatosis, in which infectious and for which cardiac complications appear to increase posttransplant mortality. An improved understanding of the underlying genetic and molecular defect will lead to advances in medical therapy and perhaps will decrease the need for liver replacement. The prospects of gene therapy are being pursued for many metabolic disorders, however until this research leads to direct clinical application, liver transplantation remains the only effective option for many patients with metabolic liver disease.

Inducible control of gene expression: prospects for gene therapy

A number of drug-regulated gene expression systems are available for controlling target gene transcription through the use of small-molecule inducing compounds. While the utility of such systems has been demonstrated in vitro and in transgenic mice, recent improvements are likely to make these systems more amenable for use in a therapeutic context, such as gene therapy. These improvements include further optimization of the antiprogestin-regulated gene switch, rendering it more sensitive to RU486, and the synthesis of nonimmunosuppressive rapamycin analogs for use in dimerization-based strategies of gene regulation.

Adenovirus Vector-Mediated Gene Transfer into Human Epileptogenic Brain Slices: Prospects for Gene Therapy in Epilepsy

As a first step in the development of a gene therapy approach to epilepsy, we evaluated the ability of adenovirus vectors to direct the transfer into and expression of a marker gene in human brain slices obtained from patients undergoing surgery for medically intractable epilepsy. Following injection of adenovirus vectors containing the Escherichia coli lacZ gene into hippocampal and cortical brain slices, lacZ mRNA, beta-galactosidase protein, and enzymatic activity were detected, confirming successful gene transfer, transcription, and translation into a functional protein. Transfected cells were predominantly glial, with some neurons expressing beta-galactosidase as well. These results support the potential of adenovirus vectors to transfer genetic information into human epileptogenic brain, resulting in expression of the gene into a functional protein. These findings also have implications for the development of gene therapy approaches to certain seizure disorders. A number of potential therapeutic approaches are discussed, including the elevation of inhibitory neurotransmitter or neuropeptide levels, expression or modulation of postsynaptic receptors, and manipulation of signal transduction systems.