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Frontiers in gene therapy: LDL receptor replacement for hypercholesterolemia
In prior chapters, we reviewed models for forecasting the health of the U.S. elderly and oldest-old population. In Chapter 4 we suggest that a major stimulus to the development of forecasting models is increased scientific knowledge about human senescence and chronic disease. As knowledge increases, the boundaries of senescence and disease become less distinct (Mooradian and Wong, 1991a,b). Models of senescence have evolved from a unidimensional, genetically fixed failure process, to multidimensional stochastic processes where polygenic determination of multiple pathologies and losses of function interact with environment (Finch, 1991; Manton, Stallard, et al., 1992). Forecasting models must keep pace with, and anticipate the future direction of, this evolution—with the complication that, even using multiple data sets, processes will be only partially observed. This chapter reviews and illustrates how biomedical research can be used to construct models, and substantive scenarios, to forecast the effects of future health interventions.
In order to sustain normal metabolic processes, the cell must recruit from its external environment an array of ions, molecules, and macromolecules. Small molecules, such as ions, water, and monosaccharides, can gain entry into the cytosol of the cell through water-filled channels. However, molecules larger than approximately 1000 Da cannot pass through these channels. Therefore, if diffusion through plasma membrane pores or channels were the only method of entry into the cell cytoplasm, larger molecules such as polypeptides, polysaccharides, and other nutrients could not be utilized by the cell for normal metabolic processes. In addition, these larger molecules are often present in the extracellular environment of the cell at much lower concentrations than ions or monosaccharides. Therefore, in order to overcome problems associated with internalizing large and scarce molecules, the cell has developed the process of receptor-mediated endocytosis. The extracellular macromolecules bind to specific, high-affinity receptors on the cell surface, thereby trapping them and concentrating these macromolecules at the plasma membrane.
Fulminant hepatic failure (FHF) is defined by the appearance of severe liver injury with hepatic encephalopathy in a previously healthy person. There are an estimated 2,000 cases of FHF in the United States yearly, representing 0.1% of all deaths and, perhaps, 6% of liver-related deaths. The causes of FHF are many, the chief ones in the United States being hepatitis A; B; non-A, non-B and drug induced liver disease. There are no specific therapies for FHF, however, liver transplantation is recommended for situations in which spontaneous recovery appears unlikely. Factors that are valuable in assessing the likelihood of spontaneous recovery are static features such as patient age and etiology of FHF and dynamic features including encephalopathy grade, prothrombin time, and serum bilirubin. Presently, approximately 7% of all liver transplants are done for FHF and the 1-year patient survival rates average 63%, somewhat less than survival rates for nonfulminant liver disease, averaging 78%. The management of patients with FHF is challenging, particularly important being monitoring and early treatment of infections, hemodynamic abnormalities, and brain edema. Innovative approaches to management and therapy include use of cytoprotective or antiviral medications, hepatic support systems, extracorporeal liver support, hepatocyte transplantation, auxiliary liver transplantation, and xenotransplantation. None of these are of proven benefit, but many are promising as a means to support the patient with FHF until spontaneous recovery occurs or a suitable liver graft is available for transplantation.
The LDL receptor's structure and function at the cellular level has been well characterized mostly by the work of Goldstein, Brown, and co-workers. A large number of mutations of the LDL receptor gene has been reported worldwide and their clinical relevance to the severity of familial hypercholesterolemia evaluated. There is ample evidence that the LDL receptor does not mediate LDL clearence only; it also is involved in the clearance of triglyceride-rich lipoproteins including postprandial lipoproteins. Several nutrients, drugs, and hormones modulate the function of the LDL receptor, through different mechanisms. Exciting new information regarding the identification of other members of the LDL receptor family has been evolving. A role of the LDL receptor and its family members in processes beyond lipoprotein clearance is emerging.
Numerous studies have reported successful allotransplantation of hepatocytes. However, none have shown long-term correction of a liver-related metabolic defect, In this study, we used a method of regional hepatocyte transplantation and subsequent induction of transplanted cell proliferation by regeneration response in the transplant-bearing liver lobes, New Zealand White rabbits were used as cell donors and Watanabe heritable hyperlipidemic (WHHL) rabbits were used as cell recipients (2x10(8) cells/rabbit). All recipient rabbits were maintained on daily cyclosporine, Two weeks after baseline serum cholesterol determination, group I WHHL rabbits (n=7) received an infusion of cells into the right lateral liver lobe, and a loose ligature was placed around the portal venous branch supplying the anterior lobe, After 1 week, to allow engraftment, the portal venous branch was ligated, which resulted in the atrophy of the affected liver parenchyma and induction of hyperplasia in the transplant-bearing liver tissue, Group II rabbits (n=6) were transplanted with New Zealand White hepatocytes without portal branch ligation (PBL) and group III rabbits (n=4) were subjected to sham transplantation (saline) and PBL, The experimental period extended to 150 days after transplantation. All WHHL rabbits transplanted with normal hepatocytes showed reduction in serum cholesterol and low-density lipoprotein (LDL) levels, Group I(PBL-stimulated) recipients demonstrated a more pronounced and sustained effect than group II animals (P<0.05). Group III controls showed only a slight, typical for aging decrease in serum cholesterol, Group I recipient livers perfused with LDL labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine perchlorate (DiI) showed much higher numbers of DiI-LDL-positive hepatocytes than those of group II recipients, In conclusion, a liver regeneration stimulus enhanced the population of transplanted hepatocytes and their functional effect in a large animal model of inborn error of liver metabolism.
This chapter contains section titled:
IntroductionThe Blood Option: Concepts and BasicsBlood Gene Expression Profiling: MethodologyBiomedical ApplicationsValidation StrategiesConclusions and PerspectivesAcknowledgments
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Since the original description of hypertrophic cardiomyopathy (HCM) in the nineteenth century, sudden cardiac death (SCD)
has been the most dreadful but fortunately uncommon manifestation of HCM. The death is tragic as it often occurs without distinct
warning signs or symptoms in apparently healthy and young athletes. Consequently, HCM has received considerable media coverage
and publicity because of occasional catastrophic death of professional athletes in the field or soon after participation in
competitive sports. However, despite the recognition of SCD as a catastrophic outcome in HCM, the efforts to resolve the problem
have been compounded by the lack of reliable predictors of the risk of SCD as well as the relatively low prevalence of HCM
in the general population and hence inadequate awareness about the disease on routine daily medical practice. The advent of
the automatic internal cardioverter/defibrillator (AICD) has somewhat alleviated the risk of SCD. However, the success depends
on early detection of those at risk, particularly, the asymptomatic individuals in order to prevent tragic events. Increased
awareness of the physicians along with robust and comprehensive means of early detection, risk stratification, and intervention
would be necessary to successfully mitigate the risk of SCD in patients with HCM.
This chapter contains section titled:
This chapter contains section titled:
Fulminant hepatic failure (FHF) is defined by the appearance of severe liver injury with hepatic encephalopathy in a previously healthy person. There are an estimated 2,000 cases of FHF in the United States yearly, representing 0.1% of all deaths and, perhaps, 6% of liverrelated deaths. The causes of FHF are many, the chief ones in the United States being hepatitis A; B; non-A, non-B and drug induced liver disease. There are no specific therapies for FHF, however, liver transplantation is recommended for situations in which spontaneous recovery appears unlikely. Factors that are valuable in assessing the likelihood of spontaneous recovery are static features such as patient age and etiology of FHF and dynamic features including encephalopathy grade, prothrombin time, and serum bilirubin. Presently, approximately 7% of all liver transplants are done for FHF and the 1-year patient survival rates average 63%, somewhat less than survival rates for nonfulminant liver disease, averaging 78%. The management of patients with FHF is challenging, particularly important being monitoring and early treatment of infections, hemodynamic abnormalities, and brain edema. Innovative approaches to management and therapy include use of cytoprotective or antiviral medications, hepatic support systems, extracorporeal liver support, hepatocyte transplantation, auxiliary liver transplantation, and xenotransplantation. None of these are of proven benefit, but many are promising as a means to support the patient with FHF until spontaneous recovery occurs or a suitable liver graft is available for transplantation. (Hepatology 1995;21:240–252).
Victor J. Dzau, Choong-Chin Liew, eds. 308 pp., illustrated. Malden, Mass: Blackwell Futura; 2007. $144.95. ISBN 978–1-4051–3394-4.
Given the rapidly growing knowledge in the field of genetics and genomics and the furious pace of translational research, the average cardiologist today is faced with information overload and a discipline that is changing in fundamental ways. The challenges are further magnified by the fact that training curricula for cardiologists have not yet bridged the gap between conventional practice of cardiovascular medicine and the futuristic trends in the discipline. In this context, this book fills a unique void, and is a must read for cardiologists, including fellows and faculty.
The …
The development of strategies for gene therapy in the liver is a challenging task because this organ is particularly involved in the manifestation of numerous genetic diseases as well as in malignant and infectious diseases. Most of the available techniques for gene transfer were already applied to hepatocytes ex vivo. Attempts to retransplant retrovirally transduced hepatocytes back to patients have shown the limits of the ex vivo approach. The obvious alternative would be the development of suitable techniques for gene transfer in vivo. Both viral and non-viral delivery systems were tested in animal models with limited success. This article summarizes the state of the art of the available techniques and vectors and discusses the essential future developments.
There has been much progress in our understanding of molecular mechanisms in the pathogenesis of inherited metabolic disorders. In addition, powerful new molecular techniques have made possible phenotypic alterations by delivery of foreign genes to target cells. As a result, concepts and methods that would have been considered purely science fiction 10 years ago can now be found in human clinical trials engaged in the treatment of these disorders. In this review, we have attempted to provide an introduction and survey of the topic of gene therapy, with specific examples of laboratory and clinical achievements to date, and highlights on potentials for applications in digestive diseases.
To facilitate clinical applications of retroviral-mediated human gene transfer, retroviral vectors must be of high titer and free of detectable replication-competent retroviruses. The purpose of this study was to optimize methods of retroviral vector production and transduction. Studies were conducted using 22 retroviral vector producer cell lines. Inactivation of retroviral vectors was greater at 37 degrees C than at 32 degrees C. A 5- to 15-fold increase of vectors was produced at 32 degrees C compared to 37 degrees C; the vector increase at 34 degrees C was intermediate. For example, PA317/G1Na.40 grew to a titer of 1.8 x 10(7) cfu/ml at 32 degrees C, compared to 5.0 x 10(5) cfu/ml at 37 degrees C. The production of retroviral vectors was scalable achieving similar results in flasks, roller bottles, or a CellCube Bioreactor. Retroviral vectors were concentrated 15-24 times with vector recovery ranging from 91 to 96% in a Pellicon tangential flow filtration system. Retroviral supernatants were successfully lyophilized. The combination of glucose or sorbitol with gelatin resulted in recovery rates of 64-83%. In studies on transduction by retroviral vectors, centrifugation of vector supernatants onto target cells significantly increased transduction efficiency as measured by vector titration for G418 resistance, fluorescence-activated cell sorting (FACS), and polymerase chain reaction (PCR) analyses. The combination of the above methods has significantly increased the growth and transduction by this vector system.
The liver is an important and attractive target for the development of gene therapy strategies. Many genetic diseases are manifested in the liver, and both infectious and malignant diseases affect this organ. Retroviral and adenoviral vectors have been shown to infect hepatocytes with varying efficiently in vitro and in vivo. The presence of unique receptors at the cellular membrane of hepatocytes has stimulated the development of transfer strategies based on receptor targeting of vectors. The results of a first clinical trial for gene therapy in the liver based on ex vivo gene delivery has shown both the feasibility and the limits of current technology. This review discusses both existing vectors and strategies and prospective developments towards liver-directed gene therapy of genetic and malignant diseases.
Smooth muscle cells (SMC) are a central cell type involved in multiple processes of coronary artery diseases including restenosis and therefore are major target cells for different aspects of gene transfer. Previous attempts to transfect primary arterial cells using different techniques like liposomes, CaPO4 and electroporation resulted in only low transfection efficiency. The development of recombinant adenoviruses dramatically improved the delivery of foreign genes into different cell types including SMC. However, cloning and identification of recombinants remain difficult and time-consuming techniques. The present study demonstrates that a complex consisting of reporter plasmid encoding firefly luciferase (pLUC), polycationic liposomes and replication-deficient adenovirus was able to yield very high in vitro transfection of primary human smooth muscle cells under optimized conditions. The technique of adenovirus-assisted lipofection (AAL) increases transfer and expression of plasmid DNA in human smooth muscle cells in vitro up to 1000-fold compared to lipofection. To verify the applicability of AAL for gene transfer into human smooth muscle cells we studied a gene therapy approach to suppress proliferation of SMC in vitro, using the prokaryotic cytosine deaminase gene (CD) which enables transfected mammalian cells to deaminate 5-fluorocytosine (5-FC) to the highly toxic 5-fluorouracil (5-FU). The effect of a transient CD expression on RNA synthesis was investigated by means of a cotransfection with a RSV-CD expression plasmid and the luciferase reporter plasmid. Western blot analysis demonstrated high expression of CD protein in transfected SMC. Cotransfected SMC demonstrated two-fold less luciferase activity in the presence of 5-FC (5 mmol/l) after 48 h compared to cells transfected with a non-CD coding plasmid. The data demonstrate that a transient expression of CD could be sufficient to reduce the capacity of protein synthesis in human SMC. This simple and effective in vitro transfection method may also be applicable to in vivo delivery of target genes to the vascular wall to inhibit SMC proliferation.
The term human gene therapy is defined as the transfer of DNA or RNA into human cells for therapeutic purposes. Significant advancements in recombinant DNA technology have led to an understanding of the molecular bases of many diseases ranging from inherited disorders to certain malignancies to infectious diseases such as acquired immunodeficiency syndrome (AIDS). The increasing ability to characterize a disease at its molecular level has made genetic interventions feasible and provides a rationale for gene therapy. The essence of gene therapy is the correction or replacement of the dysfunctioning genetic element with its normal counterpart or the specific disruption of a harmful gene product (1).
The application of gene therapy to acute inflammation has not received as much research attention as has the treatment of genetically-based diseases, cancer, and viral infections. However, gene therapy as a drug delivery system offers several theoretical and practical advantages over current protein delivery systems. These include the ability to target therapies to individual tissues or cell types, to locally produce proteins that can act intracellularly or in an autocrine, juxtacrine, or paracrine fashion, and to sustain new protein synthesis for periods up to several weeks after a single administration. Although retrovirus, herpes simplex, and adeno-associated virus have been proposed for gene therapy in cancer and in genetic diseases, nonviral and adenovirus approaches appear most applicable as drug delivery systems due to their rapid onset and short duration of transgene expression. The relative modest transduction efficiencies obtained at present with nonviral approaches, and the inherent inflammatory properties of first-generation adenovirus constructs, however, have limited their usefulness to date. The present review discusses the theoretical and practical benefits of specific gene therapy approaches for the treatment of acute inflammatory diseases, as well as our experiences with liposome:plasmid DNA and adenovirus-based approaches. Although a number of technical and theoretical hurdles remain before it can be evaluated in humans with acute inflammation, gene therapy offers a novel approach for the treatment of acute inflammation, and will likely enter the armamentarium of critical care physicians in the near future.
Index Introduction Etiology Viral hepatitis Acetaminophen Drug reactions Clinical features Encephalopathy Cerebral edema Coagulopathy Infections Metabolic changes Hemodynamic changes Diagnosis Management Intensive care Specific therapy Liver transplantation Prognostic indicators Future developments Artificial and Bioartificial support systems Hepatocyte transplantation
To develop a novel strategy of nonautologous somatic gene therapy, we now demonstrate the feasibility of culturing genetically modified fibroblasts within an immunoprotective environment and the optimal conditions required for their continued survival in vitro. When mouse Ltk(-) fibroblasts transfected with the human growth hormone gene were enclosed within permselective microcapsules fabricated from alginate-polylysine-alginate, they continued to secrete human growth hormone at the same rates as the nonencapsulated cells. They also continued to proliferate in vitro for at least 1 month even though their viability gradually declined to about 50%. The viability can be improved by controlling for (a) temperature during encapsulation, (b) duration of treatment with polylysine, (c) duration of liquefying the core alginate with sodium citrate, and (d) cell density at the time of encapsulation. The best conditions leading to improved survival and maximum proliferation of cells within the microcapsules were obtained by encapsulating the cells at 4 to 10 degrees C instead of room temperature, coating the microspheres with polylysine for 6 to 10 min instead of 20 min, liquefying the core alginate by treating with citrate for 20 min instead of 6 to 10 min, and using a concentration of 2 x 10(6) cells/mL of alginate for encapsulation. Under such conditions, normally adherent and genetically engineered mouse fibroblasts survived and proliferated optimally within the microcapsule environment. The encapsulated fibroblasts maintained their level of transgene expression while recombinant gene products such as human growth hormone could diffuse through the microcapsule membrane without impediment. The demonstration that genetically modified fibroblasts can survive and continue to deliver recombinant gene products from within these microcapsules and the optimization for their maximal viability and growth within microcapsules should increase the potential for success in using such microencapsulated recombinant cells for somatic gene therapy. (c) 1994 John Wiley & Sons, Inc.
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