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PubMedDate not supplied

Gene therapy for liver disease.

With major advances in biomedical science over the last 2 decades, the possibility of treating human disease at a genetic level has become a tantalizing possibility. As a result, a growing number of investigators are focusing on the development of techniques to deliver therapeutic genes into cells. The liver has been a model organ in the development of this gene transfer technology. This review focuses on the attributes and limitations of the current gene delivery systems that have been explored in the context of liver disease and highlights the obstacles that must be addressed before hepatic gene therapy becomes a clinical reality.

Gene Transfer Techniques↗
PubMed1995

Granulocyte-macrophage colony-stimulating factor mRNA stabilization enhances transgenic expression in normal cells and tissues.

To increase transgenic production of granulocyte-macrophage colony-stimulating factor (GM-CSF), we mutated the mRNA's 3'-untranslated region, AUUUA instability elements. Expression vectors containing human or murine GM-CSF cDNAs coding for wild-type (GM-AUUUA) or mutant versions with reiterated AUGUA repeats (GM-AUGUA) were transfected into cells in culture or animals using particle-mediated gene-transfer technology. Normal peripheral blood mononuclear cells accumulated 20-fold greater levels of GM-CSF mRNA and secreted comparably greater amounts of cytokine after transfection with hGM-AUGUA expression vectors versus hGM-AUUUA. hGM-AUGUA mRNA was fivefold more stable (t 1/2 = 95 minutes) than hGM-AUUUA mRNA (t 1/2 = 20 minutes), accounting for elevated steady-state levels. Transfection site extracts and serum samples obtained 24 hours after gene transfer of hGM-AUGUA cDNA into mouse skin contained greater than 32 ng/mL and 650 pg/mL of GM-CSF protein, respectively, compared with 0.33 ng/mL and less than 8 pg/mL for hGM-AUUUA cDNA. GM-CSF produced from mGM-AUGUA cDNA transfected into rat abdominal epidermis induced a profound neutrophil infiltrate. These data suggest a novel strategy for enhanced production of biologically active cytokines by normal cells after in vivo gene transfer.

Animals↗
PubMedDate not supplied

Science: part of the problem, most of the answer.

The history of the drug approval process in the United States includes three phases. First, the Food and Drug Act of 1906 essentially required that the labeling of drugs be truthful. The 1938 Food, Drug, and Cosmetic Act added a requirement that drugs be proven safe, and the 1962 act added the requirement that drugs be efficacious. Each of these steps required more and more sophisticated science. Subsequent to the Kefauver-Harris Amendments of 1962, the number of innovative molecular entities each year has declined, the reasons for which have been subject to great debate. This decline has paralleled decreases in innovation across almost all American industry, leading to questions about the future of our country as an industrialized society. Both the Carter and Reagan administrations attempted to address this problem in a number of ways, including cutting back on those regulations that are perceived as unnecessary. Other innovative approaches have been used, such as the establishment of an Office of Small Manufacturers Assistance in the FDA Bureau of Medical Devices, mandated by the 1976 Medical Device Amendments. The latter came about in recognition of the fact that small businesses tend to be more creative and efficient than larger industries and more adversely affected by regulation. However, the problems raised in the regulation of technology transfer almost inevitably arise because of perceived scientific questions. Such questions, in turn, can only be answered by good science performed by the sponsor and understood by the regulator. Thus, it is essential that the FDA be staffed with knowledgeable scientists who can interact easily with their peers in academia and industry. Although science is often the cause of our troubles, it is also our only hope for minimizing the costs of new drugs and other technology. In turn, minimizing such costs will maximize the opportunities for innovation.

Costs and Cost Analysis↗
PubMed2001

A flow cytometry technique for measuring chromosome-mediated gene transfer.

BACKGROUND: Using artificial chromosome expression systems (ACes), we have developed a unique and rapid screening technique to quantify delivery of foreign DNA into cells in vitro. Delivery was measured within 24 h after transfection, using flow cytometry to detect the transfer of ACes labeled with thymidine analogue. This technique can be used to optimize delivery parameters of ACes and heterologous DNA into cells and eventually tissue. METHOD: Chinese hamster ovary (CHO) cells carrying artificial chromosomes were grown in media supplemented with iododeoxyuridine (IdUrd). The 60-mb artificial chromosome was purified by flow cytometry sorting and transfected into Chinese hamster lung fibroblast cells (V79-4) or mouse connective tissue cells [LM(tk-)] using LipofectAMINE 2000trade mark, a cationic lipid, and Superfecttrade mark, a cationic dendrimer. The cells were incubated with an FITC-conjugated anti-bromodeoxyuridine (BrdUrd) antibody and analyzed by flow cytometry. IdUrd-incorporated artificial chromosome expressing green fluorescent protein (GFP) was transfected into V79-4 cells. Delivery was measured at 24 h and GFP expression was detected at 48 h. RESULTS: The delivery of intact artificial chromosomes into V79-4 and LMtk- cells was detected within 2 h and up to 48 h post-transfection. Maximum delivery rates of 20% and 14% were observed using LipofectAMINE 2000 and Superfect, respectively. Flow cytometry data correlated with microscopic observations. IdUrd incorporation resulted in less quenching after staining with Hoechst 33258 and chromomycin A3 than BrdUrd incorporation. The fluorescence intensity of the FITC-conjugated anti-BrdUrd antibody was greater with IdUrd-incorporated chromosomes than with BrdUrd-incorporated chromosomes. CONCLUSION: The results indicate that IdUrd-labeled artificial chromosomes can be detected 24 h after transfection. This efficient, sensitive, high-throughput detection technique is being used to evaluate and optimize other transfer technologies (e.g., electroporation and sonoporation), different delivery reagents, and protocols in a variety of cells in vitro. This work represents the first step in utilizing artificial chromosomes as nonviral vectors for gene therapy.

Animals↗
PubMed2003

Therapeutic cloning in the mouse.

Nuclear transfer technology can be applied to produce autologous differentiated cells for therapeutic purposes, a concept termed therapeutic cloning. Countless articles have been published on the ethics and politics of human therapeutic cloning, reflecting the high expectations from this new opportunity for rejuvenation of the aging or diseased body. Yet the research literature on therapeutic cloning, strictly speaking, is comprised of only four articles, all in the mouse. The efficiency of derivation of embryonic stem cell lines via nuclear transfer is remarkably consistent among these reports. However, the efficiency is so low that, in its present form, the concept is unlikely to become widespread in clinical practice.

Animals↗
PubMed2000

Cloning adult animals - what is the genetic age of the clones?

The rapid progress in cloning research along with its many ramifications will soon have a significant beneficial impact on basic research, agriculture, and biomedicine. However, for the nuclear transfer technology to reach its fullest potential, it is important to understand whether the cloning procedure can reverse cellular aging and generate clones with normal genetic and physiological age, similar to those produced from natural reproduction. Telomere shortening is believed to correlate with cellular aging both in vitro and in vivo. Telomere lengths in cells of cloned individuals thus may reflect their genetic age. However, controversies have developed over whether the eroded telomere in somatic cells used for nuclear transfer can be restored during the cloning process.

Aging↗
PubMedDate not supplied

Gene therapy for in vivo bone formation: recent advances.

Gene therapy has developed during the last two decades as a promising strategy for orthopaedics applications, since several different gene transfer techniques proved to be effective, both in vitro and in vivo, for the induction of bone formation. Successful results have been achieved with gene-based bone healing strategies in several preclinical studies, using different animal models. New genes and new viral and non-viral vector constructs have been developed to reduce the risks and safety issues, widening the field of possible applications and improving the potential therapeutical effects. We review the latest gene transfer technologies employed for in vivo bone formation, focusing on the recently identified network of growth factors and genes involved in the modulation of the osteogenetic process and on the variety of vectors utilized for gene delivery.

Adenoviridae↗
PubMed1994

Metabolic engineering of plant secondary products.

Plants interact with their environment by producing a diverse array of secondary metabolites. Many of these compounds are valued for their medicinal, industrial or agricultural properties. Other secondary products are toxic or otherwise undesirable and can reduce the commercial value of crops. Gene transfer technology offers new opportunities to modify directly plant secondary product synthesis through metabolic engineering. This article reviews some of the strategies which have been used to increase or decrease the synthesis of specific plant metabolites, as well as methods for expanding the biosynthetic capabilities of individual species.

Gene Expression↗
PubMed1993

Intraarticular expression of biologically active interleukin 1-receptor-antagonist protein by ex vivo gene transfer.

Gene therapy offers a radical different approach to the treatment of arthritis. Here we have demonstrated that two marker genes (lacZ and neo) and cDNA coding for a potentially therapeutic protein (human interleukin 1-receptor-antagonist protein; IRAP or IL-1ra) can be delivered, by ex vivo techniques, to the synovial lining of joints; intraarticular expression of IRAP inhibited intraarticular responses to interleukin 1. To achieve this, lapine synoviocytes were first transduced in culture by retroviral infection. The genetically modified synovial cells were then transplanted by intraarticular injection into the knee joints of rabbits, where they efficiently colonized the synovium. Assay of joint lavages confirmed the in vivo expression of biologically active human IRAP. With allografted cells, IRAP expression was lost by 12 days after transfer. In contrast, autografted synoviocytes continued to express IRAP for approximately 5 weeks. Knee joints expressing human IRAP were protected from the leukocytosis that otherwise follows the intraarticular injection of recombinant human interleukin 1 beta. Thus, we report the intraarticular expression and activity of a potentially therapeutic protein by gene-transfer technology; these experiments demonstrate the feasibility of treating arthritis and other joint disorders with gene therapy.

Animals↗
PubMed2006

Virus-based gene therapy strategies for bone regeneration.

Gene therapy has emerged as a promising strategy for the repair and regeneration of damaged musculoskeletal tissues. Application of this paradigm to bone healing has shown enhanced efficacy in preclinical animal studies compared to conventional bone grafting approaches. This review discusses current and emerging virus-based genetic engineering strategies for the delivery of therapeutic molecules which promote skeletal regeneration. Viral gene delivery vectors are discussed in the context of bone repair in order to illustrate the challenges and applications of these methods with tissue-specific examples. Moreover the concepts discussed can be broadly applied to promote healing in a wide range of tissues. We also present important considerations involved in the application of these gene therapy techniques to a variety of osteogenic (e.g. bone marrow-derived cells) and non-osteogenic (e.g. fibroblasts and skeletal myoblasts) cell types. Criteria for the selection of regenerative molecules with soluble versus intracellular modes of action and emerging combinatorial approaches are also discussed. Overall, gene transfer technologies have the potential to overcome limitations associated with existing bone grafting approaches and may enable investigators to design therapies which more closely mimic the complex spatial and temporal cascade of proteins involved in endogenous bone development and repair.

Animals↗
PubMed1997

Gene therapy of single-gene disorders: preface to the special section.

Gene therapy was introduced into clinical practice with great excitement, much publicity and considerable optimism in the early 1990s. Scientific evaluation of the early clinical trials has, however, greatly reduced the initial optimism. Follow-up studies have revealed that many early gene therapy trials mainly represented gene transfer into patients, possibly with short-term effects, but not true gene therapy where the course of the disease is permanently affected. This has lead to critical re-evaluation of the approaches taken. Clearly, more basic understanding is needed of the molecular mechanisms of the diseases treated. For this purpose, better animal models for human diseases are necessary. One of the biggest obstacles for gene therapy has been the lack of adequate vector systems. Development of new vectors for efficient and targeted delivery and uptake of therapeutic genes is a crucial area where progress needs to be made. The rationale for gene therapy depends largely on the type of disease to be treated. Recessively inherited single-gene disorders represent diseases where the concept of gene therapy--addition of a therapeutic gene to restore the lost function of two mutant alleles--is easily understood and rarely questioned. However, most gene therapy protocols are focused on multifactorial diseases such as malignancies where the therapeutic approach is quite different. While gene transfer technologies are being developed into truly effective gene therapy, the fight against inherited single-gene disorders also continues at population level by carrier screening and prenatal diagnostics where rapid methodological developments are taking place.

Alleles↗
PubMed2003

Marked enhancement in gene expression by targeting the human insulin receptor.

BACKGROUND: Exogenous genes can be delivered to cells without viral vectors using an "artificial virus" comprised of nonviral plasmid DNA encapsulated in the interior of 85 nm pegylated immunoliposomes (PIL). The liposomes are targeted to cells with receptor-specific targeting ligands such as receptor-specific peptidomimetic monoclonal antibodies. METHODS: The levels of luciferase gene expression in human or rat glioma cells are measured after targeting the PIL-encapsulated plasmid DNA via the human insulin receptor, the human epidermal growth factor receptor, or the rat transferrin receptor. The luciferase expression plasmids were either derived from pCEP4, which contains the Epstein-Barr nuclear antigen-1/oriP replication system, or from pGL2, which lacks this system for episomal replication of plasmid DNA. RESULTS: Depending on the plasmid construct used and the receptor targeted, the peak luciferase gene expression varied more than 200-fold from 1.8 +/- 0.1 to 419 +/- 31 pg luciferase per mg cell protein. With the same plasmid, the peak level of gene expression following delivery to the cell via the human insulin receptor was 100-200-fold higher than gene expression following delivery via either the epidermal growth factor receptor or the transferrin receptor. There was no gene expression if the targeting ligand on the PIL was replaced with a nonspecific isotype control antibody. CONCLUSIONS: The extent to which an exogenous gene is expressed within a cell via a nonviral, receptor-mediated gene transfer technology is determined by the receptor specificity of the targeting ligand. The highest levels of gene expression are obtained after targeting the insulin receptor, and this may derive from the nuclear targeting properties of this receptor system.

Animals↗
PubMed2001

Rapid generation of a tetracycline-inducible BCR-ABL defective retrovirus using a single autoregulatory retroviral cassette.

The development of chronic myelogenous leukemia (CML) models in mice using an inducible BCR-ABL gene has been hampered by the requirement of sequential expression of tTA (Tet repressor-VP16 fusion protein) and Tet-OP sequences in the same cells after separate transfection. This double transfection strategy is time consuming as it requires screening of many hundreds of individual clones and cannot be applied to primary hematopoietic cells. To generate a tetracycline-inducible BCR-ABL retrovirus, we have subcloned BCR-ABL p210 cDNA in the SIN-Retro-TET vector, which allows regulated expression of a gene of interest in a single autoregulatory cassette, containing both tTA and Tet OP sequences. Retroviral particles were obtained by transfecting the SIN-BCR-ABL p210 construct into the 293 cells and by VSVG pseudotyping. To determine the functionality of the retrovirus, the IL-3-dependent murine Ba/F3 cell line was retrovirally transduced and clones were grown in the absence of both IL-3 (to select for transformed cells) and a tetracycline analog, doxycycline (to induce BCR-ABL expression). Using this technique, polyclonal Ba/F3 cells and several growth factor-independent Ba/F3 clones expressing BCR-ABL were obtained within 2-3 weeks. A single dose of doxycycline added to the medium (1 microg/ml), induced in different clones, a reduction of BCR-ABL protein levels by 60-90% at 24 h, leading to cell death in the absence of IL-3. In several individual clones, BCR-ABL expression was further reduced to become almost undetectable at 48 h. The doxycycline-regulated BCR-ABL expression was stable, as many clones maintained in culture for >8 months showed a persistent inhibitory response to doxycycline addition in the medium. In in vivo experiments, subcutaneous injection of 2 x 10(6) Ba/F3-SIN p210 cells in nude mice induced visible tumors in 2 weeks and all established tumors completely regressed upon addition of doxycycline in the drinking water (200 microg/ml). To determine the functionality of the inducible BCR-ABL retrovirus in vivo, primary Lin- bone marrow cells were transduced with SIN-p210 and transplanted in lethally irradiated mice. All transplanted mice had successful hematopoietic reconstitution and BCR-ABL integration was found in the peripheral blood of seven out of 14 mice available for long-term analysis (>6 months). However, despite evidence of retrovirus-mediated gene transfer, there was no evidence of leukemia, due either to low viral titers or to the relative inefficiency of the minimal CMV promoter in primary hematopoietic cells. Thus, these results demonstrate for the first time, to our knowledge, the feasibility to generate an inducible BCR-ABL retrovirus in a single step, in the context of an immortalized cell line. Our data suggest that with further improvements of the retrovirus-mediated gene transfer technology, it might be possible to generate inducible leukemia models in mice by the use of single retroviral constructs.

Animals↗
PubMed2003

Mutagenesis by retroviral transgene insertion: risk assessment and potential alternatives.

Focus on retroviral gene transfer into hematopoietic cells has previously led to a proposed classification of side effects and discussion of issues related to combinatorial toxicity (including oncogenicity) of genetic interventions. Recent data underline that this discussion has to be broadened to any type of stable transfer technology. This review summarizes insights obtained in cell culture and animal models that were specifically designed to evaluate side effects of transgene insertion and new approaches of insertion site targeting. Ongoing research aims to reduce side effects of therapeutic transgene insertion by dose optimization and definition of contributing risk factors.

Base Sequence↗
PubMed1994

International transfers of National Health Service reforms: problems and issues.

Recent market-style reforms in the UK National Health Service (NHS) have attracted the attention of health care professionals, managers, and policy-makers from many developing and central and eastern European countries. This article asks how NHS managers and health professionals should react to such international interest. Six key issues have to be considered when introducing market-style reforms into developing and eastern European countries. First, health sector reform should be formulated on the basis of sound research and evidence. We should also take particular care in understanding how efficiency contributes to health policy objectives and the extent to which private management practices are appropriate to the public sector. We should also question the value of international technology transfer and evaluate the experiences of health sector reform in other countries. Last we should look at the way in which health sector reform is formulated and implemented. Here we highlight important questions about the suitability of replicating the UK experience in other countries.

Europe↗
PubMed2004

Targeted gene transfer in heart failure: implications for novel gene identification.

Heart failure remains an intractable disease with epidemic proportions in the Western World. While progress in conventional treatment modalities for congestive heart failure is making steady and incremental gains to reduce this disease burden, there remains a need to explore new potentially therapeutic approaches. Gene therapy, for example, was initially envisioned as a treatment strategy for inherited monogenic disorders. It is now apparent that gene therapy has broader potential that also includes acquired polygenic diseases, such as heart failure. Advances in the understanding of the molecular basis of congestive heart failure, together with the evolution of increasingly efficient gene transfer technology, has placed congestive heart failure within reach of gene-based therapy. In addition, gene-based reconstitution of a normal phenotype allows us to closely examine the behavior of a large number of transcripts as the heart fails and is rescued by genetic manipulations.

Animals↗
PubMedDate not supplied

Traffic-related injury prevention interventions for low-income countries.

Traffic-related injuries have become a major public health concern worldwide. However, unlike developed or high-income countries (HICs), many developing or low-income countries (LICs) have made very little progress towards addressing this problem. Lack of the progress in LICs is attributable, in part, to their economic situation in terms of their governments' lack of resources to invest in traffic safety, cultural beliefs regarding the fatalism of injuries, competing health problems particularly with the emergence of HIV/AIDS, distinctive traffic mixes comprising a substantial number of vulnerable road users for whom less research has been done, low literacy rates precluding motorists to read and understand road signs, and peculiar political situations occasionally predominated by dictatorship and non-democratic governments. How then can LICs tackle the challenge of traffic safety from the experiences of HICs without reinventing the wheel? This paper reviews selected interventions and strategies that have been developed to counter traffic-related injuries in HICs in terms of their effectiveness and their applicability to LICs. Proven and promising interventions or strategies such as seat belt and helmet use, legislation and enforcement of seat belt use, sidewalks, roadway barriers, selected traffic-calming designs (e.g., speed ramps/bumps), pedestrian crossing signs combined with clearly marked crosswalks, and public education and behavior modification targeted at motorists are all feasible and useable in LICs as evidenced by data from many LICs. While numerous traffic-related injury policy interventions and strategies developed largely in HICs are potentially transferable to LICs, it is important to consider country-specific factors such as costs, feasibility, sustainability, and barriers, all of which must be factored into the assessment of effectiveness in specific LIC settings. Almost all interventions and strategies that have been proven effective in HICs will need to be evaluated in LICs and particular attention paid to the effectiveness of enforcement measures. It behooves LIC governments, however, to ensure that only standard, approved safety devices like helmets are imported into their countries. Additionally, LICs may need to improvise and innovate in the traffic safety technology transfer.

Accidents, Traffic↗
PubMed2004

Organ-specific expression of the lacZ gene controlled by the opsin promoter after intravenous gene administration in adult mice.

BACKGROUND: The tissue-specific expression of an exogenous gene, under the influence of a tissue-specific promoter, has been examined in the past with pro-nuclear injections of the transgene and the development of transgenic mouse models. 'Adult transgenics' is possible with the acute expression of an exogenous gene that is administered to adult animals, providing the transgene can be effectively delivered to distant sites following an intravenous administration. METHODS: The organ specificity of exogenous gene expression in adult mice was examined with a bacterial beta-galactosidase (LacZ) expression plasmid under the influence of the bovine rhodopsin gene promoter. The 8-kb plasmid DNA was delivered to organs following an intravenous administration with the pegylated immunoliposome (PIL) non-viral gene transfer technology. The PIL carrying the gene was targeted to organs with the rat 8D3 monoclonal antibody (MAb) to the mouse transferrin receptor (TfR). RESULTS: The rhodopsin/beta-galactosidase gene was expressed widely in both the eye and the brain of adult mice, but was not expressed in peripheral tissues, including liver, spleen, lung, or heart. Ocular expression included the retinal-pigmented epithelium, the iris, and ciliary body, and brain expression was observed in neuronal structures throughout the cerebrum and cerebellum. CONCLUSIONS: The expression of trans-genes in adult animals is possible with the PIL non-viral gene transfer method. The opsin promoter enables tissue-specific gene expression in the eye, as well as the brain of adult mice, whereas gene expression in peripheral tissues, such as liver or spleen, is not observed.

Animals↗