Nucleic acid-based modulation of cardiac gene expression for the treatment of cardiac diseases. Approaches and perspectives

During the past few years major conceptual and technical advances have been made towards the therapeutic modulation of cardiac gene expression for the treatment of cardiac diseases. Among these are 1) the identification of new molecular therapy targets in cardiac disorders, often derived from genetic animal models. 2) A better understanding of the molecular and cellular determinants of cardiac gene transfer in vivo, in animal models and in first clinical trials. 3) The development of novel regulatable and long-term stable vector systems. This review is focused on nucleic acid-based modulation of cardiac calcium homeostasis as a paradigm for the new gene therapeutic approaches, since recent landmark papers have suggested this to be a molecular target of key importance in heart failure. In particular, the development of severe heart failure in the genetic MLP(-/-) animal model could be completely abolished by the targeted ablation of phospholamban (PL), a key regulator of cardiac calcium homeostasis. This impressive effect of permanent germline PL ablation provides-in conjunction with former important work on disturbed calcium handling in the failing human heart-a rationale for the gene therapeutic approach of ad hoc suppression of PL by antisense strategies (antisense RNAs, ribozymes, RNA interference) or PL variants. Based on the broad spectrum of methods employed to characterize this general strategy, PL-targeted approaches may be considered as a paradigm of future genetic treatments of cardiac disorders, although the differences between animal models and humans must be kept in mind. High safety of any such therapy will be a prerequisite for any possible clinical application and therefore novel methods to improve control are being devised: 1) The regulation of gene therapy vectors by biochemical abnormalities associated with the target disease itself (" Induction-by-Disease" gene therapy). 2) External control of vector activity by the employment of drug-sensitive promotors. In addition, the important goal of cardiac long-term stability of the therapeutic vectors has recently been achieved in animal models using vectors derived from adeno-associated viruses (AAVs).     

Current status of gene therapy for cystic fibrosis pulmonary disease

Cystic fibrosis (CF) is a common lethal genetic disorder that affects all ethnic populations; however, it is most prevalent in Caucasians. Intensive basic research over the last 20 years has resulted in a wealth of information regarding the CF gene, its protein product and the mutational basis of disease. This increased understanding has lead to the development of gene therapy for the treatment of CF pulmonary disease. Delivery of the CF gene to the airway requires direct in vivo transfer using vectors encoding for normal CF transmembrane regulator (CFTR) protein. Several vectors are currently available for CF gene transfer and include both viral (adenoviruses, adeno-associated viruses) and non-viral (liposomal) systems. Initial clinical trials with each of these vectors have demonstrated that gene transfer to the CF airway is possible. The efficiency of transfer and duration of expression, however, have been limited. The effects of gene transfer on correction of the basic ion transport defects have also been highly variable and inconsistent, irrespective of the vector. Currently, the risk of severe immunological reactions is the primary factor limiting the clinical advancement of gene therapy. Both the adenoviral and liposomal vectors are associated with significant acute inflammatory reactions. The adenoviruses and adeno-associated viruses also elicit humoral immune responses that significantly reduce the efficiency of transgene expression and increase the risk of readministration. Several strategies are under investigation to improve the efficiency of gene transfer to the CF airway. These include overcoming local barriers in the lung, circumventing the immune response and improving vector internalization and/or uptake. Application of gene transfer in the child and possibly the fetus are also potential future clinical applications of gene therapy. However, despite considerable research with gene therapy, there is little evidence to suggest that a well tolerated and effective gene transfer method is imminent and aggressive use of conventional pharmacological therapies currently offer the greatest promise in the treatment of patients with CF.     

The research of nanoparticles as gene vector for tumor gene therapy

With the development of molecular biology, the application of the gene therapy becomes a tendency in the development of oncotherapy. The gene therapy has been acknowledged as the major progress of modern medicine, also a focus in the oncotherapy research. Commonly vectors of the gene therapy mainly include two categories, namely, viral vectors and nonviral vectors. Nanoparticles gene vector of various different kinds of materials, which belong to non-viral carriers. It presents excellent abilities of adsorption, concentration and protection of DNA, which can be attributed as a main reason of the adsorption and operation of nano-gene vector on exogenous genes. In this article, we mainly reviewed the recent studies of the characteristics of nanoparticles, characteristics and transport mechanism of nanoparticles as gene vector, the progress on nanoparticles as gene vector in tumor gene therapy. Nano-gene vectors, as new drug and gene carriers, present characteristics such as the controlled-release, targeting, and the improvement of bioavailability. Nanoparticles for cancer imaging and therapy have evolved rapidly during the last decade and it is expected that more and more will become clinical practise. In the near future, as a new nanometer gene delivery vector will be in medical research and treatment play a bigger role.     

Viral vectors for vascular gene therapy

Vascular gene therapy is the focus of multiple experimental and clinical research efforts. While several genes with therapeutic potential have been identified, the best method of gene delivery is unknown. Viral vectors have the capacity to transfer genes at high efficiency rates. Several viral-based vectors have been used in experimental vascular gene therapy for in vivo and ex vivo gene transfer. Adenoviral-based vectors are being used for the induction of angiogenesis in phase 1 and 2 clinical trials. In the present review, the characteristics of the ‘ideal’ viral vector are discussed and the major types of viral vectors used in vascular gene transfer are reviewed. Basic knowledge of the use of viral vectors for direct in vivo gene transfer (adenoviral-based vectors, etc) and for ex vivo gene transfer (retroviral-based vectors) is provided. New developments in the field of viral vectorology, such as pseudotyping of retroviral vectors and targeting of other viral vectors to a specific cell type, will enhance the more rapid transition of vascular gene therapy from the experimental arena to the clinical setting.     

Self-complementary adeno-associated viral vectors for gene therapy of hemophilia B: progress and challenges

Therapies currently used for hemophilia involve injection of protein concentrates that are expensive, invasive and associated with side effects such as development of neutralizing antibodies (inhibitors) that diminish therapeutic efficacy. Gene transfer is an attractive alternative to circumvent these issues. However, until now, clinical trials using gene therapy to treat hemophilia have failed to demonstrate sustained efficacy, although a vector based on a self-complementary adeno-associated virus has recently shown promise. This article will briefly outline a novel gene-transfer approach using self-complementary adeno-associated viral vectors using hemophilia B as a target disorder. This approach is currently being evaluated in the clinic. We will provide an overview of the development of self-complementary adeno-associated virus vectors as well as preclinical and clinical data with this vector system.     

Development of novel method of non-viral efficient gene transfer into neonatal cardiac myocytes

To establish new treatment for cardiovascular disease, the development of safe and highly efficient vectors is necessary. Especially, non-viral vectors are considered to be ideal for human gene therapy, since recent adverse events with retroviral or adenoviral vectors have highlighted the issue of safety. Although we previously reported safety and high efficiency of HVJ-liposome method, we have modified the envelope of HVJ (Sendai virus). In this novel non-viral vector, the envelope of HVJ alone was utilized as a carrier to deliver proteins, genes and oligodeoxynucleotides (ODN). Thus, we optimized the transfection efficiency of HVJ-envelope vector into neonatal cardiac myocytes in this study, since cardiac myocytes is one of the most difficult cells to be transfected. HVJ-envelope, obtained after complete destruction of HVJ genome, containing FITC-labeled ODN or luciferase plasmid was incubated with cardiac myocytes. In addition, the concentration of protamine sulfate was modified (0-700 microg/ml) to increase transfection efficacy. Without HVJ-envelope vector, few cells showed fluorescence, whereas most cells demonstrated fluorescence with HVJ-envelope vector. Consistent with the high transfection efficiency of ODN, high luciferase activity was also detected using HVJ-envelope vector. Moreover, the transfection efficiency varied according to the concentration of protamine sulfate. No obvious cytotoxicity was observed in cells transfected with HVJ-envelope vector. The present study demonstrated the development of a highly efficient novel non-viral vector for cardiac myocytes, suggesting that further development may provide a new useful tool for research and clinical gene therapy in the field of cardiovascular disease.     

Onco-retroviral and lentiviral vector-based gene therapy for hemophilia: preclinical studies

Hemophilia A and B gene therapy requires long-term and stable expression of coagulation factor VIII (FVIII) or factor IX (FIX), respectively, and would need to compare favorably with protein replacement therapy. Onco-retroviral and lentiviral vectors are attractive vectors for gene therapy of hemophilia. These vectors have the potential for long-term expression because they integrate stably in the target cell genome. Whereas onco-retroviral vectors can only transduce dividing cells, lentiviral vectors can transduce a broad variety of cell types irrespective of cell division. Several preclinical and clinical studies have explored the use of onco-retroviral and, more recently, lentiviral vectors for gene therapy of hemophilia A or B. Both ex vivo and in vivo gene therapy approaches have been evaluated, resulting in therapeutic FVIII or FIX levels in preclinical animal models. Whereas in vivo gene therapy using onco-retroviral or lentiviral vectors often led to long-term FVIII or FIX expression from transduced hepatocytes, ex vivo approaches were generally hampered by either low or transient expression of FVIII or FIX levels in vivo and/or inefficient engraftment. Furthermore, immune responses against the transgene product remain a major issue that must be resolved before the full potential of these vectors eventually can be exploited clinically. Nevertheless, the continued progress in vector design combined with a better understanding of vector biology may ultimately yield more effective gene therapy approaches using these integrating vectors.     

Gene therapy of cancer

Gene therapy was initially thought of as a means to correct single gene defects in hereditary disease. In the meantime, cancer has become by far the most important indication for gene therapy in clinical trials. In the foreseeable future, the best way to achieve reasonable intratumoral concentrations of a transgene with available vectors is direct intratumoral injection with or without the aid of various techniques such as endoscopy or CT-guidance. At present, viral and non-viral methods of gene transfer are used either in vivo or ex vivo/in vitro. The most important viral vectors currently in use in clinical trials comprise retroviruses, adenoviruses, adeno-associated viruses, and herpes viruses. None of the available vectors satisfies all the criteria of an ideal gene therapeutic system, and vectors with only minimal residues of their parent viruses ("gutless vectors") as well as completely "synthetic viral vectors" will gain more and more importance in the future. Non-viral gene therapy methods include liposomes, injection of vector-free DNA ("naked DNA"), protein-DNA complexes, delivery by "gene gun," calcium-phosphate precipitation, electroporation, and intracellular microinjection of DNA. The first clinical trial of gene therapy for cancer was performed in 1991 in patients with melanoma, and since then more than 5000 patients have been treated worldwide in more than 400 clinical protocols. With the exception of a case of fatal toxicity in a young man with hereditary liver disease treated intrahepatically with high doses of adenovirus, side effects have been rare and usually mild in all these studies and expression of the transgene could be demonstrated in patients in vivo. However, despite anecdotal reports of therapeutic responses in some patients, unequivocal proof of clinical efficacy is still lacking for most of the varied approaches to gene therapy in humans. As well as our only fragmentary understanding of the molecular pathophysiology of many diseases, the principal reason for the present lack of clinical success of gene therapy is the very low transduction and expression efficiency in vivo of available vectors. Despite the complexities of gene therapy for cancer, the numerous different approaches can be subdivided into three basic concepts: (1) strengthening of the immune response against a tumour, (2) repair of cell cycle defects caused by losses of tumour suppressor genes or inappropriate activation of oncogenes, and (3) suicide gene strategies. In addition, the importance of gene marker studies and gene therapeutic protection of normal tissue are briefly covered in this review.     

Preclinical gene therapy studies for hemophilia using adeno-associated virus (AAV) vectors

Gene therapy offers a potential cure for hemophilia and several gene transfer vectors have been evaluated for their ability to treat this disease. This article reviews the studies that have been performed to evaluate the ability of recombinant adeno-associated virus (AAV) vectors to achieve safely the sustained expression of clotting factors following intramuscular, intravenous, and intrahepatic delivery to several animal models. These routes of administration are all effective in providing sustained and therapeutic levels of factor IX (FIX), although the levels vary. Intrahepatic delivery is more efficacious than intravenous administration, which is superior to intramuscular delivery. The recent development of efficient factor VIII (FVIII) expression cassettes has made AAV-based gene therapy for hemophilia A also within reach. Although no acute toxicity has been observed with any route of administration, an increased risk of antibody formation against FIX has been noted following intramuscular delivery. Biodistribution studies concluded that the vector disseminates to most tissues in a dose-dependent and time-dependent manner, but the majority of the vector resides in the targeted tissue. In addition, the risk of germline transmission has been shown to be low or absent. The relatively recent isolation of new AAV serotypes has resulted in the identification of vectors that have enhanced tropism for certain tissues. This combined with the potential of these new vectors to evade the immune response to AAV2, makes them attractive candidates for gene therapy. Although much progress has been made using AAV to treat hemophilia, there are several outstanding issues that need to be addressed. Delivery of AAV to large animals has not been reproducible, which could be due to nonoptimized delivery and/or immune responses to the vector or transgene product. In addition, a complete understanding of the biology of these vectors is required to assess their long-term safety. Solving these issues will lead to the development of a successful gene therapy product.     

Adenovirus-mediated gene transfer

The introduction of foreign genetic material into somatic cells in intact organisms is an important investigational technique that holds considerable promise as a therapeutic tool. Although successful gene transfer has been achieved by the use of both cell-mediated and direct techniques, most strategies have been limited either by constraints on the type, accessibility, and growth state of the target cell population, or by the low efficiency of genetic modification. Among the available vectors for somatic cell gene transfer, recombinant adenoviruses have several properties that make them particularly attractive for direct, in vivo introduction of foreign genes into adult animals and people. Simple techniques for the efficient generation and propagation of recombinant adenoviruses have been developed, and early studies employing recombinant adenoviral vectors demonstrate their potential for broad experimental and eventual clinical application. To exploit this potential properly, a number of important issues, including the efficiency of genetic modification of a targeted cell population, stability of foreign gene expression, effects of host immune response, and cell-type specific targeting of gene transfer, remain to be addressed.     

Improving adenovirus based gene transfer: strategies to accomplish immune evasion

Adenovirus (Ad) based gene transfer vectors continue to be the platform of choice for an increasing number of clinical trials worldwide. In fact, within the last five years, the number of clinical trials that utilize Ad based vectors has doubled, indicating growing enthusiasm for the numerous positive characteristics of this gene transfer platform. For example, Ad vectors can be easily and relatively inexpensively produced to high titers in a cGMP compliant manner, can be stably stored and transported, and have a broad applicability for a wide range of clinical conditions, including both gene therapy and vaccine applications. Ad vector based gene transfer will become more useful as strategies to counteract innate and/or pre-existing adaptive immune responses to Ads are developed and confirmed to be efficacious. The approaches attempting to overcome these limitations can be divided into two broad categories: pre-emptive immune modulation of the host, and selective modification of the Ad vector itself. The first category of methods includes the use of immunosuppressive drugs or specific compounds to block important immune pathways, which are known to be induced by Ads. The second category comprises several innovative strategies inclusive of: (1) Ad-capsid-display of specific inhibitors or ligands; (2) covalent modifications of the entire Ad vector capsid moiety; (3) the use of tissue specific promoters and local administration routes; (4) the use of genome modified Ads; and (5) the development of chimeric or alternative serotype Ads. This review article will focus on both the promise and the limitations of each of these immune evasion strategies, and in the process delineate future directions in developing safer and more efficacious Ad-based gene transfer strategies.     

Survival of the fittest: in vivo selection and stem cell gene therapy

Stem cell gene therapy has long been limited by low gene transfer efficiency to hematopoietic stem cells. Recent years have witnessed clinical success in select diseases such as X-linked severe combined immunodeficiency (SCID) and ADA deficiency. Arguably, the single most important factor responsible for the increased efficacy of these recent protocols is the fact that the genetic correction provided a selective in vivo survival advantage. Since, for most diseases, there will be no selective advantage of gene-corrected cells, there has been a significant effort to arm vectors with a survival advantage. Two-gene vectors can be used to introduce the therapeutic gene and a selectable marker gene. Efficient in vivo selection strategies have been demonstrated in clinically relevant large-animal models. Mutant forms of the DNA repair-enzyme methylguanine methyltransferase in particular have allowed for efficient in vivo selection and have achieved sustained marking with virtually 100% gene-modified cells in large animals, and with clinically acceptable toxicity. Translation of these strategies to the clinical setting is imminent. Here, we review how in vivo selection strategies can be used to make stem cell gene therapy applicable to the treatment of a wider scope of genetic diseases and patients.     

The use of nonhuman primate models to improve gene transfer into haematopoietic stem cells

Primitive haematopoietic progenitor and stem cells (HSC) have been pursued as highly desirable targets for genetic therapy as technology allowing safe and controllable transfer of exogenous genes into eukaryotic cells was developed a decade ago. Retroviral vectors have been used for the majority of preclinical and clinical studies directed at these cells, because these vectors have a number of the necessary properties, including chromosomal integration, helper-free production systems, and lack of toxicity. Until recently, however, results with these vectors in clinical trials and large animal models indicated efficiency of gene transfer as a major hurdle to be overcome. We have focused on using the rhesus macaque autologous transplantation model to optimize gene transfer to primitive haematopoietic cells, and investigate questions regarding in vivo stem cell behaviour, in a system with proven predictive value for human haematopoiesis. By optimization of transduction conditions using standard vectors, gene transfer efficiency to primitive repopulating cells has reached the clinically relevant range of 5-20% long-term. Alternative vector systems, have also yielded promising results. We have also found that relatively simple manipulation of cell cycle status prior to reinfusion of marked cells results in significantly improved engraftment of transduced cells: this finding may have an impact particularly in the nonablative setting. The high level marking has permitted insertion site analysis and clonal tracking in vivo. Inverse PCR and/or a ligation-mediated PCR procedure have demonstrated that a large number of transduced clones (over 50) contribute to multiple lineages in vivo for up to at least 2 years post-transplantation. Thus far we have little evidence for rapid clonal succession or lineage-restricted engraftment of transduced cells. These and other advances should result in successful gene therapy for a variety of acquired and congenital disorders affecting HSCs and their progeny lineages.     

Progress in Chimeric Vector and Chimeric Gene Based Cardiovascular Gene Therapy

Gene therapy 15 to deliver and exPress a protee-tive exo罗nous罗ne into the somatie eells Of a pa-tient in order to eorreet a defeetive gene.It rePre-sents a new way to treat human diseases.Cardiovas-eular disease 15 the leading eause of death in theworld一wide.Gene theraPy for eardiovascular diseasesas a novel approaeh 15 being developed.A key stepof gene therapy 15 to transfer genes.New veetors,novel target genes identified·and ex详rimental models(e·9.,ehronie isehemie model,animal model…     

Toward gene therapy for disorders of globin synthesis

Inherited disorders of hemoglobin remain desirable targets for genetically based therapies. That stem cell replacement reverses the phenotype of both thalassemia and sickle cell anemia has been well established through allogeneic bone marrow transplantation studies, yet significant toxicities and finite donor availability limit this approach to a minority of affected individuals. Genetically based strategies that have as their goal addition of a normal copy of the human beta-globin gene along with key regulatory sequences to autologous hematopoietic stem cells represent a viable alternative to allogeneic transplantation, but this approach has been impeded by formidable obstacles over the last decade. Large animal models have become the standard for the development of clinically relevant gene addition strategies, and significant progress in the techniques used to deliver potentially therapeutic genes has been achieved. The clinical application of such strategies may be close at hand, at least for disorders in which modest level, constitutive expression is sufficient to correct the phenotype. For the thalassemias and hemoglobinopathies, complex, regulated, lineage specific expression of the beta-globin gene at relatively high levels will be required. The discovery of the beta-globin locus control region renewed interest in the thalassemias and sickle cell anemia as targets for gene transfer, but difficulties in attaining high-titer vectors along with a tendency toward rearrangement when segments of the locus control region (LCR) were incorporated into retroviral vectors stalled further progress. Recent advances in vector construction have circumvented this problem and others limiting both gene transfer efficiency and regulation of transgene expression, offering new hope for clinical application.     

Gene therapy as a therapeutic approach for the treatment of rheumatoid arthritis: innovative vectors and therapeutic genes

In recent years, significant progress has been made in the treatment of rheumatoid arthritis (RA). In addition to conventional therapy, novel biologicals targeting tumour necrosis factor-alpha have successfully entered the clinic. However, the majority of the patients still has some actively inflamed joints and some patients suffer from side-effects associated with the high systemic dosages needed to achieve therapeutic levels in the joints. In addition, due to of the short half-life of these proteins there is a need for continuous, multiple injections of the recombinant protein. An alternative approach might be the use of gene transfer to deliver therapeutic genes locally at the site of inflammation. Several viral and non-viral vectors are being used in animal models of RA. The first gene therapy trials for RA have already entered the clinic. New vectors inducing long-term and regulated gene expression in specific tissue are under development, resulting in more efficient gene transfer, for example by using distinct serotypes of viral vectors such as adeno-associated virus. This review gives an overview of some promising vectors used in RA research. Furthermore, several therapeutic genes are discussed that could be used for gene therapy in RA patients.