Antonio Liras

Future research and therapeutic applications of human stem cells: general, regulatory, and bioethical aspects

There is much to be investigated about the specific characteristics of stem cells and about the efficacy and safety of the new drugs based on this type of cells, both embryonic as adult stem cells, for several therapeutic indications (cardiovascular and ischemic diseases, diabetes, hematopoietic diseases, liver diseases). Along with recent progress in transference of nuclei from human somatic cells, as well as iPSC technology, has allowed availability of lineages of all three germ layers genetically identical to those of the donor patient, which permits safe transplantation of organ-tissue-specific adult stem cells with no immune rejection. The main objective is the need for expansion of stem cell characteristics to maximize stem cell efficacy (i.e. the proper selection of a stem cell) and the efficacy (maximum effect) and safety of stem cell derived drugs. Other considerations to take into account in cell therapy will be the suitability of infrastructure and technical staff, biomaterials, production costs, biobanks, biosecurity, and the biotechnological industry. The general objectives in the area of stem cell research in the next few years, are related to identification of therapeutic targets and potential therapeutic tests, studies of cell differentiation and physiological mechanisms, culture conditions of pluripotent stem cells and efficacy and safety tests for stem cell-based drugs or procedures to be performed in both animal and human models in the corresponding clinical trials. A regulatory framework will be required to ensure patient accessibility to products and governmental assistance for their regulation and control. Bioethical aspects will be required related to the scientific and therapeutic relevance and cost of cryopreservation over time, but specially with respect to embryos which may ultimately be used for scientific uses of research as source of embryonic stem cells, in which case the bioethical conflict may be further aggravated.

Recombinant proteins in therapeutics: haemophilia treatment as an example

One of the most spectacular advances in the history of scientific knowledge was the discovery of deoxyribonucleic acid (DNA) by Watson and Crick in 1953. This enabled certain proteins to be prepared in this way for their therapeutic use in clinical practice. Today, in the first decade of the 21st century, hundreds of therapeutic proteins have been produced recombinantly and about 50 of them have been approved for clinical use. Because of the specific procedure used for obtaining these products, which is based on expressing a atherapeutica gene from a fragment of DNA in a cell to produce a functional protein that is free from any human or animal component, they are especially acleana and thus the therapy of choice for many current diseases. The immediate question is: why are recombinant products not used more extensively given their high efficacy and maximum safety? In short, we are faced with an interesting but also unfortunate paradox of pharmacology that greater progress in therapeutic procedures is not always associated with greater introduction of those resources that are safest, for the simple reason that they are more costly.

Induced human pluripotent stem cells and advanced therapies: future perspectives for the treatment of haemophilia?

Human induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field. These iPSCs from somatic cells have been reprogrammed with the introduction of transcription factors and are capable to differentiate into cells from all three germ layers. These strategies require retrovirus transduction or transfection of plasmid vectors strategy without viral transduction. Another promising strategy is based on direct delivery of the reprogramming proteins, addition of signal transduction inhibitors and chemical promoter cell survival. The main advantages of iPSCs cells are that they have not included in the debate over the ethics of embryonic stem cell. Current therapy of haemophilia is based on factor VIII (FVIII) or factor IX (FIX) replacement therapy including prophylactic or demand protocols of fixed-dose, and as future alternative, gene and cell therapy. Gene therapy can be made by using viral vectors, mainly lentiviral (LVV) and adeno-associated viruses (AAV) in adult stem cells and autologous fibroblasts, platelets or haematopoietic stem cells, and transfer using non-viral vectors (NVV). Cell therapy for haemophilia is based, mainly, in transplantation of healthy cells to replace the deficient function, for example, the transplantation of liver sinusoidal endothelial cells or endothelial progenitor cells derived from iPSCs. Recently, as first time in haemophilia, endothelial progenitor cells derived from iPSCs cells express FVIII protein effectively, engraft within the hepatic parenchyma, and functionally integrate to provide the therapeutic benefit for a phenotypic correction in haemophilia. Advanced therapies, gene and cell therapy and tissue engineering or iPSCs technology, can provide a potential clinical application in the treatment of haemophilia and other monogenic disorders. Because to date there are not relevant results for phenotypic correction in haemophilia, iPSCs technology could represent a potential alternative based on cellular therapy.

Advanced therapies for the treatment of hemophilia: future perspectives

Monogenic diseases are ideal candidates for treatment by the emerging advanced therapies, which are capable of correcting alterations in protein expression that result from genetic mutation. In hemophilia A and B such alterations affect the activity of coagulation factors VIII and IX, respectively, and are responsible for the development of the disease. Advanced therapies may involve the replacement of a deficient gene by a healthy gene so that it generates a certain functional, structural or transport protein (gene therapy); the incorporation of a full array of healthy genes and proteins through perfusion or transplantation of healthy cells (cell therapy); or tissue transplantation and formation of healthy organs (tissue engineering). For their part, induced pluripotent stem cells have recently been shown to also play a significant role in the fields of cell therapy and tissue engineering. Hemophilia is optimally suited for advanced therapies owing to the fact that, as a monogenic condition, it does not require very high expression levels of a coagulation factor to reach moderate disease status. As a result, significant progress has been possible with respect to these kinds of strategies, especially in the fields of gene therapy (by using viral and non-viral vectors) and cell therapy (by means of several types of target cells). Thus, although still considered a rare disorder, hemophilia is now recognized as a condition amenable to gene therapy, which can be administered in the form of lentiviral and adeno-associated vectors applied to adult stem cells, autologous fibroblasts, platelets and hematopoietic stem cells; by means of non-viral vectors; or through the repair of mutations by chimeric oligonucleotides. In hemophilia, cell therapy approaches have been based mainly on transplantation of healthy cells (adult stem cells or induced pluripotent cell-derived progenitor cells) in order to restore alterations in coagulation factor expression.

Cartilage restoration in haemophilia: advanced therapies

Summary.  Current treatment of joint cartilage lesions is based either on conventional techniques (bone marrow stimulation, osteochondral autograft or allograft transplantation) or on newly developed techniques (chondrocyte implantation and those based on cell therapy that use bioreactors, growth factors, mesenchymal stem cells [MSCs] and genetically modified cells). The aim of this article is to review the therapeutic strategies above mentioned and to determine whether the chondral damage seen in haemophilia could benefit from any of them. The different conventional techniques have shown similar results whereas autologous chondrocyte implantation, which is in common use at the present time, has not been shown to produce any conclusive results or to lead to the formation of hyaline cartilage. MSCs hold promise for the repair of joint cartilage given their differentiation capacity and the therapeutic effect. The use of bioreactors and growth factors, which stimulate cartilage formation, may optimize such strategies in the context of reimplantation of chondrocytes, differentiated MSCs and cartilage progenitor cells. The aim of cell therapy is restoration of function through the repair of damaged tissue or the stimulation of growth factor synthesis. Implantation of autologous chondrocytes or MSCs was up to now able to address only highly localized chondral lesions. Adequate control of the differentiation process as well as the use of growth factors and appropriate bioreactors could transform cell‐based therapies into a more efficient and longer term treatment even for patients with haemophilia. Nevertheless, raising false expectations in these patients should be avoided. There are a number of approaches to cartilage restoration in haemophilic arthropathy, which are currently being explored for other joint related degenerative disorders. If it can be proven to be effective for the disorders in which clinical trials are ongoing and costs could be limited, it might be an useful palliative approach to haemophilic arthropathy. However, we still have a long way to go for use in haemophilic arthropathy.

Gene therapy for haemophilia…yes, but…with non‐viral vectors?

Summary.  High‐purity plasma‐derived and recombinant factors are currently safe and efficient treatment for haemophilia. The mid‐term future of haemophilia treatment will involve the use of modified recombinant factors to achieve advantages such as decreased immunogenicity in inhibitor formation and enhanced efficacy as a result of their longer half‐life. In the long‐term, gene therapy and cell therapy strategies will have to be considered. Achievements in cell therapy to date have been using embryonic stem cells and hepatic sinusoidal endothelial cells. Current gene therapy strategies for haemophilia are based on gene transfer using adeno‐associated viruses and non‐viral vectors. Gene therapy for haemophilia is justified because it is a chronic disease and because a very regular factor infusion is required that may involve fatal risks and because it is very expensive. Haemophilia is a very good candidate for use of gene therapy protocols because it is a monogenic disease, and even low expression is able to achieve reversion from a severe to a moderate phenotype. The current trends in haemophilia using adeno‐associated viral vectors are safe but also involve immunogenicity problems. The other alternatives are non‐viral vectors. There have been in recent years relevant advances in non‐viral transfection that raise hope for considering this possibility. Several research groups are opting for this experimental alternative. An expression over 5%, representing a moderate phenotype, for a few months with a high safety, regarding vector, transfected cells, and implantation procedure, would already be a great success. This may represent an intermediate protocol in which the expression levels and times obtained are lower and shorter respectively as compared to viral vectors, but which provide a potential greater patient safety. This may more readily win acceptance among both patients and haematologists because fatal events in the past due to HIV/HCV infection may constrain the implementation of viruses as vectors.

Treatment for hemophilia: recombinant versus plasma-derived coagulation factors - controversy and debate forever? An ethical medical challenge?

Author for correspondence:Department of Physiology,Biological Sciences School,Complutense University ofMadrid, Cell Therapy andRegenerative Medicine Unit,La Paz University Hospital HealthResearch Institute-IdiPAZ,Madrid, 28040, SpainandRoyal Foundation ‘VictoriaEugenia’ of Haemophilia,Madrid, 28040, SpainTel.: +34 630068924Fax: +34 91637965aliras@hotmail.com

Induced Pluripotent Stem Cells: TherapeuticApplications in Monogenic and Metabolic Diseases, andRegulatory and Bioethical considerations.

The potential use of stem cells in advanced therapies such as tissue engineering, regenerative medicine, cell therapy and gene therapy by virtue of their significant therapeutic potential and clinical applications has aroused keen interest among scientists [1,2]. Cell therapy is based on the transplantation of living cells into an organism with a view to repairing tissue or restoring a lost or deficient function. Stem cells are the most frequently used cells for such purposes given their ability to differentiate into other more specialized cells [3].

Advanced therapies for hemophilia: reality or fantasy?

The article is about the future therapeutic products based on advanced therapies such as gene and cell therapy and tissue engineering or induced pluripotent stem cell technology may offer innumerable potential clinical applicationsfor the treatment of several monogenic disorders including hemophilia.

Mission of World Federation of Haemophilia, Biotechnology and treatment for all? The paradox.

Today, the hard reality of haemophilia in the world is that 70% of all patients with haemophilia are not diagnosed, and 75% are not treated. These diagnostic and treatment deficiencies obviously derive from a series of shortcomings fundamentally related to a lack of infrastructures, deficient health education, and precarious training and specialization on the part of physicians and specialists. This is generally so because data from the World Federation of Haemophilia (WFH) indicate that haemophilia is not a priority concern for governments, and that moreover, the cost of treatment proves inaccessible in some cases. The unfortunate result is that many patients die in childhood, while others suffer life-long disability and great impairment of quality of life. In the light of this worldwide situation of the disease, the WFH [1] ‐ through each of the Member Federations ‐ has a series of clear objectives. A general objective is to try to improve and maintain the care of patients with hereditary bleeding disorders throughout the world, while a more specific objective is to promote a global diagnosis of the disease, as it is essential to detect the problem to adopt solutions. In this context, since the World Haemophilia Congress in 2004, there has been a 14% increase in the number of patients diagnosed with haemophilia and other coagulation disorders throughout the world. Achievement of the objectives implicates the patients and their families, the healthcare professionals ‐ physicians, nurses, orthopaedists, physiotherapists, social workers, dentists, laboratory technicians, etc. ‐ hospital services in general, and particularly the haemophilia and hemotherapy units, the government authorities, Ministries of Health,