Gene therapy and metabolic engineering

Gene therapy involves the introduction of normal, healthy genes into cells to correct the underlying cause of a wide variety of inherited and acquired diseases. Future progress in developing effective clinical protocols involving gene therapy for the treatment of cellular dysfunction associated with disease may incorporate metabolic engineering. Metabolic engineering can be applied to gene therapy for the successful identification of disease genes; elucidation of disease pathways; development of safe and efficient gene-delivery systems; and regulation and control of gene expression. Cystic fibrosis, cancer, and diabetes are reviewed as examples of diseases where gene therapy approaches are being studied.     

Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges

In vivo gene replacement for the treatment of inherited disease is one of the most compelling concepts in modern medicine. Adeno-associated virus (AAV) vectors have been extensively used for this purpose and have shown therapeutic efficacy in a range of animal models. Successful translation to the clinic was initially slow, but long-term expression of donated genes at therapeutic levels has now been achieved in patients with inherited retinal disorders and haemophilia B. Recent exciting results have raised hopes for the treatment of many other diseases. As we discuss here, the prospects and challenges for AAV gene therapy are to a large extent dependent on the target tissue and the specific disease.     

Injection of recombinant human type VII collagen restores collagen function in dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa (DEB) is a family of inherited mechano-bullous disorders that are caused by mutations in the type VII collagen gene and for which ex vivo gene therapy has been considered. To develop a simpler approach for treating DEB, we evaluated the feasibility of protein-based therapy by intradermally injecting human recombinant type VII collagen into mouse skin and a DEB human skin equivalent transplanted onto mice. The injected collagen localized to the basement membrane zone of both types of tissues, was organized into human anchoring fibril structures and reversed the features of DEB disease in the DEB skin equivalent.     

An inducible mouse model for epidermolysis bullosa simplex: implications for gene therapy

The Dowling-Meara variant of epidermolysis bullosa simplex (EBS-DM) is a severe blistering disease inherited in an autosomal-dominant fashion. Here we report the generation of a mouse model that allows focal activation of a mutant keratin 14 allele in epidermal stem cells upon topical administration of an inducer, resulting in EBS phenotypes in treated areas. Using laser capture microdissection, we show that induced blisters healed by migration of surrounding nonphenotypic stem cells into the wound bed. This observation provides an explanation for the lack of mosaic forms of EBS-DM. In addition, we show that decreased mutant keratin 14 expression resulted in normal morphology and functions of the skin. Our results have important implications for gene therapy of EBS and other dominantly inherited diseases.     

Rescue from Photoreceptor Degeneration in the rd Mouse by Human Immunodeficiency Virus Vector-Mediated Gene Transfer

Retinitis pigmentosa (RP) is the most common inherited retinal disease, in which photoreceptor cells degenerate, leading to blindness. Mutations in the rod photoreceptor cGMP phosphodiesterase beta subunit (PDEbeta) gene are found in patients with autosomal recessive RP as well as in the rd mouse. We have recently shown that lentivirus vectors based on human immunodeficiency virus (HIV) type 1 achieve stable and efficient gene transfer into retinal cells. In this study, we evaluated the potential of HIV vector-mediated gene therapy for RP in the rd mouse. HIV vectors containing a gene encoding a hemagglutinin (HA)-tagged PDEbeta were injected into the subretinal spaces of newborn rd mouse eyes. One to three rows of photoreceptor nuclei were observed in the eyes for at least 24 weeks postinjection, whereas no photoreceptor cells remained in the eyes of control animals at 6 weeks postinjection. Expression of HA-tagged PDEbeta in the rescued photoreceptor cells was confirmed by two-color confocal immunofluorescence analysis using anti-HA and anti-opsin antibodies. HIV vector-mediated gene therapy appears to be a promising means for the treatment of recessive forms of inherited retinal degeneration.     

The role of mast cells in the development of skin fibrosis in tight-skin mutant mice

Chronic inflammatory conditions can evolve a fibrotic phenotype often associated with an increase in the number of mast cells (MC) near or within the granulation tissue. Despite the potential of MC to mediate fibrosis, it is unclear whether these cells play a central role in the pathogenesis of ibrosis or whether their presence is simply circumstantial. The tight-skin (Tsk) mouse develops an inherited fibrotic disease (sharing many similarties with the human disease scleroderma, systemic sclerosis) in which the lesions are associated with increased numbers and heightened granule release implicating MC in the pathogenesis of fibrosis. Despite their close association with the skin fibrosis of Tsk mice, the precise role of the MC in the pathogenesis of this inherited disease is unknown. Therefore, to assess directly whether MC are key elements in the pathogenesis of Tsk fibrosis, we generated MC deficient mice carrying the Tsk locus by utilizing selective interbreeding between Tsk and mutant mice deficient in mast cells (W, dominant white-spotting). We found that in the absence of MC, the early natural history of Tsk fibrosis was not altered. Furthermore, in older (5–7 months) Tsk mice, we found that the number of cutaneous MC was correlated with a more pronounced fibrosis. Therefore, we conclude that Tsk skin lesions are a pleiotropic manifestation of the Tsk gene in which MC are ivolved/recruited by an uncharacterized mechanism and that subsequent proliferation and activation of MC leads to augmentation of fibrosis.     

Gene therapy: progress and predictions

The first clinical gene delivery, which involved insertion of a marker gene into lymphocytes from cancer patients, was published 25 years ago. In this review, we describe progress since then in gene therapy. Patients with some inherited single-gene defects can now be treated with their own bone marrow stem cells that have been engineered with a viral vector carrying the missing gene. Patients with inherited retinopathies and haemophilia B can also be treated by local or systemic injection of viral vectors. There are also a number of promising gene therapy approaches for cancer and infectious disease. We predict that the next 25 years will see improvements in safety, efficacy and manufacture of gene delivery vectors and introduction of gene-editing technologies to the clinic. Gene delivery may also prove a cost-effective method for the delivery of biological medicines.     

Capturing epidermal stemness for regenerative medicine

The skin is privileged because several skin-derived stem cells (epithelial stem cells from epidermis and its appendages, mesenchymal stem cells from dermis and subcutis, melanocyte stem cells) can be efficiently captured for therapeutic use. Main indications remain the permanent coverage of extensive third degree burns and healing of chronic cutaneous wounds, but recent advances in gene therapy technology open the door to the treatment of disabling inherited skin diseases with genetically corrected keratinocyte stem cells. Therapeutic skin stem cells that were initially cultured in research or hospital laboratories must be produced according strict regulatory guidelines, which ensure patients and medical teams that the medicinal cell products are safe, of constant quality and manufactured according to state-of-the art technology. Nonetheless, it does not warrant clinical efficacy and permanent engraftment of autologous stem cells remains variable. There are many challenges ahead to improve efficacy among which to keep telomere-dependent senescence and telomere-independent senescence (clonal conversion) to a minimum in cell culture and to understand the cellular and molecular mechanisms implicated in engraftment. Finally, medicinal stem cells are expansive to produce and reimbursement of costs by health insurances is a major concern in many countries.     

Exacerbation, then clearance, of mutation-proven Darier's disease of the skin after radiotherapy for bronchial carcinoma: a case of radiation-induced epidermal differentiation?

We investigated a radiotherapy-induced flare and subsequent clearance of skin lesions of a patient with the rare, dominantly inherited genodermatosis, Darier's disease (DD). The DD gene, ATP2A2, was recently isolated and shown to be a cation pump responsible for regulating intracellular calcium homeostasis. A severe exacerbation of Darier's skin lesions developed within the radiation field when 40 Gy of palliative thoracic external-beam radiation therapy and concurrent chemotherapy (cisplatin and hydroxyurea) were delivered for non-small cell lung cancer. The DD lesions subsequently completely cleared from irradiated skin, as they did when a subsequent course of radiation alone was given for a loco-regional tumor recurrence. The two radiation therapy-treated areas of skin remained free from lesions of the skin disorder until the patient's death from progressive lung cancer 9 months later. The nucleotide sequence of the patient's ATP2A2 gene was determined by PCR-based cycle sequencing. We identified four nucleotide sequence variants in the ATP2A2 gene in this patient. Three were probable polymorphisms and the other appeared to be a novel disease-causing mutation (R751Q), situated in the transmembrane portion of the ATP2A2 protein. This finding confirmed the clinical diagnosis. Since epidermis turns over every 3-4 weeks, total and persistent clearance of the DD lesions by chemoradiotherapy suggests that this treatment induced sustained differentiation of the DD-affected skin by an unknown mechanism. Oncologists treating malignant disease in patients with DD should anticipate temporary deterioration in DD-involved irradiated skin. Radiation therapy has therapeutic potential in severe DD.     

Gene therapy for diseases of the nervous system.

Advances in molecular biology and recombinant DNA technologies have contributed to our understanding of the molecular basis of many diseases. Now the possibility of gene transfer into normal cells to produce a gene product of therapeutic potential, or into diseased cells to correct the pathologic alteration, promises to revolutionize medical practice. In contemporary medicine, many therapeutic strategies focus on the link between a biochemical deficiency and the ensuing disorder. The treatment of noninfectious disease is often based on replacement therapy; medication is given to compensate for biochemical defects and to prevent or reverse the progression of disease. Although conventional therapies seldom alter the fundamental cause of a disease, gene therapy potentially could correct, at a molecular level, the genetic abnormalities contributing to its pathogenesis. Treatment directed at specific molecular alterations associated with the development of neurologic disease provides expectations of more effective and less toxic therapy. The development of gene therapy for nervous system tumors has progressed rapidly and may be prototypical in the development of therapies for inherited and acquired disorders of the nervous system. We describe possible strategies for using gene therapy to treat nervous system disorders, and we review recent advances in gene therapy for nervous system tumors.     

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.     

Pathways to improving skin regeneration

A significant proportion of the human population suffers from some form of skin disorder, whether it be from burn injury or inherited skin anomalies. The ideal treatment for skin disorders would be to regrow skin tissue from stem cells residing in the individual patient's skin. Locating these adult stem cells and elucidating the molecules involved in orchestrating the production of new skin cells are important steps in devising more-efficient methods of skin production and wound healing via the ex vivo expansion of patient keratinocytes in culture. This review focuses on the structure of the skin, the identification of skin stem cells, and the role of Notch, Wnt and Hedgehog signalling cascades in regulating the fate of epidermal stem cells.     

Fabry's disease (alpha-galactosidase-A deficiency): recent therapeutic innovations

Fabry disease (FD, OMIM 301500) is an X-linked inherited disorder of metabolism due to mutations in the gene encoding alpha-galactosidase A, a lysosomal enzyme. The enzymatic defect leads to the accumulation of neutral glycosphingolipids throughout the body, particularly within endothelial cells. Resulting narrowing and tortuosity of small blood vessels with endothelial dysfunction lead to tissue ischaemia and infarction. Inability to prevent the progression of glycosphingolipid deposition causes significant morbidity and mortality from early onset strokes, cardiomyopathy and renal failure in adulthood. Medical management is symptomatic and consists of partial pain relief with analgesic drugs (gabapentin, carbamazepine), antihypertensive drugs, whereas renal transplantation or dialysis is available for patients experiencing end-stage renal failure. However, the ability to produce high doses of alpha-galactosidase A in vitro has opened the way to preclinical studies in the mouse model, and to the development of the first clinical trials in patients with Fabry disease. Enzyme replacement therapy has recently been validated as a therapeutic agent for Fabry disease patients. Long term safety and efficacy of replacement therapy are currently being investigated. Substrate deprivation and gene therapy may also prove future alternative therapeutic options.     

After sun reversal of DNA damage: enhancing skin repair

UV-induced DNA damage has been directly linked to skin cancer, and DNA repair is an important protection against this neoplasm. This is illustrated by the genetic disease xeroderma pigmentosum wherein a serious defect in DNA repair of cyclobutane pyrimidine dimers dramatically increases the rate of skin cancer. In other instances in which skin cancer rates are elevated, deficits in DNA repair may also be one of the causal factors. For example, solid organ transplant patients have elevated rates of skin cancer that are correlated with the dose and length of exposure to immunosuppressive drugs (predominantly cyclosporine A (CsA) and ascomycin (FK506)-related tacrolimus). We have found that treatment of cultured epidermal cells with CsA or ascomycin inhibits their removal of DNA damage by about 20% at 24 h. In a further example, people with a polymorphism in the DNA repair gene 8-oxo-guanine glycosylase (OGG1) have an increased risk of skin cancer. We have found that the cells with this variant polymorphism have an increased sensitivity of about 20% to a broad range of cytotoxic agents. The DNA deficits caused by immunosuppressive drugs or the OGG1 polymorphism can be overcome by the delivery of DNA repair enzymes in liposomes. The data suggests that deficits in DNA repair, even if they are not as severe as in the case of XP, may contribute to increased rates of cancer, and that topical therapy with DNA repair enzymes may be a promising avenue for after-sun protection.     

Oral curcumin mitigates the clinical and neuropathologic phenotype of the Trembler-J mouse: a potential therapy for inherited neuropathy

Mutations in myelin genes cause inherited peripheral neuropathies that range in severity from adult-onset Charcot-Marie-Tooth disease type 1 to childhood-onset Dejerine-Sottas neuropathy and congenital hypomyelinating neuropathy. Many myelin gene mutants that cause severe disease, such as those in the myelin protein zero gene (MPZ) and the peripheral myelin protein 22 gene (PMP22), appear to make aberrant proteins that accumulate primarily within the endoplasmic reticulum (ER), resulting in Schwann cell death by apoptosis and, subsequently, peripheral neuropathy. We previously showed that curcumin supplementation could abrogate ER retention and aggregation-induced apoptosis associated with neuropathy-causing MPZ mutants. We now show reduced apoptosis after curcumin treatment of cells in tissue culture that express PMP22 mutants. Furthermore, we demonstrate that oral administration of curcumin partially mitigates the severe neuropathy phenotype of the Trembler-J mouse model in a dose-dependent manner. Administration of curcumin significantly decreases the percentage of apoptotic Schwann cells and results in increased number and size of myelinated axons in sciatic nerves, leading to improved motor performance. Our findings indicate that curcumin treatment is sufficient to relieve the toxic effect of mutant aggregation-induced apoptosis and improves the neuropathologic phenotype in an animal model of human neuropathy, suggesting a potential therapeutic role in selected forms of inherited peripheral neuropathies.     

Liposome-Mediated Gene Transfer into Normal and Dystrophin-Deficient Mouse Myoblasts

Abstract: A range of tissue types has now been targeted for development of gene therapeutic procedures both to correct genetic defects and to treat acquired disease. In particular, skeletal muscle holds great importance, not exclusively for the treatment of inherited muscle disorders but also as a platform for the expression of heterologous recombinant proteins, destined to immunise the host or to serve some systemic therapeutic goal. With respect to the X-linked myopathy Duchenne muscular dystrophy (DMD), several gene therapy protocols are being developed that focus on complementing primary genetic defects in the DMD gene by introducing copies of recombinant gene constructs into muscle cells both ex vivo and in vivo. In the present study the potential use of a range of polycationic liposomes as physical gene delivery systems for skeletal muscle has been examined. Using a LacZ reporter gene under optimised conditions up to 40% transfection efficiencies were obtained with the mouse myoblast cell line C2C12. With primary cultures of normal and dystrophin-deficient mdx mouse muscle, up to 10% transfection efficiency was obtained with reporter gene constructs, and high levels of recombinant human dystrophin expression were observed following transfer of dystrophin cDNA gene constructs. These in vitro studies indicate that cationic liposomes can be used to deliver recombinant genes to muscle cells at high efficiency and form a basis to expand investigations into in vivo expression of recombinant dystrophin protein either by direct intramuscular gene transfer or via implantation of transfected myoblasts.     

Gene therapy of Gaucher's and Fabry's diseases: current status and prospects

Gaucher disease and Fabry disease are lysosomal storage disorders characterized by the accumulation of sphingolipids. In both cases, the goal of gene therapy is to permanently provide tissues with enzyme levels allowing to avoid storage of the undigested substrates. Different gene therapy strategies must however be designed as Gaucher disease is due to a deficiency in the membrane-associated enzyme glucocerebrosidase, whereas Fabry disease is caused by a deficiency in the soluble enzyme alpha-galactosidase. Indeed, a soluble enzyme can be provided to tissues is trans by gene-modified cells whereas gene transfer has to target the most affected cells in the case of membrane-bound enzymes. Thus, in non-neurological Gaucher disease (type 1), the hematopoietic tissue has to be targeted as the deficiency affects the monocyte/macrophage lineage. Following promising preclinical studies, clinical protocols have been initiated to explore the feasibility and safety of retroviral transfer of the glucocerebrosidase gene into CD34+ cells from patients with type 1 Gaucher disease. Although gene-marked cells were detected in vivo, the level of corrected cells was very low, a finding indicating that improved vectors along with partial myeloablation may be necessary. Here, lentiviral vectors should enable more gene transduction into the hematopoietic target cells. As concerns the diffuse neurological lesions in types 2 and 3 of Gaucher disease, they will probably be especially difficult to target by gene therapy because of the non soluble nature of glucocerebrosidase. Finally, over the last few years, Fabry disease has become a compelling target for gene therapy as an etiology-based treatment strategy. Indeed, several recent studies aiming at creating a large in vivo source of alpha-galactosidase have yielded positive long-term results in the Fabry knock-out mouse model.     

Apoptotic process in cystic fibrosis cells

Cystic fibrosis (CF) is a recessively inherited disease caused by genetic lesions in CF transmembrane conductance regulator (CFTR) gene. CF is characterized by exaggerated inflammation, progressive tissue damage, and chronic bacterial colonization, mainly in the respiratory tract. The mechanisms underlying these pathological changes are increasingly well understood. However, apoptotic dysfunction in CF disease is still debated since studies report controversial results. Nonetheless, it is clear that apoptosis participates to onset of pathology and concerns various types of cells with variable susceptibility. Apoptosis is a physiological process necessary for the preservation of homeostasis of epithelial organization and function for clearance of inflammatory cells. Increased susceptibility to apoptosis in epithelial cells and failed apoptosis in neutrophils would contribute to the self-perpetuating inflammatory cycle in CF. Also, retention of mutated CFTR in the endoplasmic reticulum participates to inflammation which may trigger apoptosis. Independently of the sensibility to apoptosis of CF cells, it has been shown that clearance of apoptotic cells, due in part to decrease in efferocytosis, is flawed and that accumulation of such cells may contribute to ongoing inflammation in CF patients. Despite great advance in understanding CF pathophysiology, there is still no cure for the disease. The most recent therapeutic strategies are directed to target CFTR protein using cell and gene therapy as well as pharmacotherapy.     

Epidermal stem cells and ex vivo cutaneous gene therapy: application to xeroderma pigmentosum

Ex vivo cutaneous gene therapy is an alternative treatment for recessively inherited diseases with cutaneous traits. It relies on the transfer in cultured epidermal keratinocytes of the wild-type allele of the gene whose mutation is responsible for the disease. As for severely burnt patients, epithelial sheets developed from genetically corrected cells may then be grafted back to the patients. Long term correction and graft take depend on the genetic correction of stem cells. Success of such an approach has recently been reported in the case of one patient suffering from a severe case of junctional epidermolysis bullosae. Here we report a method for safely selecting keratinocytes populations after genetic manipulation. The method is non invasive and non immunogenic and allows high enrichment of genetically manipulated stem keratinocytes. This could perhaps contribute to ex vivo gene therapy approaches of cancer prone genodermatoses such as xeroderma pigmentosum.