Gene therapy for metachromatic leukodystrophy

Metachromatic leukodystrophy (MLD) is an inherited metabolic disease which is characterized by a deficiency of arylsulfatase A (ASA). This deficiency causes progressive accumulation of cerebroside sulfate in oligodendrocytes (OL) in the brain, resulting in dysmyelination. Approaches being developed by the authors to treating MLD are based on direct delivery of ASA genes into the brain. In the present report, it has been shown that the recombinant adenovirus (Adex 1SRLacZ) was able to transduce the OL very efficiently. Moreover, primary fibroblasts from MLD patients were exposed to recombinant adenovirus expressing the ASA gene (Adex1SRASA) and the cells expressed the transgene. The influence of overexpression of ASA on the activity of other sulfatases was also tested in fibroblasts from patients with MLD using a retrovirus vector (MFG-ASA). It was demonstrated that the overexpression of ASA reduces the activity of various sulfatases by a small amount but does not induce an accumulation of glycosaminoglycan. These results indicate that the influence of ASA overexpression on other sulfatases is different from that of the N-acetygalactosamine-4-sulfatase overexpression in a previous report. It was concluded that the correction of ASA deficiency by a recombinant adenovirus that potentially could be used to transfer the gene to the brain, and gene therapy for MLD based on gene transfer of the ASA gene to mutant cells will be feasible because the overexpression of ASA in cells does not lead to profound deficiency of other sulfatases or result in a new phenotype.     

Airway gene therapy and cystic fibrosis

Airway disease in cystic fibrosis (CF) is the major cause of death and is presently inadequately treatable, but genetic therapies offer the hope that such life-long disease will be curable, or at least satisfactorily treated. Normal pathogen defences that have evolved on airway surfaces also prevent the various gene vectors now available from producing effective gene transfer. Nevertheless, findings from basic research and human clinical trials are revealing how these barriers might be overcome or circumvented, with benefits to therapeutic efficacy and patient safety. Though progress is slower than expected or desired, the therapeutic rewards will be great when safe and effective gene therapy for CF airway disease becomes a clinical reality.     

Long-term enzymatic and phenotypic correction in the phenylketonuria mouse model by adeno-associated virus vector-mediated gene transfer

Phenylketonuria (PKU) is an autosomal recessive metabolic disorder caused by a deficiency of phenylalanine hydroxylase (PAH). The accumulation of phenylalanine leads to severe mental and psychomotor retardation, and hypopigmentation of skin and hair. Low-phenylalanine diet therapy can prevent irreversible damage if instituted from birth. However, poor compliance with the strict lifelong dietary therapy leads to various neurologic and behavioral problems. To develop a safe and promising gene therapy method for PKU, we investigated whether a recombinant adeno-associated virus could be used as a PAH gene transfer vector to reduce the excessive phenylalanine level in the PKU mouse model. A recombinant adeno-associated virus vector encoding the human PAH gene (rAAV-hPAH), driven by EF1-alpha promoter, was infused into PAH-deficient mice, Pah(enu2), via the hepatic portal vein. Two weeks after injection, the plasma phenylalanine level dramatically decreased to 360 microM in male PKU mice, accompanied by the coat color changing to black. The mean plasma phenylalanine level of untreated PKU mice was 1800 microM. The PAH enzyme activities of treated mice increased to 10-17% of wild-type mice. No signs of liver toxicity were observed after gene transfer. The biochemical and phenotypic corrections were sustained for up to 25 wk (25-wk detection period). In contrast, the treatment was less effective in female PKU mice. These results indicate that recombinant adeno-associated virus vector-mediated gene therapy can be a useful therapeutic candidate for patients with PKU. Further studies are needed to clarify the differences in PKU pathogenesis in males and females, and to explore alternative administration routes besides hepatic portal vein injection.     

Preliminary studies related to anti-interleukin-1β therapy in children with newly diagnosed type 1 diabetes

Sumpter KM, Adhikari S, Grishman EK, White PC. Preliminary studies related to anti‐interleukin‐1β therapy in children with newly diagnosed type 1 diabetes. Background: Interleukin‐1β (IL‐1β) may play a role in the pathogenesis of type 1 diabetes, but there are no data regarding the efficacy of agents antagonizing IL‐1β in patients with this disorder. We characterized the effects of IL‐1β on gene expression in peripheral blood mononuclear cells (PBMC) and the clinical and gene expression effects of a short course of recombinant IL‐1 receptor antagonist protein, anakinra, on children with newly diagnosed diabetes. Methods: PBMC from healthy adult volunteers were exposed to IL‐1β for 24 h in vitro. Gene expression was analyzed via microarray. Fifteen children within 1 wk of diagnosis of type 1 diabetes received daily anakinra for 28 d and were followed for 6 months. Blood was drawn for microarray analysis before and after anakinra treatment. Insulin secretory capacity was assessed by mixed‐meal tolerance testing (MMTT) at 3–4 wk and 7 months after diagnosis. Hemoglobin A1c (HbA1c) and insulin doses were periodically recorded. Data were compared with two historical control groups of children with newly diagnosed diabetes. Results: Although in vitro exposure to IL‐1β caused many changes in PBMC gene expression, gene expression did not change significantly after anakinra therapy in diabetes patients. Anakinra‐treated patients had similar HbA1c and MMTT responses, but lower insulin requirements 1 and 4 months after diagnosis compared to controls, and lower insulin‐dose‐adjusted A1c 1 month after diagnosis. Conclusions: Anakinra therapy is well tolerated in children with newly diagnosed type 1 diabetes. Further studies are needed to demonstrate biological effects.     

Somatische Gentherapie bei X-chromosomal vererbtem schwerem kombiniertem Immundefekt (X-SCID)

Background

Patients with X-SCID suffer from a severe deficiency of cellular and humoral immunity. Without specific treatment they usually die within the 1st year of life. The patients can be cured by bone marrow or stem cell transplantation. A small number of patients have successfully been treated with somatic gene therapy, using retroviral in vitro transfection of the gene to the patients' own stem cells.

Gene therapy

A 6-month-old boy with Pneumocystis carinii pneumonia, recurrent infections, skin mycosis, and failure to thrive was genetically confirmed as having X-SCID. Lacking an HLA-identical donor, he received gene therapy as the tenth patient in a study in Paris: 18 weeks after treatment he showed extensive reconstitution of his immune system. The impressive initial success of gene therapy in X-SCID made it a considerable alternative to haploidentical transplantation. A serious setback was the recent occurrence of two cases of lymphoproliferative disease among the 11 patients treated in Paris, which are probably causatively associated with the gene therapy.     

CNS-directed gene therapy for lysosomal storage diseases

Lysosomal storage diseases (LSDs) are a group of inherited metabolic disorders usually caused by deficient activity of a single lysosomal enzyme. As most lysosomal enzymes are ubiquitously expressed, a deficiency in a single enzyme can affect multiple organ systems, including the central nervous system (CNS). At least 75% of all LSDs have a significant CNS component. Approaches such as bone marrow transplantation (BMT) or enzyme replacement therapy (ERT) can effectively treat the systemic disease associated with LSDs in some patients. However, CNS disease remains a major challenge. Gene therapy represents a promising approach for the treatment of CNS disease as it has the potential to provide a permanent source of the deficient enzyme. Direct intracranial injection of viral gene transfer vectors has resulted in reduced lysosomal storage and functional improvement in certain small (rodent) and large (canine and feline) animal models of LSDs. The addition of protein transduction domains (PTDs) to the recombinant enzymes increased the distribution of enzyme and the extent of correction. Therapeutic levels of lysosomal enzymes can also be delivered to distant sites in the brain by anterograde and retrograde axonal transport. Finally, combining disparate approaches such as BMT and CNS-directed gene therapy can increase treatment efficacy in LSDs with severe CNS disease that are refractory to more conventional approaches. CONCLUSION: The development of gene transfer vectors that mediate persistent expression in vivo, the addition of PTDs, a better understanding of lysosomal enzyme trafficking and combining different therapies provide hope that the CNS component of LSDs can be effectively treated.     

Perinatal stem-cell and gene therapy for hemoglobinopathies

Most genetic diseases of the lymphohematopoietic system, including hemoglobinopathies, can now be diagnosed early in gestation. However, as yet, prenatal treatment is not available. Postnatal therapy by hematopoietic stem cell (HSC) transplantation from bone marrow, mobilized peripheral blood, or umbilical cord blood is possible for several of these diseases, in particular for the hemoglobinopathies, but is often limited by a lack of histocompatible donors, severe treatment-associated morbidity, and preexisting organ damage that developed before birth. In-utero transplantation of allogeneic HSC has been performed successfully in various animal models and recently in humans. However, the clinical success of this novel treatment is limited to diseases in which the fetus is affected by severe immunodeficiency. The lack of donor cell engraftment in nonimmunocompromised hosts is thought to be due to immunologic barriers, as well as to competitive fetal marrow population by host HSCs. Among the possible strategies to circumvent allogeneic HLA barriers, the use of gene therapy by genetically corrected autologous HSCs in the fetus is one of the most promising approaches. The recent development of strategies to overcome failure of efficient transduction of quiescent hematopoietic cells using new vector constructs and transduction protocols opens new perspectives for gene therapy in general, as well as for prenatal gene transfer in particular. The fetus might be especially susceptible for successful gene therapy approaches because of the developing, expanding hematopoietic system during gestation and the immunologic naiveté early in gestation, precluding immune reaction towards the transgene by inducing tolerance. Ethical issues, in particular regarding treatment safety, must be addressed more closely before clinical trials with fetal gene therapy in human pregnancies can be initiated.     

Gene therapy: progress in childhood disease

The recent sequencing of the human genome combined with the development of massively high throughput genetic analysis technologies is driving unprecedented growth in our knowledge of the molecular basis of disease. While this has already had a major impact on our diagnostic power, the therapeutic benefits remain largely unrealised. This review examines progress in the exciting and challenging field of gene therapy. In particular we focus on the treatment of genetic disease in infants and children where the most significant successes have been observed to date, despite the majority of trial participants being adults. Notably, gene transfer to the haematopoietic compartment has provided the clearest examples of therapeutic benefit, particularly in the context of primary immunodeficiencies. The triumphs and tribulations of these successes are explored, and the key challenges confronting researchers as they seek to further advance the field are defined and discussed.     

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??Gene therapy for thalassemia is based on gene-modified autologous hematopoietic stem cell transplantation. At present??preclinical and clinical studies have proved the safety and efficacy of lentivirus vector gene therapy??but there are still many factors limiting its clinical application??such as the quantity and quality of effective stem cells??the efficiency of gene transduction??level of gene expression??and toxicity of pre-transplantation preconditioning regimen.     

Gene therapy

DNA technology opens up new approaches to the treatment of some hereditary diseases. By means of such techniques as retrovirus-mediated gene transfer, cloned human genes may be introduced into the cell genome to correct the defect present in these inherited disorders. Several ethical issues have been raised in the discussion of human gene therapy. There is general consensus that germline gene therapy should not be applied in humans, as the gene manipulation could be passed on to subsequent generations. As reviewed here, laboratories are focusing on concepts relating to somatic gene therapy. This approach involves the insertion of genetic material into somatic cells and is comparable with organ transplantation.     

Somatische Gentherapie der Mukoviszidose Aktueller Forschungsstand

Background. Numerous gene mutations associated with hereditary disorders have been identified. In cystic fibrosis the hereditary defect is attributed to mutations in one single gene, the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Conventional therapies of CF have dramatically increased the life expectancy of afflicted individuals. However, the ultimate incurability of this disease calls for novel and better therapeutic strategies. As cystic fibrosis is believed to be caused by mutations in one single gene, it has appeared to be the ideal candidate for one of the most tempting approaches in clinical therapy, namely gene therapy. Laboratory protocols for the introduction of genes into various tissues have been developed and applied over the last years. The ease of gene transfer under laboratory conditions gave rise to the hope that rapid advances in gene transfer protocols under clinical settings could be achieved as well. Clinical trials. 25 clinical trials of gene therapy for cystic fibrosis have been initiated using viral and non-viral vectors for gene transfer. The outcome of the CF gene therapy studies as well as of those for other diseases have clearly demonstrated that gene transfer and gene therapy in humans is a much more complex and challenging task than originally thought. Still, the encouraging results achieved in animal models and the rapid progress in vector technology justify the hope that the novel genetic therapies will be applied successfully to the benefit of patients suffering from cystic fibrosis.     

以免疫球蛋白和T细胞受体基因重排为标志定量检测MLL基因重排儿童急性淋巴细胞白血病的微小残留病和预后关系

目的 探讨MLL基因重排儿童急性淋巴细胞白血病（ALL）微小残留病（MRD）与临床特征的关系及其对预后的指导作用。方法 以2003年4月至2009年12月首都医科大学附属北京儿童医院血液肿瘤中心收治的MLL基因重排的ALL患儿为研究对象。以免疫球蛋白和T细胞受体基因重排、MLL融合转录本为标志,定量PCR方法监测MRD水平。以诱导治疗结束时MRD水平≥10-4为MRD阳性组,<10-4为MRD阴性组。卡方检验和Kaplan-Meier生存分析分别比较MRD阳性和阴性组临床特征和无事件生存率（EFS）的差异。结果 14例ALL患儿在诱导治疗结束时检测了MRD水平,MRD阳性组患儿初诊时外周血WBC计数显著高于MRD阴性组,对泼尼松实验治疗反应显著低于MRD阴性组。MRD阴性组5年EFS显著优于MRD阳性组,100% vs （37.5±17.1）%,P=0.022。结论 诱导治疗结束时MRD水平有助于对MLL基因重排儿童ALL进行预后分组,指导个体化治疗、改善预后。     

Non-viral transfer approaches for the gene therapy of mucopolysaccharidosis type II (Hunter syndrome)

Aims : Hunter syndrome is a rare X-linked lysosomal storage disorder caused by the deficiency of the housekeeping enzyme iduronate-2-sulphatase (IDS). Deficiency of IDS causes accumulation of undegraded dermatan and heparan-sulphate in various tissues and organs. Approaches have been proposed for the symptomatic therapy of the disease, including bone marrow transplantation and, very recently, enzyme replacement. To date, gene therapy strategies have considered mainly retroviral and adenoviral transduction of the correct cDNA. In this paper, two non-viral somatic gene therapy approaches are proposed: encapsulated heterologous cells and muscle electro-gene transfer (EGT). Methods : Hunter primary fibroblasts were co-cultured with either cell clones over-expressing the lacking enzyme or with the same incorporated in alginate microcapsules. For EGT, plasmid vector was injected into mouse quadriceps muscle, which was then immediately electro-stimulated. Results : Co-culturing Hunter primary fibroblasts with cells over-expressing IDS resulted in a three- to fourfold increase in fibroblast enzyme activity with respect to control cells. Fibroblast IDS activity was also increased after co-culture with encapsulated cells. EGT was able to transduce genes in mouse muscle, resulting in at least a tenfold increase in IDS activity 1–5 weeks after treatment.
Conclusion : Although preliminary, results from encapsulated heterologous cell clones and muscle EGT encourage further evaluations for possible application to gene therapy for Hunter syndrome.     

A novel mutation in the NF1 gene in two siblings with neurofibromatosis type 1 and bilateral optic pathway glioma

We present the clinical and ophthalmological findings, genetic analysis, and therapy of two siblings with NF1 and bilateral OPG. In genetic analysis, a heteroduplex profile was detected in exon 4b of the NF1 gene for the affected patients and mother. Sequencing of the DNA samples identified a C > T nucleotide change in exon 4b (c484CAG > TAG). This nonsense mutation resulted in a change of glutamine to a stop codon (Q162X) and is a novel NF1 gene alteration.     

Gene transfer and gene therapy]

The application of gene transfer technology to human gene therapy has been intensively investigated during the last decade. The first human clinical trials in adenosine deaminase deficiency and metastatic melanoma recently demonstrated the feasibility and safety of using retroviral gene transduction for human gene therapy. Many problems remain to be solved for a better understanding of the functioning of genes to be transferred, a better efficiency of gene transfer and genetic correction by site-specific targeting in vivo, a better knowledge of the biological properties of target cells such as totipotent hematopo?etic stem cells. Clinical developments are expected in genetic diseases (immunodeficiency, thalassemia, hemophilia) and non genetic disorders (lymphokine gene therapy for cancer).     

Gene therapy for phenylketonuria

Classical phenylketonuria (PKU) is an autosomal recessive disorder caused by a deficiency of hepatic phenylalanine hydroxylase (PAH). Three different vector systems have been developed to examine the potential of somatic gene therapy for the treatment of PKU. Recombinant retroviral vectors and DNA/protein complexes can efficiently transduce PAH-deiicient hepatocytes in vitro, but their present phenylketonuria, retrovirus application is limited by their low transduction efficiency in vivo. In contrast, infusion of a recombinant adenoviral vector expressing the human PAH cDNA into the portal circulation of PAH-deficient mice restores 10-80% of normal hepatic PAH activity and completely normalizes serum phenylalanine levels. At present, this effect is transient and re-administration has no further effect. However, this result suggests that PKU can be completely corrected by somatic gene therapy as more persistent vectors are developed.     

Gene therapy for disorders affecting children, progress and potential

For over two decades gene therapy has been actively pursued as a treatment modality for the inherited diseases that affect the paediatric population, however, it is still to make a real impact in the clinic. There are many reasons for this including inadequate technology and a lack of understanding of the biological complexities that impact on the efficiency of gene delivery and its outcomes, both positive and negative. However, recent progress is now addressing these issues and indicates that these problems can be overcome, and that gene therapy will play a significant role in the treatment of at least some of these disorders. This review will first give a short overview of relevant gene delivery technologies, what strategies can be used and which diseases are potential targets for gene therapy, and then illustrate several specific diseases for which gene therapy is actively being developed.     

Prim?re Immundefekte

Patients with deficiencies of the immune system usually present with recurrent and often severe infections. A possible underlying molecular defect can be suspected from either the pattern of infections (organ or systemic infections) or the types of microorganisms identified. An acute infection should be treated with broad spectrum antibiotics and the regimen must be adapted after identification of the pathogens involved. After successful treatment of initial infections precautions for relapses need to be considered because the underlying defect still persists. Both immunoreconstitutive and antimicrobial agents may be useful for secondary prevention. Antibody deficiency syndromes can be successfully treated by regular replacement of immunoglobulins. Other severe especially cellular diseases can only be cured by stem cell transplantation. Very few genetically well characterized disorders have been successfully treated by somatic gene therapy in only a few patients. The risk for adverse events, especially development of leukemia, however, shows that gene therapy is still experimental and that safer procedures to correct the genetic defect need to be developed.     

Contribution of antineoplastic biotherapy in the treatment of leukemia in children]

Improvements in the chemotherapeutic and transplant regimens have had a significant impact in improving survival rates for pediatric leukemia. However, there are still major problems to address including what options are available for patients with chemoresistant disease and what strategies are available to avoid toxicity associated with highly cytotoxic treatment regimens. Gene and immunotherapy protocols hold great promise. Using gene transfer of a marker gene, a number of biologic issues in the therapy of leukemia have been addressed. For example, by gene marking autologous bone marrow grafts it has been possible to demonstrate that infused marrow contributes to relapse in acute and chronic myeloid leukemias. In the allogeneic transplant setting, genetically modified T-cells have proven valuable for the prophylaxis and treatment of viral diseases and may have an important role in preventing or treating disease relapse. Gene transfer is also being used to modify tumor function, enhance immunogenicity, and confer drug-resistance to normal hematopoietic stem cells. With the continued scientific advancements in this field, gene therapy will almost certainly have a major impact on the treatment of pediatric leukemia in the future.     

Molecular analysis of the SMN and NAIP genes in Iranian spinal muscular atrophy patients

Background:  Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder characterized by degeneration of spinal cord anterior horn cells, leading to muscular atrophy. SMA is clinically classified into three subgroups based on the age of onset and severity. The majority of patients with SMA have homozygous deletions of exons 7 and 8 of the survival motor neuron ( SMN ) gene. The purpose of the present study was to determine the frequency of SMN and neuronal apoptosis inhibitory protein ( NAIP ) gene deletions in Iranian SMA patients. Experience in prenatal diagnosis of SMA in this population is also reported.
Methods:  To study the frequency of deletions of SMN and NAIP genes in an Iranian sample group, 75 unrelated SMA patients (54 type I, eight type II and 13 type III) were analyzed according to the methods described by van der Steege et al and Roy et al .
Results:  Homozygous deletion of SMN1 exons 7 and/or 8 were identified in 68 out of 75 patients (90%). Deletion of exon 5 of the NAIP gene was found in 40/54 of type I, 2/8 of type II and 1/13 of type III patients.
Conclusions:  Deletion of the SMN1 gene is a major cause of SMA in Iran, and NAIP gene deletions were common in the present patients with type I SMA. Also, the incidence of NAIP deletion is higher in more severe SMA.