Current Gene Therapy (v.14, #6)

Gene Therapy for Wiskott-Aldrich Syndrome by Marita Bosticardo, Francesca Ferrua, Marina Cavazzana, Alessandro Aiuti (413-421).
The Wiskott-Aldrich Syndrome (WAS) is a monogenic X-linked primary immunodeficiency characterised alsoby thrombocytopenia, eczema, and a high susceptibility to develop tumours and autoimmunity. WAS patients have a severelyreduced life expectancy, unless they undergo a successful HLA-matched haematopoietic stem cell (HSC) transplantation.However, several WAS patients lack a compatible donor and complications, such as autoimmunity, can arise ina significant fraction of HSC transplanted patients. Administration of WAS gene-corrected autologous HSC represents analternative therapeutic approach, potentially applicable to all WAS patients. To this aim, several gene therapy approachesfor WAS using initially γ -retroviral vectors (RVs) and subsequently HIV-based lentiviral vectors (LVs) have been developed.In the present review, we will first describe the results of the preclinical studies conducted in the murine model ofWAS and then discuss the outcome of different phase I/II clinical trials using RV or LV- transduced HSC. Both genetherapy approaches led to restored WASP expression, correction of functional defects and clinical improvement. WhileRV-mediated gene therapy was associated with a high occurrence of leukaemia, results obtained in the first patientstreated with LV-based HSC gene therapy indicate a safer risk-benefit profile.

Gene/Cell Therapy Approaches for Immune Dysregulation Polyendocrinopathy Enteropathy X-Linked Syndrome by Laura Passerini, Francesca R. Santoni de Sio, Matthew H. Porteus, Rosa Bacchetta (422-428).
Immune dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome is a rare autoimmunedisease due to mutations in the gene encoding for Forkhead box P3 (FOXP3), a transcription factor fundamental for thefunction of thymus-derived (t) regulatory T (Treg) cells. The dysfunction of Treg cells results in the development of devastatingautoimmune manifestations affecting multiple organs, eventually leading to premature death in infants, if notpromptly treated by hematopoietic stem cell transplantation (HSCT). Novel gene therapy strategies can be developed forIPEX syndrome as more definitive cure than allogeneic HSCT. Here we describe the therapeutic approaches, alternative toHSCT, currently under development. We described that effector T cells can be converted in regulatory T cells by LVmediatedFOXP3-gene transfer in differentiated T lymphocytes. Despite FOXP3 mutations mainly affect a highly specificT cell subset, manipulation of stem cells could be required for long-term remission of the disease. Therefore, we believethat a more comprehensive strategy should aim at correcting FOXP3-mutated stem cells. Potentials and hurdles of bothstrategies will be highlighted here.

Safety of Gene Therapy: New Insights to a Puzzling Case by Michael Rothe, Axel Schambach, Luca Biasco (429-436).
Over the last few years, the transfer of therapeutic genes via gammaretro- or lentiviral vector systems hasproven its virtue as an alternative treatment for a series of genetic disorders. The number of approved phase I/II clinicaltrials, especially for rare diseases, is steadily increasing, but the overall hurdles to become a broadly acceptable therapyremain numerous. The efforts by clinicians and basic scientists have tremendously improved the knowledge availableabout feasibility and biosafety of gene therapy. Nonetheless, despite the generation of a plethora of clinical and preclinicalsafety data, we still lack sufficiently powerful assays to predictively assess the exact levels of toxicity that might be observedin any given clinical gene therapy. Insertional mutagenesis is one of the major concerns when using integratingvectors for permanent cell modification, and the occurrence of adverse events related to genotoxicity, in early gene therapytrials, has refrained the field of gene therapy from emerging further. In this review, we provided a comprehensiveoverview on the basic principles and potential co-factors concurring in the generation of adverse events reported in genetherapy clinical trials using integrating vectors. Additionally, we summarized the available systems to assess genotoxicityat the preclinical level and we shed light on the issues affecting the predictive value of these assays when translating theirresults into the clinical arena. In the last section of the review we briefly touched on the future trends and how they couldincrease the safety of gene therapy employing integrating vector technology to take it to the next level.

Gene Therapy for Haemophagocytic Lymphohistiocytosis by Claire Booth, Marlene Carmo, H. Bobby Gaspar (437-446).
Haemophagocytic lymphohistiocytosis (HLH) describes a severe and often fatal immunodysregulatory disordercaused primarily by the uncontrolled activation and proliferation of T cells and macrophages. A number of genetic defectsmainly involving defective granule exocytosis and effector cell cytotoxicity have been identified and well characterised atthe molecular and cellular level. These conditions have limited therapeutic options and given the predominant restrictionof the causative gene to the haematopoietic system, they have become attractive targets for haematopoietic cell gene therapy.Pre clinical studies in murine models of HLH due to perforin deficiency have shown correction of the disease phenotypeas a result of autologous haematopoietic stem cell (HSC) gene transfer using lentiviral vectors. In a murine model ofX-linked lymphoproliferative disease (XLP1), HSC gene transfer is able to correct the immunological manifestations ofthe disease. These encouraging murine studies have led to further work to develop clinically applicable strategies. An alternativeapproach is to correct defective T cells as this approach is safer than HSC gene therapy and may allow early controlof the HLH through the engraftment of functional gene modified effector T cells. Both strategies are now in developmentand a gene therapy option for certain genetic forms of HLH may soon enter clinical trials.

Gene Therapy for Chronic Granulomatous Disease: Current Status and Future Perspectives by Kerstin B. Kaufmann, Maria Chiriaco, Ulrich Siler, Andrea Finocchi, Janine Reichenbach, Stefan Stein, Manuel Grez (447-460).
Several Phase I/II clinical trials aiming at the correction of X-linked CGD by gene transfer into hematopoieticstem cells (HSCs) have demonstrated the therapeutic potential of gene modified autologous HSCs for the treatment ofCGD. Resolution of therapy-resistant bacterial and fungal infections in liver, lung and spinal canal of CGD patients wereclearly documented in all trials. However, clinical benefits were not sustained over time due to the failure of gene transducedcells to engraft long-term. Moreover, severe adverse effects were observed in some of the treated patients due to insertionalmutagenesis leading to the activation of growth promoting genes and to myeloid malignancy. These setbacksfostered the development of novel safety and efficacy improved vectors that have already entered or are about to enter theclinics. Meanwhile, ongoing research is constantly refining the CGD disease phenotype, including the definition of factorsthat may explain the unique engraftment phenotype observed in CGD gene therapy trials. This review provides a condensedoverview on the current knowledge of the molecular pathomechanisms and clinical manifestations of CGD andsummarizes the lessons learned from clinical gene therapy trials, the preclinical progress in vector design and the futureperspectives for the gene therapy of CGD.

TALEN-Mediated Generation and Genetic Correction of Disease-Specific Human Induced Pluripotent Stem Cells by Sivaprakash Ramalingam, Narayana Annaluru, Karthikeyan Kandavelou, Srinivasan Chandrasegaran (461-472).
Generation and precise genetic correction of patient-derived hiPSCs have great potential in regenerative medicine.Such targeted genetic manipulations can now be achieved using gene-editing nucleases. Here, we report generationof cystic fibrosis (CF) and Gaucher's disease (GD) hiPSCs respectively from CF (homozygous for CFTRΔF508 mutation)and Type II GD [homozygous for β-glucocerebrosidase (GBA) 1448T>C mutation] patient fibroblasts, using CCR5-specific TALENs. Site-specific addition of loxP-flanked Oct4/Sox2/Klf4/Lin28/Nanog/eGFP gene cassette at the endogenousCCR5 site of patient-derived disease-specific primary fibroblasts induced reprogramming, giving rise to bothmonoallele (heterozygous) and biallele CCR5-modified hiPSCs. Subsequent excision of the donor cassette was done bytreating CCR5-modified CF and GD hiPSCs with Cre. We also demonstrate site-specific correction of sickle cell disease(SCD) mutations at the endogenous HBB locus of patient-specific hiPSCs [TNC1 line that is homozygous for mutated β-globin alleles (βSS)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutatedβS to the wild-type βA in one of the HBB alleles, while the other allele remained a mutant phenotype. After excision of theloxP-flanked DNA cassette from the SCD-corrected hiPSC lines using Cre, we obtained secondary heterozygous βSAhiPSCs, which express the wild-type (βA) transcript to 30-40% level as compared to uncorrected (βSS) SCD hiPSCswhen differentiated into erythroid cells. Furthermore, we also show that TALEN-mediated generation and genetic correctionof disease-specific hiPSCs did not induce any off-target mutations at closely related sites.

Epigenetics and Periodontal Disease: Hope to Tame the Untameable by Vishakha Grover, Anoop Kapoor, Ranjan Malhotra, Sonia Sachdeva (473-481).
Epigenetics means gene expression alterations which occur due to the biochemical changes of the nucleotidesmodifying structure of DNA rather than the changes in the genetic code itself as in case of mutations. The epigenome,consisting of chromatin and its modifications, acts as a link between the inherited genome and the changes imposed by theenvironment. Over the past decade, there has been mounting evidence suggestive of associations between epigeneticmodifications and various human conditions such as aging, and most common human diseases viz. cancer, cardiovasculardiseases, diabetes, rheumatoid arthritis, HIV etc and the clearest evidence as the central mechanism for common multifactorialdiseases, has been identified with the factors involved in the inflammatory response. Periodontal disease, basicallyan immune-inflammatory affliction, being a multifactorial complex disease, owing to its high prevalence, chronicity andwide ranging systemic effects, essentially calls for a better comprehension of the underlying disease mechanisms, so as todevelop and decipher the novel methodologies to combat this disease. The current paper aims to visualize periodontal diseasefrom an epigenetic perspective, featuring the contemporary evidence supported literature and tends to explore thepossibilities to find some explanations for perio-systemic health links, individualized and improvised diagnostic tools forearlier detection and ways to halt the disease and help regeneration and reconstruction of the lost periodontal attachmentapparatus with the biology based approaches.