Lung gene therapy—How to capture illumination from the light already present in the tunnel

AbstractGene therapy has been considered as the most ideal medical intervention for genetic diseases because it is intended to target the cause of diseases instead of disease symptoms. Availability of techniques for identification of genetic mutations and for in vitro manipulation of genes makes it practical and attractive. After the initial hype in 1990s and later disappointments in clinical trials for more than a decade, light has finally come into the tunnel in recent years, especially in the field of eye gene therapy where it has taken big strides. Clinical trials in gene therapy for retinal degenerative diseases such as Leber's congenital amaurosis (LCA) and choroideremia demonstrated clear therapeutic efficacies without apparent side effects. Although these successful examples are still rare and sporadic in the field, they provide the proof of concept for harnessing the power of gene therapy to treat genetic diseases and to modernize our medication. In addition, those success stories illuminate the path for the development of gene therapy treating other genetic diseases. Because of the differences in target organs and cells, distinct barriers to gene delivery exist in gene therapy for each genetic disease. It is not feasible for authors to review the current development in the entire field. Thus, in this article, we will focus on what we can learn from the current success in gene therapy for retinal degenerative diseases to speed up the gene therapy development for lung diseases, such as cystic fibrosis

Temporal and Tissue Specific Regulation of RP-Associated Splicing Factor Genes PRPF3, PRPF31 and PRPC8—Implications in the Pathogenesis of RP

Genetic mutations in several ubiquitously expressed RNA splicing genes such as PRPF3, PRP31 and PRPC8, have been found to cause retina-specific diseases in humans. To understand this intriguing phenomenon, most studies have been focused on testing two major hypotheses. One hypothesis assumes that these mutations interrupt retina-specific interactions that are important for RNA splicing, implying that there are specific components in the retina interacting with these splicing factors. The second hypothesis suggests that these mutations have only a mild effect on the protein function and thus affect only the metabolically highly active cells such as retinal photoreceptors.We examined the second hypothesis using the PRPF3 gene as an example. We analyzed the spatial and temporal expression of the PRPF3 gene in mice and found that it is highly expressed in retinal cells relative to other tissues and its expression is developmentally regulated. In addition, we also found that PRP31 and PRPC8 as well as snRNAs are highly expressed in retinal cells.Our data suggest that the retina requires a relatively high level of RNA splicing activity for optimal tissue-specific physiological function. Because the RP18 mutation has neither a debilitating nor acute effect on protein function, we suggest that retinal degeneration is the accumulative effect of decades of suboptimal RNA splicing due to the mildly impaired protein

Overcoming Immunological Challenges to Helper-Dependent Adenoviral Vector-Mediated Long-Term CFTR Expression in Mouse Airways

Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and CF patients require life-long treatment. Although CFTR modulators show a great potential for treating most CF patients, some individuals may not tolerate the treatment. In addition, there is no effective therapy for patients with some rare CFTR mutations, such as class I CF mutations, which lead to a lack of CFTR protein production. Therefore, other therapeutic strategies, such as gene therapy, have to be investigated. Currently, immune responses to gene therapy vectors and transgene products are a major obstacle to applying CF gene therapy to clinical applications. In this study, we examined the effects of cyclophosphamide on the modulation of host immune responses and for the improvement of the CFTR transgene expression in the repeated delivery of helper-dependent adenoviral (HD-Ad) vectors to mouse lungs. We have found that cyclophosphamide significantly decreased the expression of T cell genes, such as CD3 (cluster of differentiation 3) and CD4, and reduced their infiltration into mouse lung tissues. We have also found that the levels of the anti-adenoviral antibody and neutralizing activity as well as B-cell infiltration into the mouse lung tissues were significantly reduced with this treatment. Correspondingly, the expression of the human CFTR transgene has been significantly improved with cyclophosphamide administration compared to the group with no treatment. These data suggest that the sustained expression of the human CFTR transgene in mouse lungs through repeated vector delivery can be achieved by transient immunosuppression

Overcoming Immunological Challenges to Helper-Dependent Adenoviral Vector-Mediated Long-Term CFTR Expression in Mouse Airways

Cystic Fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and CF patients require life-long treatment. Although CFTR modulators show a great potential for treating most CF patients, some individuals may not tolerate the treatment. In addition, there is no effective therapy for patients with some rare CFTR mutations, such as class I CF mutations, which lead to a lack of CFTR protein production. Therefore, other therapeutic strategies, such as gene therapy, have to be investigated. Currently, immune responses to gene therapy vectors and transgene products are a major obstacle to applying CF gene therapy to clinical applications. In this study, we examined the effects of cyclophosphamide on the modulation of host immune responses and for the improvement of the CFTR transgene expression in the repeated delivery of helper-dependent adenoviral (HD-Ad) vectors to mouse lungs. We have found that cyclophosphamide significantly decreased the expression of T cell genes, such as CD3 (cluster of differentiation 3) and CD4, and reduced their infiltration into mouse lung tissues. We have also found that the levels of the anti-adenoviral antibody and neutralizing activity as well as B-cell infiltration into the mouse lung tissues were significantly reduced with this treatment. Correspondingly, the expression of the human CFTR transgene has been significantly improved with cyclophosphamide administration compared to the group with no treatment. These data suggest that the sustained expression of the human CFTR transgene in mouse lungs through repeated vector delivery can be achieved by transient immunosuppression

TALEN-Mediated Gene Targeting for Cystic Fibrosis-Gene Therapy

Cystic fibrosis (CF) is an inherited monogenic disorder, amenable to gene-based therapies. Because CF lung disease is currently the major cause of mortality and morbidity, and the lung airway is readily accessible to gene delivery, the major CF gene therapy effort at present is directed to the lung. Although airway epithelial cells are renewed slowly, permanent gene correction through gene editing or targeting in airway stem cells is needed to perpetuate the therapeutic effect. Transcription activator-like effector nuclease (TALEN) has been utilized widely for a variety of gene editing applications. The stringent requirement for nuclease binding target sites allows for gene editing with precision. In this study, we engineered helper-dependent adenoviral (HD-Ad) vectors to deliver a pair of TALENs together with donor DNA targeting the human AAVS1 locus. With homology arms of 4 kb in length, we demonstrated precise insertion of either a LacZ reporter gene or a human cystic fibrosis transmembrane conductance regulator (CFTR) minigene (cDNA) into the target site. Using the LacZ reporter, we determined the efficiency of gene integration to be about 5%. In the CFTR vector transduced cells, we were able to detect CFTR mRNA expression using qPCR and function correction using fluorometric image plate reader (FLIPR) and iodide efflux assays. Taken together, these findings suggest a new direction for future in vitro and in vivo studies in CF gene editing

Subretinal gene delivery using helper-dependent adenoviral vectors

Abstract This study describes the successful delivery of helper-dependent adenoviral vectors to the mouse retina with long term and robust levels of reporter expression in the retina without apparent adverse effects. Since these vectors have a large cloning capacity, they have great potential to extend the success of gene therapy achieved using the adeno-associated viral vector