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Early efforts in cystic fibrosis (CF) gene therapy faced major challenges in delivery efficiency and sustained therapeutic gene expression. Recent advancements in engineered site-specific endonucle...

The COVID-19 pandemic has affected more than 120 million people and resulted in over 2.8 million deaths worldwide. Several COVID-19 vaccines have been approved for emergency use in humans and are being used in many countries. However, all of the approved vaccines are administered by intramuscular injection and this may not prevent upper airway infection or viral transmission. Here, we describe intranasal immunization of a COVID-19 vaccine delivered by a novel platform, the helper-dependent adenoviral (HD-Ad) vector. Since HD-Ad vectors are devoid of adenoviral coding sequences, they have a superior safety profile and a large cloning capacity for transgenes. The vaccine (HD-Ad_RBD) codes for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and intranasal immunization induced robust mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost regimen, resulted in complete protection of the upper respiratory tract against SARS-CoV-2 infection. As such, intranasal immunization based on the HD-Ad vector promises to provide a powerful platform for constructing highly effective vaccines targeting SARS-CoV-2 and its emerging variants.

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.

Cystic fibrosis (CF) is a genetic disorder affecting multiple organs, including the pancreas, hepatobiliary system and reproductive organs; however, lung disease is responsible for the majority of morbidity and mortality. Management of CF involves CF transmembrane conductance regulator (CFTR) modulator agents including corrector drugs to augment cellular trafficking of mutant CFTR as well as potentiators that open defective CFTR channels. These therapies are poised to help most individuals with CF, with the notable exception of individuals with class I mutations where full-length CFTR protein is not produced. For these mutations, gene replacement has been suggested as a potential solution.In this work, we used a helper-dependent adenoviral vector (HD-CFTR) to express CFTR in nasal epithelial cell cultures derived from CF subjects with class I CFTR mutations.CFTR function was significantly restored in CF cells by HD-CFTR and reached healthy control functional levels as detected by Ussing chamber and membrane potential (FLIPR) assay. A dose–response relationship was observed between the amount of vector used and subsequent functional outcomes; small amounts of HD-CFTR were sufficient to correct CFTR function. At higher doses, HD-CFTR did not increase CFTR function in healthy control cells above baseline values. This latter observation allowed us to use this vector to benchmarkin vitroefficacy testing of CFTR-modulator drugs.In summary, we demonstrate the potential for HD-CFTR to informin vitrotesting and to restore CFTR function to healthy control levels in airway cells with class I or CFTR nonsense mutations.

Summary Background Lifestyle interventions can delay the onset of type 2 diabetes in people with impaired glucose tolerance, but whether this leads subsequently to fewer complications or to increased longevity is uncertain. We aimed to assess the long-term effects of lifestyle interventions in people with impaired glucose tolerance on the incidence of diabetes, its complications, and mortality. Methods The original study was a cluster randomised trial, started in 1986, in which 33 clinics in Da Qing, China, were randomly assigned to either be a control clinic or provide one of three interventions (diet, exercise, or diet plus exercise) for 6 years for 577 adults with impaired glucose tolerance who usually receive their medical care from the clinics. Subsequently, participants were followed for up to 30 years to assess the effects of intervention on the incidence of diabetes, cardiovascular disease events, composite microvascular complications, cardiovascular disease death, all-cause mortality, and life expectancy. Findings Of the 577 participants, 438 were assigned to an intervention group and 138 to the control group (one refused baseline examination). After 30 years of follow-up, 540 (94%) of 576 participants were assessed for outcomes (135 in the control group, 405 in the intervention group). During the 30-year follow-up, compared with control, the combined intervention group had a median delay in diabetes onset of 3·96 years (95% CI 1·25 to 6·67; p=0·0042), fewer cardiovascular disease events (hazard ratio 0·74, 95% CI 0·59–0·92; p=0·0060), a lower incidence of microvascular complications (0·65, 0·45–0·95; p=0·025), fewer cardiovascular disease deaths (0·67, 0·48–0·94; p=0·022), fewer all-cause deaths (0·74, 0·61–0·89; p=0·0015), and an average increase in life expectancy of 1·44 years (95% CI 0·20–2·68; p=0·023). Interpretation Lifestyle intervention in people with impaired glucose tolerance delayed the onset of type 2 diabetes and reduced the incidence of cardiovascular events, microvascular complications, and cardiovascular and all-cause mortality, and increased life expectancy. These findings provide strong justification to continue to implement and expand the use of such interventions to curb the global epidemic of type 2 diabetes and its consequences. Funding US Centers for Disease Control and Prevention, WHO, Chinese Center for Disease Control and Prevention, World Bank, Ministry of Public Health of the People's Republic of China, Da Qing First Hospital, China–Japan Friendship Hospital, and National Center for Cardiovascular Diseases & Fuwai Hospital.

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.

Lung is an immune sensitive organ with presence of all immune cells at the frontline of immunity. Under chronic infection conditions, inducible Bronchus-associated lymphoid tissue (iBALT) can be formed to defend foreign infectious organisms. We wonder whether viral vectors for gene therapy could induce iBALT formation and whether this process can be controlled through immune modulation. To test these ideas, we repeatedly administered a helper-dependent adenoviral (HD-Ad) vector to mouse lungs and examined iBALT formation. HD-Ad vectors are based on adenovirus but with all viral coding sequences deleted and is one option for large gene delivery. In our previous studies, HD-Ad vectors have exhibited high in vivo efficiency in transduction of mouse, rabbit and pig airway epithelial cells, including stem/progenitor cells such as basal cells. HD-Ad vectors expressing therapeutic gene encoding cystic fibrosis transmenbrane conductance regulator (CFTR) rescue CFTR function in CF patient primary cells. In our mouse studies, we found that HD-Ad vectors induced iBALT formation and antibody production. We used cyclophosphamide or HD-Ad expressing IL-10 to modulate the host immune responses and to examine the effects on iBALT formation. Comparing with control group (HD-Ad vectors only), the mice treated with cyclophosphamide or IL-10 expressing HD-Ad vector showed a significant reduction of iBALT formation and infiltration of inflammatory cells, including CD4+ and B220+ as well as production of anti-Ad antibodies and neutralizing antibodies. Our results indicate that transient modulation significantly mitigated immunopathology and extended transgene expression in mouse airways following readministration of HD-Ad vectors.

A major challenge in developing gene-based therapies for airway diseases such as cystic fibrosis (CF) is sustaining therapeutic levels of transgene expression over time. This is largely due to airway epithelial cell turnover and the host immunogenicity to gene delivery vectors. Modern gene editing tools and delivery vehicles hold great potential for overcoming this challenge. There is currently not much known about how to deliver genes into airway stem cells, of which basal cells are the major type in human airways. In this study, helper-dependent adenoviral (HD-Ad) vectors were delivered to mouse and pig airways via intranasal delivery, and direct bronchoscopic instillation, respectively. Vector transduction was assessed by immunostaining of lung tissue sections, which revealed that airway basal cells of mice and pigs can be targeted in vivo. In addition, efficient transduction of primary human airway basal cells was verified with an HD-Ad vector expressing green fluorescent protein. Furthermore, we successfully delivered the human CFTR gene to airway basal cells from CF patients, and demonstrated restoration of CFTR channel activity following cell differentiation in air-liquid interface culture. Our results provide a strong rationale for utilizing HD-Ad vectors to target airway basal cells for permanent gene correction of genetic airway diseases.

Gene 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.

ZNF403, also known as GGNBP2 (gametogenetin binding protein 2), is a highly conserved gene implicated in spermatogenesis. However, the exact biological function of ZNF403 is not clear. In this study, we identified the role of ZNF403 in cell proliferation and cell-cycle regulation by utilizing short hairpin RNA (shRNA)-mediated knockdown. ZNF403-specific shRNA expressing helper-dependent adenoviral vector (HD-Ad-ZNF403-shRNA) was constructed and transduced human cell lines. ZNF403 mRNA and protein expression levels were inhibited as evidenced by real-time PCR and western blot analyses. Noticeably, we found that knockdown of ZNF403 expression suppressed cell proliferation compared to the non-target shRNA and vector controls. Furthermore, cell-cycle analysis demonstrated that downregulation of ZNF403 promoted G2/M cell-cycle arrest in a dose-dependent manner. Moreover, human cell-cycle real-time PCR array revealed that ZNF403 knockdown influenced the expression profile of genes in cell-cycle regulation. Among these genes, western blot analysis confirmed the protein up-regulation of p21 and down-regulation of MCM2 in response to the ZNF403 knockdown. Additionally, knockdown of ZNF403 also showed an anti-carcinogenetic effect on anchorage-independent growth by colony formation assay and tumor cell migration by wound-healing assay with human laryngeal cancer cell line Hep-2 cells. Altogether, our findings suggest an essential role of ZNF403 in cell proliferation and provide a new insight into the function of ZNF403 in regulating the G2/M cell-cycle transition.

Objective: The purpose of this study was to establish a comprehensive evaluation system to assess the risk factors of lung cancer for the general population. Methods: With the method of evidence-based medicine, risk factors of lung cancer were identified and their risk assignments were calculated to design the Self-evaluation Scoring Questionnaire for High-risk Individuals of Lung Cancer. Studies including more than 10 000 subjects were carried the out to confirm the questionnaire's value. Results: The questionnaire consisted of 15 risk factors and their risk assignments, such as sex, age, smoking, female passive smoking, previous illness histories, exposure to harmful gases, mental depression and genetic susceptibility. In the population application, data from 30 lung cancer patients revealed its desired reliability and validity. The next pre-investigation, including 94 patients and 252 controls, confirmed its differentiating power, and encouraged a much larger-scale survey with 2161 subjects to determine the threshold (T) to identify high-risk individuals, the threshold was 116 points. According to this criterion, 1537 high-risk volunteers and 6556 controls were recruited to participate in a 3-year follow-up study from 2007 to 2009. There were 31 cases of lung cancer detected in the high-risk group, with a detection rate of 2.02%, significantly higher than that of the controls (5/6556, 0.08%), indicating an excellent predictive value of the questionnaire. Conclusions: The Self-evaluation Scoring Questionnaire for High-risk Individuals of Lung Cancer was a good means for evaluating the risks of lung cancer.

After two decades of ups and downs, gene therapy has recently achieved a milestone in treating patients with Leber's congenital amaurosis (LCA). LCA is a group of inherited blinding diseases with retinal degeneration and severe vision loss in early infancy. Mutations in several genes, including RPE65, cause the disease. Using adeno-associated virus as a vector, three independent teams of investigators have recently shown that RPE65 can be delivered to retinal pigment epithelial cells of LCA patients by subretinal injections resulting in clinical benefits without side effects. However, considering the whole field of gene therapy, there are still major obstacles to clinical applications for other diseases. These obstacles include innate and immune barriers to vector delivery, toxicity of vectors and the lack of sustained therapeutic gene expression. Therefore, new strategies are needed to overcome these hurdles for achieving safe and effective gene therapy. In this article, we shall review the major advancements over the past two decades and, using lung gene therapy as an example, discuss the current obstacles and possible solutions to provide a roadmap for future gene therapy research.

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.

Airway inflammation is the hallmark of many respiratory disorders, such as asthma and cystic fibrosis. Changes in airway gene expression triggered by inflammation play a key role in the pathogenesis of these diseases. Genetic linkage studies suggest that ESE-2 and ESE-3, which encode epithelium-specific Ets-domain-containing transcription factors, are candidate asthma susceptibility genes. We report here that the expression of another member of the Ets family transcription factors ESE-1, as well as ESE-3, is upregulated by the inflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) in bronchial epithelial cell lines. Treatment of these cells with IL-1beta and TNF-alpha resulted in a dramatic increase in mRNA expression for both ESE-1 and ESE-3. We demonstrate that the induced expression is mediated by activation of the transcription factor NF-kappaB. We have characterized the ESE-1 and ESE-3 promoters and have identified the NF-kappaB binding sequences that are required for the cytokine-induced expression. In addition, we also demonstrate that ESE-1 upregulates ESE-3 expression and downregulates its own induction by cytokines. Finally, we have shown that in Elf3 (homologous to human ESE-1) knockout mice, the expression of the inflammatory cytokine interleukin-6 (IL-6) is downregulated. Our findings suggest that ESE-1 and ESE-3 play an important role in airway inflammation.

Sustained expression of the CFTR gene is a major challenge to gene therapy with either viral or nonviral vectors with immune response to vector and transgene products. One strategy to achieve sustained CFTR expression is to modulate the host immune system through transient immunosuppression. In this study, we examined cyclophosphamide (cytoxan), dexamethasone (Dex), and a combination of cyclosporin, methylprednisolone, and azathioprine (combination) for their effects on long-term expression of the human CFTR delivered with helper-dependent adenoviral vectors in mouse airways. We found that cyclophosphamide significantly enhanced long-term expression of the transgenic human CFTR and the reporter gene LacZ by reducing host immune responses. Dex administration greatly reduced neutralizing antibody production but had no effect on transgene expression. Treatment with a combination of cyclosporin A, azathioprine, and methylprednisolone affected neither CFTR gene expression nor inflammation. Our data suggest that transient immunosuppression might be a strategy to improve sustained expression in gene therapy.

There have been significant advancements in the field of retinal gene therapy in the past several years. In particular, therapeutic efficacy has been achieved in three separate human clinical trials conducted to assess the ability of adeno-associated viruses (AAV) to treat of a type of Leber's congenital amaurosis caused by RPE65 mutations. However, despite the success of retinal gene therapy with AAV, challenges remain for delivering large therapeutic genes or genes requiring long DNA regulatory elements for controlling their expression. For example, Stargardt's disease, a form of juvenile macular degeneration, is caused by defects in ABCA4, a gene that is too large to be packaged in AAV. Therefore, we investigated the ability of helper dependent adenovirus (HD-Ad) to deliver genes to the retina as it has a much larger transgene capacity.Using an EGFP reporter, our results showed that HD-Ad can transduce the entire retinal epithelium of a mouse using a dose of only 1 × 105 infectious units and maintain transgene expression for at least 4 months. The results demonstrate that HD-Ad has the potential to be an effective vector for the gene therapy of the retina.

Paraoxonase (PON) is an HDL-bound enzyme capable of hydrolyzing lipid peroxides and believed to be in part responsible for the protective effect of HDL against LDL oxidation. Its activity is mainly determined by a gene polymorphism of the PON 1 gene (Glu-Arg 192). Low activity has been related to an elevated incidence of myocardial infarction. In several case-control studies, however, the high activity B allele is paradoxically more prevalent in patients. We have re-investigated this relationship, using carotid intima-media thickness (IMT) as a surrogate continuous variable for macroangiopathy. Genotypes were determined in 197 non insulin-dependent diabetic patients (HbAlc 8.8+/-0.15%, BMI 28.3+/-0.36). IMT, measured by high resolution mode B ultrasound, was the same for all genotypes (AA: 0.83+/-.013, AB 0.82+/-.017 and BB: 0.81+/-.034 mm). Bearers of the B allele displayed higher Lp(a) concentration (AA: 197+/-28, AB: 221+/-26, BB: 225+/-45 mg/l, P=0.024) with a significant linear trend (P0.005). Multiple regression showed age and systolic blood pressure, but not Lp(a), to be the main determinants of IMT variability without the contribution of the PON genotype. No consistent differences could be found between genotypes in the peroxidizability of LDL (lag-time, rate of diene production and maximal concentration). Our data support the view that there is no association between the early changes of atherosclerosis as defined by carotid IMT and variation in codon 192 of PON 1.

Cystic Fibrosis (CF) is a common life-threatening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial chloride channel. As a chronic, lifelong disease, CF should be best treated with a continue level of CFTR expression. Pulmonary gene therapy may ultimately cure the CF lung disease. Thus, efficient long-term expression of the delivered CFTR gene in targeted cell types is essential to achieve this goal either by repeated application or with a long-duration expression system. Even though novel approaches to target airway progenitor/stem cells with gene editing may be more attractive, the efficiency for targeting specific progenitor cells will still be a challenge in airway delivery. Repeated administration of viral vectors may be required to increase the percentage of cells to be targeted and to boost long-term therapeutic effects. However, immune responses to viral proteins are the greatest barrier to repeated vector administration. Here, we demonstrated that repeated transduction of human CFTR gene in mouse airway can be successfully achieved with helper-dependent adenoviral (HD-Ad) vector through transient immunosuppression.We showed previously that cyclophosphamide significantly enhanced human CFTR expression in mice received HD-Ad vector readministration and sustained expression through inhibition of host immune reactions. In this study, we examined cyclophosphamide effects on expression of the human CFTR in multiple rounds of HD-Ad vector delivery to mouse airways. Four group mice were nasally delivered with an HD-Ad-K18-CFTR vector at 1.5×1010 vector particles per mouse at day 0, day 70 and day 120. Two groups were treated with cyclophosphamide 6 hours before and 4 day and 8 days after vector delivery each time (except the last round of vector delivery for mice sacrificed at day 123). The other two groups without treatment were used as controls. One treated group and one control group were sacrificed at day 123, and the others at day 153 for detecting transgene expression and immune reaction. We found that cyclophosphamide has significantly improved the expression of CFTR (4.1 times higher) compared to the control group as determined by real-time qPCR 3 days after last delivery. And the CFTR expression level was 3.1 times higher 30 days after last vector transduction. Cyclophosphamide also significantly reduced the levels of antiadenoviral and neutralizing antibodies in both BALF and serum as well as prevented leukocyte infiltration in lung tissues. This data suggested that efficient repeated transduction of human CFTR gene with HD-Ad vector can be achieved by transient immunopuression in mouse lung.

The standard definition of high-risk individuals for lung cancer was not uniform and the value of chest digital radiography (DR) in lung cancer screening was still unproven. The aim of this study was to assess whether the original questionnaire named as "Self-evaluation Scoring Questionnaire for High-risk Individuals of Lung Cancer" combined with DR examinations could detect early stage of lung cancer effectively. The Self-evaluation Scoring Questionnaire for High-risk Individuals of Lung Cancer had been designed in previous studies. Subjects with scores over 116 points were regarded as high-risk individuals and underwent the current DR scans at least once a year from 2007 to 2009. Noncalcified nodules with a diameter over 30 mm, along with enlarged pulmonary hilus and atelectasis, were considered to be positive and subjected to further special examinations. Efficacy of the scoring questionnaire combined with DR scans was estimated by 3-year results. Among 1,537 subjects, 13, 11, and 7 were diagnosed with lung cancer in the first, second, and third year, respectively, indicating the detection rate of 2.02 % (31/1,537). In addition, 77.42 % (24/31) of the patients were in stage I and 51.61 % (16/31) were adenocarcinomas. For the 31 cases, 28 were defined as detected cancers, while the other three were interval ones, only accounting for 0.20 % (3/1,504) of individuals with negative judgments. The protocol of Self-evaluation Scoring Questionnaire for High-risk Individuals of Lung Cancer combined with DR scans is a cost-effective and safe approach to detect early stage of lung cancer.

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.

Background Clinical studies have shown that gene therapy is a promising approach for treating such genetic diseases as the eye disease, Leber's congenital amaurosis. Development of gene therapy approaches for treating chronic inflammatory diseases is, however, more challenging because it requires the production of anti-inflammatory molecules at the diseased tissues only when they are needed. Methods We designed such a system by modifying the human interleukin (IL)-6 gene promoter to direct transgene expression and delivered the system into cultured cells as well as mouse lungs using a helper-dependent adenoviral vector. Results We have demonstrated both in vitro and in vivo that the reporter LacZ or human IL-10 gene can be induced by inflammatory stimuli. Conclusions The results obtained indicate that the inflammation inducible gene expression system based on the modified human IL-6 gene promoter has the potential to be used for developing gene therapy for treating inflammatory diseases. Copyright © 2010 John Wiley & Sons, Ltd.

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

In spite of the extensive research in the field of gene therapy, host immune responses continue to be the major barrier in translating basic research to clinical practice. Helper-dependent adenoviral (HD-Ad) vectors show great potential for pulmonary gene therapy, but the knowledge of pulmonary immune responses toward these vectors is very limited. In this study, we show that HD-Ad vectors are potent stimulators of dendritic cell (DC) maturation, thus leading to stimulation of T cell proliferation with ∼6% of naive CD4+ T cells from pulmonary mediastinal lymph node responding to HD-Ad-treated DCs. In contrast to the belief that HD-Ad vectors are unable to prime adaptive immune response, we show for the first time, through in vivo pulmonary studies in mice, that HD-Ad vectors can prime CD4+ and CD8+ T cell responses in the lung at high and substantially low doses. This indicates cross-presentation of HD-Ad-derived epitopes by DCs to prime CD8+ T cell responses. To assess the basis of pulmonary T cell response against HD-Ad vectors, we examined the response of conventional DCs (cDCs) and plasmacytoid DCs (pDCs) in the lung. In response to HD-Ad delivery, there is induction of maturation in both cDC and pDC subsets, but it is the cDCs, not pDCs, that migrate rapidly to draining lymph nodes within the first 2 days after vector delivery to prime adaptive immune response against these vectors. These findings have implications for development of strategies to prevent adaptive immune responses against gene therapy vectors.

Mutations in PRPF3, a gene encoding the essential pre-mRNA splicing factor Hprp3p, have been identified in patients with autosomal dominant retinitis pigmentosa type 18 (RP18). Patients with RP18 have one of two single amino acid substitutions, Pro493Ser or Thr494Met, at the highly conserved Hprp3p C-terminal region. Pro493Ser occurs sporadically, whereas Thr494Met is observed in several unlinked RP families worldwide. The latter mutation also alters a potential recognition motif for phosphorylation by casein kinase II (CKII). To understand the molecular basis of RP18, we examined the consequences of Thr494Met mutation on Hprp3p molecular interactions with components of the U4/U6.U5 small nuclear ribonucleoprotein particles (snRNPs) complex. Since numerous mutations causing human diseases change pre-mRNA splice sites, we investigated whether Thr494Met substitution affects the processing of PRPF3 mRNA. We found that Thr494Met does not affect PRPF3 mRNA processing, indicating that the mutation may exert its effect primarily at the protein level. We used small hairpin RNAs to specifically silence the endogenous PRPF3 while simultaneously expressing HA-tagged Thr494Met. We demonstrated that the C- but not N-terminal region of Hprp3p is indeed phosphorylated by CKII in vitro and in cells. CKII-mediated Hprp3p phosphorylation was significantly reduced by Thr494Met mutation. Consequently, the Hprp3p C-terminal region is rendered partially defective in its association with itself, Hprp4p, and U4/U6 snRNA. Our findings provide new insights into the biology of Hprp3p and suggest that the loss of Hprp3p phosphorylation at Thr494 is a key step for initiating Thr494Met aberrant interactions within U4/U6 snRNP complex and that these are likely linked to the RP18 phenotype.

Innate immune responses form the first line of defense against foreign insults and recently significant advances have been made in our understanding of the initiation of innate immune response along with its ability to modulate inflammation. In airway diseases such as asthma, COPD and cystic fibrosis, over reacting of the airway innate immune responses leads to cytokine imbalance and airway remodeling or damage. Helper-dependent adenoviral vectors have the potential to deliver genes to modulate airway innate immune responses and have many advantages over its predecessors. However, there still are a few limitations that need to be addressed prior to their use in clinical applications.

Cystic Fibrosis (CF) is a common life-threatening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial chloride channel. Gene therapy for CF lung disease has been tested extensively. However, sustained expression of the CFTR gene is a major challenge to gene delivery with either viral or no viral vectors. One strategy to improve the persistent CFTR expression is to modulate host immune responses by transient immunosuppression.We showed previously that cyclophosphamide significantly enhanced human CFTR expression in mice received HD-Ad vector readministration, through inhibition of host immune reactions. In this study, we examined cyclophosphamide, dexmethoson, and combination of cyclosporine, methylprednsolone and azathioprine, for their effects on long term expression of the human CFTR delivered with helper-dependent adenoviral (HD-Ad) vectors to mouse airways. Four group mice were nasally delivered with an HD-Ad-K18-CFTR vector at 1.5×10E10 particles per mouse. Three groups were treated with immunesuppresants and one group without any treatment was used as a control. The immunosuppressants were administrated twice, at 6 hours before and 48 hours after nasal delivery of the HD-Ad-CFTR vector. Expression of the CFTR gene was assessed 4 months after vector administration. We found that cyclophosphamide has significantly improved the sustained expression of the CFTR gene (2.5 times higher) compared to the no treatment group as determined by real-time qPCR. It alsogreatly reduced the levels of anti-adenoviral and neutralizing antibodies in both BALF and serum. However, cyclophosphamide did not show major effects on the innate immune reaction to HD-Ad vectors, such as NK cell infiltration and activation, 4 days after vector delivery. Dexmethesone also greatly reduced neutralizing antibodies production, but not affected transgene expression. A combination of cyclosporine, methylprednsolone and azathioprine which is used to suppress immune reactions in lung transplantation, also did not affect CFTR gene expression, nor immune reactions following HD-Ad-CFTR vector delivery. These data demonstrate that transient administration of cyclophosphamide can significantly improve sustained HD-Ad-mediated transgene expression in mice and it is more effective than dexmethsone or the combination of cyclosporine, methylprednsolone and azathioprine.

Adenovirus-based vectors are promising vehicles for gene replacement therapy due to their ability to efficiently transduce a wide variety of proliferating and non-proliferating cells. Over the past decade, different versions of adenoviral vectors (Ads) have been developed. These vectors can be classified into two major categories, based on whether the viral coding sequences are partially (first or second-generation Ads) or completely deleted (helper-dependent or gutted Ads). Both types of Ads have been tested in a variety of gene delivery studies, and major obstacles to their clinical application have been identified. Currently, innate and adaptive host immune responses to Ads remain major challenges, limiting both the initial viral dose and the effectiveness of subsequent administrations. Recent developments in vector design and delivery methods have improved the potential of Ads for successful gene therapy application.

High density lipoprotein (HDL)-associated paraoxonase (PON) seems to play a major role in the protection of low density lipoprotein (LDL) against peroxidation by HDL, and the partly purified enzyme exerts a dose-dependent protective effect. A common polymorphism of the human gene (192 Q→R) modulates paraoxonase activity but purified enzyme from either genotype is equally effective against LDL peroxidation. The inhibition of Cu2+-induced LDL peroxidation by HDL was monitored by lipid peroxide assay and change in LDL electrophoretic mobility. We show that HDL from type 2 diabetic patients with the QQ or RR genotype (n = 12 for each) reduce, to the same extent, both peroxide production (by 60.6 ± 20.0 and 63.9 ± 23.5%) and relative change in mobility (61.3 ± 21.8 and 61.4 ± 26.5%) despite a 6-fold difference in paraoxonase activity (47.4 ± 4.4 vs. 299.7 ± 23.7 U/l, P < 0.0001). Protection was, however, related to paraoxonase activity, but with a different efficiency in each group corresponding to a better protection per unit of enzyme in the QQ genotype group. Inactivation of PON activity by heating (56°C, 10 min) or by EDTA was totally without effect on protection, which remained correlated with the paraoxonase activity measured prior to inactivation. In summary, these results suggest that the protein bearing both paraoxonase and arylesterase activities also possesses a third thermostable property, closely associated with paraoxon hydrolysis activity and unaffected by PON genetic variability.—Cao, H., A. Girard-Globa, F. Berthezene, and P. Moulin. Paraoxonase protection of LDL against peroxidation is independent of its esterase activity towards paraoxon and is unaffected by the Q→R genetic polymorphism. J. Lipid Res. 1999. 40: 133–139.

Our progress in understanding mammalian gene function has lagged behind that of gene identification. New methods for mammalian gene functional analysis are needed to accelerate the process. In yeast, the powerful genetic shuffle system allows deletion of any chromosomal gene by homologous recombination and episomal expression of a mutant allele in the same cell. Here, we report a method for mammalian cells, which employs a helper-dependent adenoviral (HD-Ad) vector to synthesize small hairpin (sh) RNAs to knock-down the expression of an endogenous gene by targeting untranslated regions (UTRs). The vector simultaneously expresses an exogenous version of the same gene (wild-type or mutant allele) lacking the UTRs for functional analysis. We demonstrated the utility of the method by using PRPF3, which encodes the human RNA splicing factor Hprp3p. Recently, missense mutations in PRPF3 were found to cause autosomal-dominant Retinitis Pigmentosa, a form of genetic eye diseases affecting the retina. We knocked-down endogenous PRPF3 in multiple cell lines and rescued the phenotype (cell death) with exogenous PRPF3 cDNA, thereby creating a genetic complementation method. Because Ad vectors can efficiently transduce a wide variety of cell types, and many tissues in vivo, this method could have a wide application for gene function studies.