Hearing preservation after inner ear gene therapy: the effect of vector and surgical approach

Over seventy studies have examined the potential of gene therapy in the inner ear. For the most part, they have focused on adenoviral vectors and delivery into the cochlea. Most studies have emphasized looking at the expression of marker genes driven by a CMV promoter and have used first-generation adenoviral constructs. E1/E3/E4 deleted adenoviral vectors carrying the green fluorescent protein (GFP) gene were injected into the round window, the basal turn of the cochlea (via a cochleostomy) or into the superior semicircular canal. Hearing was then tested 24 h after viral gene transfer. Large vector titers in small volumes of fluid were well tolerated with the round window approach resulting in complete hearing preservation with transfer of GFP to hair cells and spiral ganglion cells. Injection of comparable doses of vector into a basal turn cochleostomy resulted in high-frequency hearing loss. Addition of a pancaspase inhibitor protected hearing when larger volumes of fluid were administered to the inner ear.     

腺相关病毒介导自杀基因系统对头颈肿瘤细胞体外杀伤效应

目的探讨腺相关病毒(adeno-associated virus,AAV)介导的I型人单纯疱疹病毒胸苷激酶基因(herpes simplex virus thymidine kinase,HSV-tk)联合羟甲基无环鸟苷(ganciclovir,GCV)系统(HSV- tk／GCV)对头颈肿瘤细胞系的敏感性,以及组蛋白脱羧酶抑制剂FR901228对AAV-HSV-tk／GCV系统头颈肿瘤细胞系的体外杀伤效应的影响。方法采用AAV各血清型转染三种头颈肿瘤细胞系(黑色素瘤细胞系B-16,人喉鳞状细胞癌细胞系HEp-2和人上颔窦癌细胞系NKO-1),检测荧光表达强度。进一步应用AAV携载HSV-tk／GCV系统感染细胞系,以及与FR901 228联合应用,XTT法检测肿瘤杀伤效应。实时定量PCR检测细胞中tk基因的含量及其表达。结果经统计学分析显示,不同血清型AAV对三种细胞系转染率有显著差异。AAV1型对B-16,AAV2型对HEp-2以及AAV3型对NKO-1转导效率高。AAV介导的HSV-tk／GCV系统对三种细胞系均具有明显的杀伤效应,FR901228增加了细胞内tk基因的含量及其表达,同时明显提高了肿瘤杀伤效应,其LD50明显小于对照组。结论头颈肿瘤的细胞系对AAV介导的HSV-tk/GCV系统均高度敏感,联合FR901 228增强对肿瘤的杀伤效应,可望成为头颈肿瘤基因治疗的新途径。     

Gene transfer in human vestibular epithelia and the prospects for inner ear gene therapy.

Transfer of exogenous genetic material into the mammalian inner ear using viral vectors has been characterized over the last decade. A number of different viral vectors have been shown to transfect the varying cell types of the nonprimate mammalian inner ear. Several routes of delivery have been identified for introduction of vectors into the inner ear while minimizing injury to existing structures and at the same time ensuring widespread distribution of the agent throughout the cochlea and the rest of the inner ear.These studies raise the possibility that gene transfer may be developed as a potential strategy for treating inner ear dysfunction in humans. Furthermore, a recent report showing successful transfection of excised human vestibular epithelia offers proof of principle that viral gene transfer is a viable strategy for introduction and expression of exogenous genetic material to restore function to the inner ear. Human vestibular epithelia were harvested from patients undergoing labyrinthectomy, either for intractable Ménière's disease or vestibular schwannoma resection, and cultured for as long as 5 days. In those experiments, recombinant, multiply-deleted, replication-deficient adenoviral vectors were used to transfect and express a reporter gene as well as the functionally relevant gene, wild-type KCNQ4, a potassium channel gene that when mutated causes the autosomal dominant HL DFNA2.Here, we review the current state of viral-mediated gene transfer in the inner ear and discuss different viral vectors, routes of delivery, and potential applications of gene therapy. Emphasis is placed on experiments demonstrating viral transfection of human inner ear tissue and implications of these findings and for the future of gene therapy in the human inner ear.     

pAdKDR-tk/GCV自杀基因系统联合γ射线放疗鼻咽癌的体内实验

目的:探讨pAdKDR-tk/GCV自杀基因系统联合γ射线放疗人鼻咽癌的杀伤效应。方法:分别以pAdKDR-tk/GCV、60Coγ射线放疗及两者联合治疗裸鼠人鼻咽癌移植瘤模型,比较疗效;利用肿瘤生长曲线及肿瘤抑瘤率评价该自杀基因系统联合放疗的疗效。结果:单纯基因治疗与放疗对鼻咽癌CNE-2细胞裸鼠皮下移植瘤生长的抑瘤率分别为58.43%和70.88%,而基因治疗与放疗联合应用的抑瘤率达到84.39%,与前两者比较,差异有统计学意义(P<0.01);第21天基因治疗与放疗联合治疗组肿瘤平均体积仅为对照组的13.5%,明显低于单纯基因治疗与放疗。结论:pAdKDR-tk/GCV自杀基因系统联合放疗的疗效较单一治疗方案有显著的提高,为进一步开展靶向肿瘤血管内皮自杀基因联合放疗研究奠定了良好的理论和临床基础。     

Novel adenoviral gene delivery system targeted against head and neck cancer

OBJECTIVES: The clinical applicability of adenovirus-mediated gene therapy is limited by the lack of tumor-targeted strategies. Ubiquitous expression of the coxsackie-adenovirus receptor, the native binding site for adenovirus, broadens viral tropism and increases systemic toxicity. Adenoviruses can be genetically engineered to target tumor-specific cell surface biomarkers. Here, we present a novel recombinant adenovirus vector (Ad5-Flag-LDS) that demonstrated a marked targeting bias against Hsp47, a biomarker for head and neck squamous cell carcinoma (HNSCC). METHODS: Cell surface expression of Hsp47 was determined in six human HNSCC cell lines and in negative and positive control cells. Colocalization of LDS and Hsp47 was assessed by immunocytochemistry in Ad5-Flag-LDS-transfected cells, and subsequent transgene expression was determined. The contribution of the Hsp47 biomarker in mediating targeted gene transfer was evaluated with a blocking assay. Ad5-Flag-LDS-targeting efficacy in a mixed cell population was determined by immunofluorescence. RESULTS: HNSCC cells had significantly higher Hsp47 biomarker density than control cell lines. After Ad5-Flag-LDS transfection, significant colocalization was found between the LDS peptide and Hsp47 biomarker, indicating that viral entry occurred via Hsp47-LDS binding. This unique tumor-targeted entry feature significantly enhanced gene transfer relative to an untargeted adenoviral vector. Blockade of Hsp47 biomarkers abrogated transgene expression, indicating that Hsp47 has a dominant role in Ad5-Flag-LDS targeting. Ad5-Flag-LDS-targeting selectivity was maintained in a cell mixture, producing greater transgene expression in Hsp47-expressing cells. CONCLUSIONS: The enhanced targeting achieved with Ad5-Flag-LDS highlights a potential strategy for clinically applicable targeted gene therapy against HNSCC or any tumor type expressing Hsp47.     

Limitations of adenovirus-mediated interleukin-2 gene therapy for oral cancer

OBJECTIVE/HYPOTHESIS: Adenoviral interleukin-2 (AdV-IL-2) gene therapy has previously not proven effective in treating established murine oral cancer. We hypothesize that the intratumoral level of IL-2 expression is a major limiting factor in treatment outcome. METHODS: A microscopic disease and established oral cancer murine model was used to test this hypothesis. IL-2 gene transfer was performed with a recombinant adenovirus vector. RESULTS: Tumor cells were transduced in vitro with AdV-IL-2 and subsequently implanted into the floor of the mouth in C3H/HeJ mice. IL-2 expression in vitro ranged from 990 to 1,050 pg/10(6) tumor cells. This microscopic disease treatment resulted in either complete tumor regression or a dramatic decrease in tumor progression. Cytolytic T-cell (CTL) assays demonstrated a predominance of CD8-specific, T-cell-mediated tumor killing. Reducing IL-2 expression by half with a mixture of 1:1 transduced to nontransduced tumor cells eliminated the antitumor effect and decreased the CTL response. These findings support the presence of a critical "threshold" of IL-2 expression. Adenovirus repurification and amplification allowed isolation of a twofold-higher-titer AdV-IL-2 vector. Treatment of established tumors with the higher-titer AdV-IL-2 at a new maximal dose of 1.4 x 10(9) plaque-forming units (pfu) increased in vivo IL-2 expression to 1,127 pg/10(6) cells and generated a significant antitumor response. Complete regression of established tumors, however, could not be achieved, and we noted a decrease in IL-2 expression well below the threshold at 1 week after treatment. Upon repeat maximal AdV-IL-2 injection in vivo, a greater antitumor effect and increased CTL response was seen, but also, 28% of the animals died of IL-2 toxicity. CONCLUSION: Although limited by expression and toxicity as a single-treatment strategy for established tumors, AdV-IL-2 gene therapy should be considered a potential component of combination therapy strategies.     

Combination radiation and adenovirus-mediated P16(INK4A) gene therapy in a murine model for head and neck cancer

Since adenoviral P16(INK4A) gene transfer has shown limited promise in preclinical studies, we developed a strategy combining ionizing radiation (x-rays) with Ad-P16 gene therapy. Both radiation (2 Gy) and Ad-P16 alone demonstrated anti-tumor responses. The combination of radiation and Ad-P16 in vitro showed an augmented therapeutic response. Two head and neck squamous cell carcinoma (HNSCC) cell lines treated with Ad-P16 demonstrated positive staining to annexin V and terminal deoxynucleotidyl-transferase, suggesting apoptosis. When the HNSCC cell lines were grown in nude mice, the anti-tumor response by Ad-P16, radiation, and combination therapy ranged from limited benefit of the single-agent therapy to a statistically significant additive response to combination therapy. Our data suggest that Ad-P16 gene therapy may prove effective in treating human HNSCC that is refractory to radiation therapy.     

白细胞介素—2基因与单纯疱疹病毒胸苷激酶基因联合治疗小鼠头颈鳞状细胞癌的研究

OBJECTIVE: To assess the efficacy of combined interleukin-2 (IL-2) and herpes simplex virus thymidine kinase (HSV-TK) gene therapy for murine head and neck squamous cell carcinoma (HNSCC). METHODS: Ad IL-2 or/and Ad HSV-TK were injected into the tumor tissues directly after the murine HNSCC model was established. DL312 or PBS was used as control and ganciclovir (GCV) was used at 25 mg/kg for 7 days in Ad HSV-TK gene treatment groups. Tumor size was measured before and after treatment to evaluate the response to treatment. Cytotoxic T-lymphocyte (CTL) and natural killer (NK) assays were performed and IL-2 expressions were also measured after IL-2 gene transfection. RESULTS: HNSCC tumor growth was significantly inhibited following combined IL-2 and HSV-TK gene therapy as compared to other groups (P < 0.05). Increased levels of IL-2 protein expression was found in combined and single IL-2 treated groups. The combination and IL-2 treated groups produced greater activities of CTL and NK than that of the controls. CONCLUSION: IL-2 gene therapy can efficiently induce antitumor immunity of the host and enhance antitumor effects of HSV-TK. Combined IL-2 and HSV-TK gene therapy could significantly inhibit HNSCC tumor growth in the murine model.     

白细胞介素-2基因与单纯疱疹病毒胸苷激酶基因联合治疗小鼠头颈鳞状细胞癌的研究

目的观察白细胞介素-2(interleukin-2,IL-2)基因与单纯疱疹病毒胸苷激酶(herpes simplex virus thymidine kinase,HSV-TK)基因联合治疗小鼠头颈鳞状细胞癌的疗效.方法建立小鼠头颈鳞状细胞癌动物模型后在荷瘤部位分别注射表达小鼠IL-2基因的重组腺病毒(recombinantadenovirus, Ad)和表达HSV-TK基因的重组腺病毒Ad HSV-TK及联合注射组,对照组分别注射不带目的基因的腺病毒或磷酸盐缓冲液(phosphatic buffered solution,PBS).对注射有Ad HSV-TK基因组和联合治疗组每日腹腔注射抗病毒药物更西罗韦(ganciclovir,GCV) 25 mg/kg体重,每日2次连续7 d.观察肿瘤大小变化并检测脾脏自然杀伤细胞和颈淋巴结细胞毒性T淋巴细胞的活性,探讨其抗肿瘤机制.结果 Ad IL-2和Ad HSV-TK联合治疗组,肿瘤生长明显受抑制,疗效显著优于单独治疗组和对照组(P<0.05),在注射有Ad IL-2基因的治疗组中,IL-2蛋白水平明显升高,自然杀伤细胞活性和细胞毒性T淋巴细胞杀伤活性增强,肿瘤组织中可见大片坏死,并含有大量的CD+4、CD+8淋巴细胞浸润.结论 Ad IL-2基因治疗可提高肿瘤局部和全身的抗肿瘤免疫应答,能加强自杀基因AdHSV-TK的抗肿瘤效果,两者联合应用能显著抑制小鼠头颈鳞状细胞癌的生长.     

Intravenous delivery of 5′-iododeoxyuridine during hyperfractionated radiotherapy for locally advanced head and neck cancers: Results of a pilot study

Objectives: Locally advanced cancers of the head and neck require aggressive treatment, often with limited effectiveness and significant toxicity and morbidity. This pilot study was designed to assess tolerance using combined hyperfractionated radiotherapy and the halogenated pyrimidine radiosensitizer 5′-iododeoxyuridine (IdUrd). Study Design: This was a prospective single-arm study open to patients with advanced head and neck cancers that had a poor chance of control with conventional radiation therapy. Patients were treated with hyperfractionated radiation therapy at standard doses in combination with an IdUrd infusion and observed for tumor response and normal tissue tolerances. Methods: Radiation therapy was delivered in fractions of 1.2 Gy or 1.5 Gy twice daily to a total dose in the range of 70 to 76 Gy. IdUrd was delivered as an intravenous infusion (1000 mg/m ² per day) for a maximum of 14 days at the beginning and then again during the middle of the radiotherapy. Results: Twelve patients with advanced squamous cell lesions were enrolled and 11 were observed to have complete clinical remissions. Seven patients remained clinically free of local disease at the time of death or most recent follow-up. Acute toxicities, usually hematologic or mucosal, were severe and all patients required treatment modifications and considerable supportive care. Conclusions: Although a high rate of response was achieved using this regimen, the toxicities are prohibitive. The kinetic profile of IdUrd incorporation suggests the need for future studies using repetitive short courses of IdUrd.     

Lymphoepithelial carcinoma in parotid gland related to EBV infection: A case report

Lymphoepithelial carcinoma commonly occurs at the nasopharynx and rarely occurs at other sites in the head and neck region. It is well known to occur at limited patients of local area as Asia or Arctic Circle. Related to this point, it is pointed out that this tumor has strong relation with Epstein–Barr Virus (EBV) infection.In this time, we experienced to treat lymphoepithelial carcinoma with metastatic cervical lymph nodes occurring at parotid gland. The morbidity ratio of this tumor is less than one percent of all parotid gland tumors. Moreover, we proved the infection of EBV to tumor cell by in situ hybridization (ISH).Incidentally, because it is considered that this tumor has well sensitivity against irradiation or anti-tumor drugs, prognosis of this tumor is better than that of other head and neck tumors with different pathological type. Actually, we tried to perform chemotherapy twice in (Nedaplatin (CDGP) 60 mg/m² × day 2 and 5-FU 600 mg/m² × day 5) and to irradiate about 70 Gy dose against parotid gland and cervical lymph nodes. It could not find local recurrence or metastasis as of now after five years from treatment.     

The Effects of Epoetin Alfa on Transfusion Requirements in Head and Neck Cancer Patients: A Prospective,Randomized, Placebo‐Controlled Study

Objective To evaluate the efficacy of perioperative recombinant human erythropoietin (r‐HuEPO, epoetin alfa) in stimulating hematopoiesis and reducing allogeneic blood transfusion requirements in major head and neck cancer surgery. Study Design Double‐blinded, placebo‐controlled, randomized, prospective clinical trial. Methods Fifty‐eight patients undergoing surgical resection of head and neck tumors at the University of Iowa hospitals completed this study. Patients were required to have a pre‐study hemoglobin ≥10.0 g/dL and ≤13.5 g/dL. Group 1 (29 patients) received three doses of 600 IU/kg epoetin alfa before surgery. Group 2 (29 patients) received a placebo. All patients received oral iron supplementation (150 mg FeSO₄ twice per day). Results The epoetin alfa group demonstrated a significant increase in baseline to day‐of‐surgery mean hemoglobin (0.57 g/dL, P = .016), hematocrit (2.04%, P = .015), and reticulocyte count (95.3 × 10³ cells/mm³, P = <.001), whereas there was no significant change in these hematologic variables in the placebo group. The percent of patients who avoided transfusion in the epoetin alfa group was 34.5% versus 17.2% in the placebo group. Patients requiring allogeneic blood transfusions received an average of 3.16 units in the epoetin alfa group and 4.12 units in the placebo group. Conclusion In this single institution study, we demonstrated a significant improvement in hematopoietic parameters and a trend toward decreased transfusion requirements using perioperative epoetin alfa in a head and neck cancer patient population. Further studies may delineate additional benefits in treating qualified patients with epoetin alfa during therapy for head and neck malignancies.     

Viral-mediated gene transfer to study the molecular physiology of the Mammalian inner ear

Several classes of viral vectors including adenovirus, adeno-associated virus, herpes simplex virus, lentivirus and vaccinia virus have been reported to infect cells of the inner ears of mammals and may be useful for protein manipulation and therapeutic purposes. We have screened a few of these for use as vectors to mediate gene transfer into the sensory hair cells of organotypic cultures from the neonatal mouse cochlea and utricle. Recombinant, replication-deficient adenovirus has emerged as a useful vector for several reasons: ease of vector generation at high titer; efficient hair cell specific infection; robust expression of reporter genes and minimal toxicity. Previously, we characterized adenovirus infected hair cells using a vector that carried the gene for green fluorescent protein (GFP). We screened GFP-positive cells electrophysiologically and found that although hair cells survive adenoviral vector infection, their mechanosensitivity was compromised. Until recently this has limited the scope of adenovirus application to the problems of inner ear physiology and pathophysiology. However, a modified adenoviral vector, now available, has been reported to have reduced ototoxicity in vivo. The modifications include the deletion of the adenoviral genes E1, E3, the viral polymerase, and the preterminal protein. We are currently working to characterize viral-mediated gene transfer into hair cells of the cultured mouse utricle using this new modified adenoviral vector. We have found that hair cells infected with the modified vector have intact hair bundles and robust mechanotransduction.     

Feasibility of inner ear gene transfer after middle ear administration of an adenovirus vector

BACKGROUND: Several groups demonstrated in animal experiments that gene transfer is a feasible tool for inner ear intervention. Various approaches for inoculation of vectors have been successfully used for inner ear gene therapy. One possible way to reduce the risk of hearing loss following the opening of the cochlea for application of the vector into the perilymphatic space is to deliver vectors through the round window. This study was designed to determine whether middle ear application of an adenoviral vector is a feasible approach to inoculate vectors and lead to transduction of cells in the inner ear. METHODS: A unilateral middle ear application of an adenoviral vector was performed in 4 guinea pigs directly on the round window membrane (RWM) and in 4 additional animals by placing a cotton patch soaked with the vector solution on the RWM. The expression of a reporter gene (lacZ) was used to localize vector-transduced cells. RESULTS: Only one out of 8 animals showed cochlear expression of the reporter gene, whereas all 8 animals showed strong lacZ expression in the middle ear mucosa, in the RWM and in the mucosa surrounding the stapes. CONCLUSION: Our results indicate that the RWM presents a close barrier, almost completely preventing the adenovirus to diffuse into the perilymphatic space. Therefore middle ear application of an adenoviral vector cannot be used to induce inner ear gene transfer. However, middle ear application of a viral vector may be useful for developing treatment for diseases of the middle ear mucosa.     

Prediction model for hearing outcome in patients with idiopathic sudden sensorineural hearing loss

The aim of this study is to develop a regression model for predicting hearing outcome in patients with idiopathic sudden sensorineural hearing loss (ISSNHL). A total of 174 consecutive patients with ISSNHL (average of the hearing levels at 250, 500, 1,000, 2,000, and 4,000 Hz was ≥40 dB; time from onset to treatment was ≤30 days) were retrospectively analyzed. They received steroid administration (400 mg/day of hydrocortisone sodium succinate followed by tapered doses) in combination with hyperbaric oxygen therapy. The hearing improvement rate compared to the unaffected contralateral ear was calculated. Correlations between the hearing improvement rate and four prognostic factors (patient’s age, days from onset to treatment, initial hearing level, and the presence of vertigo) were examined by simple and multiple regression analyses. In the simple regression analysis, significant correlations were observed between the hearing improvement rate and all four prognostic factors. In the multiple regression analysis, the correlation was significant for patient’s age, days from onset to treatment, and the presence of vertigo with partial correlation coefficients of −0.221, −0.324, and −0.329, respectively, but was not significant for the initial hearing level. We subsequently formulated a multiple regression equation for predicting the hearing improvement rate. The multiple correlation coefficient was 0.495 with a p value of 1.42 × 10⁻⁹. Using this regression model, the hearing improvement rate is still difficult to predict with 95% probability, but is predictable with 70% probability.     

Untersuchungen zum Generations-cyclus maligner Tumoren im HNO-Bereich und ihre Bedeutung für die cytostatische Therapie

Summary More than 40 patients with malignant tumors of head and neck were investigated on cytostatic therapy. In these patients advances tumor stage was manifested. Therefore a major curative effect of therapy was not expected. In the beginning of cytostatis surgical and radiological possibilities were exhausted.Theoretical basis of the performed therapy were animal experiments of H. O. Klein in which evidence of cell cinetics for cytostatic therapy was demonstrated. The tumorcell cycle was determined autoradiographically with the double labeling method (H₃- and C₁₄-Thymidine) in vitro in most of the patients.A slow proliferation rate of tumor cells regularly was found. So a high dose oytostatic treatment (high dose monodrug therapy) appeared not to be optimal. Therefore we prefered in our cases a combination therapy with Cyclophosphamide (2–3 mg/kg body weight/day) and Methotrexate^® (2.5–5 mg/day). Methotrexate^® was given on only 3 days of the week to avoid severe haematological complications. In other cases without these complications the substance was administered dayly. In drug incompatibility other oytostatic substances were used.In most of the patients tumor development could be retarded, stopped, or a regression could be induced. Only in some cases no effect was observed. Details of cytostatic therapy, evaluation of toxic side effects and complications, also time and success of therapy are demonstrated in diapositives.     

The role of viral infection in sudden deafness (author's transl)

A series of 35 patients with various degrees of sudden deafness were investigated in regard to recent or coexistent viral infection. The virological studies included 1) viral isolation by nasopharyngeal and faecal cultures; 2) viral serology for 20 different species of virus and the Paul Bunnel test; and 3) immunocomplex tests. In only two patients could a viral infection (a viral parotitis and an adenoviral infection) be proven (i.e. 6%). Immunocomplexes were found in 15 of 22 patients. Our studies, using conventional viral diagnostic tests, do not support the belief that viral infection is a common cause of sudden deafness. To solve the question whether viral infections play a part in the occurrence of sudden deafness these diagnostic aids must be complemented with modern immunological methods.     

Enhancement of Cisplatin sensitivity in squamous cell carcinoma of the head and neck transfected with a survivin antisense gene

OBJECTIVE: To study a new method for treating squamous cell carcinoma of the head and neck using a survivin antisense gene. DESIGN: An adenoviral vector encoding surviving antisense was used for in vitro and in vivo experiments. KB cells were treated with pAd.CMV[cytomegalovirus]-antisurvivin. Western blot analysis, in vitro cytotoxic assay, and in vivo experiment were performed. SETTING: In vitro and in vivo study of head and neck cancer cell line KB. SUBJECTS: Male, 5-week-old BALB/c nude mice. MAIN OUTCOME MEASURES: Expression of survivin was assessed using Western blot analysis. The effect of antisurvivin to KB cells was measured by cytotoxic assay (in vitro) and tumor volume (in vivo). RESULTS: In the in vitro experiments, transduction of the survivin antisense gene caused a nearly 12-fold increase in the sensitivity of KB cells to cisplatin, as reflected by the 50% inhibitory concentration. In in vivo experiments in nude mice, tumor growth was more inhibited by the combination of cisplatin and survivin antisense gene transduction compared with either alone. CONCLUSION: These findings suggest that survivin targeting with adenoviral antisense vectors might be used for selective therapy of squamous cell carcinomas of the head and neck.