Jonathan B. Zuckerman

A phase I study of adenovirus-mediated transfer of the human cystic fibrosis transmembrane conductance regulator gene to a lung segment of individuals with cystic fibrosis

A third-generation adenoviral vector containing recombinant human cystic fibrosis transmembrane conductance regulator (CFTR) gene was delivered by bronchoscope in escalating doses to the conducting airway of 11 volunteers with cystic fibrosis. Assessments of dose-limiting toxicity (DLT), efficiency of gene transfer, and cell-mediated and humoral immune responses to vector administration were performed. DLT, manifest by flulike symptoms and transient radiographic infiltrates, was seen at 2.1 x 10(11) total viral particles. A highly specific assay for gene transfer was developed using in situ hybridization with an oligoprobe against unique vector sequence. Detectable gene transfer was observed in harvested bronchial epithelial cells (<1%) 4 days after vector instillation, which diminished to undetectable levels by day 43. Adenovirus-specific cell-mediated T cells were induced in most subjects, although only mild increases in systemic humoral immune response were observed. These results demonstrate that gene transfer to epithelium of the lower respiratory tract can be achieved in humans with adenoviral vectors but that efficiency is low and of short duration in the native CF airway.

Gene Therapy for Lung Disease: Hype or Hope?

Approximately 10 years ago, advances in molecular genetics and gene transfer technology made possible the development of gene therapy, modification of the genetic makeup of cells for therapeutic purposes. Although gene therapy was originally proposed for treatment of inherited recessive disorders in which transfer of a normal copy of a single defective gene might prevent the development of disease or slow its progression (1), it soon became clear that reasonable targets for gene therapy extended beyond traditional genetic disorders into the realm of acquired diseases, such as vascular diseases and cancer. The concept of gene therapy now encompasses the treatment of any pathophysiologic state on the basis of the transfer of genetic material, including complementary DNA, full-length genes, RNA, or oligonucleotides. Lung diseases are prominent among the disorders for which human gene therapy protocols have been approved (2). From a technical perspective, delivery to the lung can be accomplished by multiple approachesthrough the airway, by direct transthoracic injection, or through the dual pulmonary blood supply. From a disease perspective, the genetic basis for the two most common fatal inherited lung disorders, cystic fibrosis and 1-antitrypsin deficiency, is known, and current treatment regimens are supportive but not curative (3, 4). Several other pulmonary disorders, such as lung cancer, mesothelioma, pulmonary vascular diseases, radiation-induced lung injury, transplantation-induced lung injury, asthma, and pulmonary inflammation, are potential targets for gene therapy. Study Selection English-language articles were identified by searching the MEDLINE database from 1966 to the present using the terms gene therapy, lung, lung cancer, cystic fibrosis, 1 -antitrypsin deficiency, mesothelioma, retrovirus, adenovirus, adeno-associated virus, lentivirus, vaccinia virus, and liposome. Selected references cited in identified articles were reviewed. We focused on articles that described clinical trials. Because of space limitations, the references include excellent review articles that discuss the many in vitro and animal model experiments that form the scientific background for the clinical studies. Vectors Used in Gene Therapy Efficient gene transfer is a basic requirement for effective gene therapy. Various viral and nonviral gene transfer vectors are currently available (5-8). As summarized in Table 1, each vector has certain advantages in DNA-carrying capacity, types of cells targeted, efficiency of in vivo gene transfer, duration of expression, and induction of inflammation. It has become clear that no one gene delivery strategy will be suitable for all candidate disorders. Table 1. Characteristics of Gene Therapy Vectors The most widely used vector in lung gene therapy has been replication-incompetent recombinant adenovirus . This vector system offers many advantages, including high-efficiency transduction in a wide variety of target cells (including nondividing cells) and high levels of expression of the delivered transgene (8, 9). Of note, these vectors are stable in vivo, permitting direct delivery of the gene to many tissue sites, including the lung parenchyma and pleural space. The two primary disadvantages of adenoviruses are that they result in only transient gene expression and that when virions are used for direct in vivo applications, they elicit a prominent local and systemic inflammatory response. This inflammatory response, which includes an early innate immune response resulting in cytokine release and a late acquired immune response resulting in the generation of neutralizing antiadenoviral antibodies and cytotoxic T lymphocytes (10-15), has been the primary source of adenoviral vector toxicity and has limited the amount of vector that can be delivered. Gene Therapy for Inherited Pulmonary Disorders Cystic Fibrosis Cystic fibrosis results from mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) (4). The CFTR seems to play an important role in regulating the vectorial movement of fluid and electrolytes across various tissues and probably contributes to processes as diverse as mucus secretion, sialylation of surface receptors, sulfation of glycoconjugates, and local host defense (4, 16, 17). An early finding that made gene therapy for cystic fibrosis a potentially achievable goal was the observation that in a polarized epithelial sheet, expression of CFTR in as few as 6% to 10% of airway epithelial cells lacking CFTR restored normal chloride transport properties (18, 19). Because respiratory problems are responsible for most of the morbidity and mortality in persons with cystic fibrosis, the bulk of gene therapy research in cystic fibrosis has focused on methods of transfer to airway epithelium. Three vector systems have been used in clinical trials to date: adenovirus, adeno-associated virus, and liposomal/DNA complexes. Adenovirus To date, results of seven phase I clinical trials that used an adenoviral vector carrying the CFTR transgene have been published (Table 2). In the initial studies, the CFTR transgene was delivered by direct instillation in the nose or lung (20-22). In a randomized, vehicle-controlled, blinded study in 12 patients, Knowles and coworkers (23) studied gene transfer to nasal epithelium. Although no significant side effects occurred, gene transfer was inefficient and significant inflammation was noted at a dose of 2 1010 plaque-forming units, an estimated multiplicity of infection of 1000 (that is, 1000 infectious units of virus per cell). Furthermore, the investigators could not detect correction of sodium or chloride transport as measured by using nasal electrical potential difference. Zabner and colleagues (24) reported on the effect of repeated administration of recombinant adenoviral vector CFTR applied to nasal epithelium. Six patients received five escalating doses of adenovirus over at least 28 days. A humoral immune response was stimulated, gene transfer was inefficient, and functional correction was variable and seemed to be attenuated by subsequent administration of vector. To improve gene transfer, Bellon and coworkers (25) used an adenoviral vector CFTR (identical to that used by Crystal and associates [21]) to deliver aerosolized recombinant CFTR sequentially to the nasal epithelium and lung in 6 patients. Recombinant transcripts and CFTR protein were detected up to 15 days later in epithelial cells harvested from the nose and lung, although the amounts detected were low. In an effort to reduce immunogenicity, Zuckerman and colleagues (26) delivered a more crippled form of recombinant adenovirus, in which the E1 and E4 regions were deleted, to the lower respiratory tracts of 11 patients. As in the previous studies, gene transfer efficiency was low; gene transfer was detected in less than 1% of harvested bronchial epithelial cells at 4 days and 0% at 42 days. Systemic humoral immune responses to adenovirus were relatively mild, but cell-mediated responses (measured by lymphoproliferative responses to adenovirus) were stimulated in all patients. Dose-limiting toxicity was noted at 1011 viral particles. Table 2. Clinical Trials of Gene Therapy for Cystic Fibrosis Adeno-Associated Virus Adeno-associated viruses have been considered as gene therapy for cystic fibrosis for some time, but they were only recently used in human trials because of technical constraints related to production issues (32, 33). Adeno-associated virus is attractive because preclinical studies have suggested that long-term (up to 6 months) transgene expression can be obtained (32, 34) with little inflammatory response (35). Two phase I clinical studies have been approved by the National Institutes of Health, and to date, a summary of the results of one has been published. The first study (27, 28) involved a randomized, nonblinded, dose-escalation protocol in which a vector suspension was instilled into the maxillary sinuses of 10 patients with cystic fibrosis who had previously undergone bilateral endoscopic antrostomies (Table 2). Vector-derived DNA was detected in all participants who received more than 104 replication units. Although vector-derived RNA could not be detected in any of the specimens, transepithelial potential difference responses consistent with CFTR-mediated chloride transport were observed up to 14 days after instillation in patients receiving more than 5 104 replication units. The second study, which is ongoing, is examining dose-escalated administration of adeno-associated virus CFTR, both intranasally and to a single lobe of the lung (by bronchoscope); toxicity is the primary end point (36). Although the results from the sinus study are encouraging, questions remain about the efficiency of transduction of differentiated respiratory epithelium (37-39) and the long-term safety of adeno-associated virus vectors (40). Liposomes The liposomal vectors in clinical use are generally composed of cationic lipids mixed with cholesterol and dioleoylphosphatidylethanolamine. The liposome/DNA complexes were originally thought to be less immunogenic than viral vectors, although as more efficient complexes have been designed, it seems that these vectors can also generate significant inflammatory responses, probably because of the inherent immunogenicity of bacterial-derived DNA (41, 42). The results of three clinical trials (Table 2) have been published to date. Caplen and colleagues (29) conducted a double-blind, placebo-controlled study in which 9 patients with cystic fibrosis received nasal spray application of liposome/DNA complex and 6 such patients received control liposomes. The investigators observed vector-derived RNA in 5 of 8 patients and a transient, partial normalization of the response to low chloride perfusion during nasal potential difference measurements in the treated patients with cystic fibrosis (compared with controls

Pooled analysis of two large randomised phase III inhaled mannitol studies in cystic fibrosis

BACKGROUND To evaluate safety and efficacy of inhaled mannitol treatment in subgroups of a large global CF population. METHODS Data were pooled from two multicentre, double-blind, randomised, controlled, parallel group phase III studies in which 600 patients inhaled either mannitol (400 mg) or control (mannitol 50 mg) twice a day for 26 weeks. RESULTS Both the mean absolute change in FEV(1) (mL) and relative change in FEV(1) by % predicted from baseline for mannitol (400 mg) versus control were statistically significant (73.42 mL, 3.56%, both p<0.001). Increases in FEV(1) were observed irrespective of rhDNase use. Significant improvements in FEV1 occurred in adults but not children (6-11) or adolescents (aged 12-17). Pulmonary exacerbation incidence was reduced by 29% (p=0.039) in the mannitol (400 mg) group. CONCLUSIONS Sustained six-month improvements in lung function and decreased pulmonary exacerbation incidence indicate that inhaled mannitol is an important additional drug in the treatment of CF.

Piperacillin-induced immune hemolytic anemia in an adult with cystic fibrosis.

We report a case of drug-induced immune hemolytic anemia (DIIHA) in an adult female with cystic fibrosis (CF), complicating routine treatment of a pulmonary exacerbation with intravenous piperacillin-tazobactam. Workup revealed a positive direct antiglobulin test (DAT) due to red blood cell (RBC)-bound IgG and C3 and piperacillin antibodies detectable in the patients serum. The potential influence of CF transmembrane conductance regulator mutations on the severity of DIIHA is discussed. This report illustrates the importance of early identification of DIIHA, a rare complication of a commonly utilized medication in CF.

Successful Meropenem Desensitization in a Patient with Cystic Fibrosis

Objective To report a case of successful meropenem desensitization in a patient with cystic fibrosis (CF) with documented hypersensitivity to multiple antibiotics including carbapenems. Case Summary A 20-year-old white man with CF was admitted to the hospital for treatment of an acute pulmonary exacerbation caused by multidrug-resistant Burkholderia cepacia complex and methicillin-resistant Staphylococcus aureus (MRSA). Past treatments of his CF exacerbations were complicated by urticarial eruptions following administration of β-lactams, including meropenem, and ototoxicity from aminoglycosides. Skin testing revealed hypersensitivity reactions to β-lactam antibiotics including penicillin, piperacillin/tazobactam, ceftazidime, and imipenem. A literature review (MEDLINE, January 3, 2002) and communication with the manufacturer of meropenem revealed no specific information on desensitizing patients to this agent. Because of meropenems activity against B. cepacia complex alone and in combination with other antimicrobials, a desensitization protocol was adapted and applied to meropenem in an effort to provide the most beneficial treatment available. A 12-dose escalation protocol was successfully employed without incident. Discussion Antimicrobial therapy is limited in CF patients by susceptibility profiles of common infecting organisms (e.g., Pseudomonas spp., B. cepacia complex, MRSA). Unfortunately, host responses may further reduce the utility of many effective antibiotic classes due to hypersensitivity and/or adverse reactions. Desensitization is a useful alternative that allows the administration of beneficial medications to patients with documented allergy histories. Conclusions Meropenem is an important treatment option in the CF population, particularly due to its activity against B. cepacia complex. Successful desensitization using a dose-escalation protocol in patients with a documented carbapenem allergy will allow the most beneficial therapy to continue.

LUNG TRANSPLANTATION FOR CYSTIC FIBROSIS

Lung transplantation currently stands as the only therapeutic option that carries the potential to restore patients with advanced cystic fibrosis to a more normal state of health. Nonetheless, the procedure carries significant risk and median survival following transplantation is only 5 years. This article discusses the currently achievable outcomes and the common short-comings of transplantation. Strategies to optimize outcomes through appropriate patient selection, use of living donors, and novel research initiatives are discussed.

Iron supplementation does not worsen respiratory health or alter the sputum microbiome in cystic fibrosis

BACKGROUND Iron supplementation for hypoferremic anemia could potentiate bacterial growth in the cystic fibrosis (CF) lung, but clinical trials testing this hypothesis are lacking. METHODS Twenty-two adults with CF and hypoferremic anemia participated in a randomized, double-blind, placebo-controlled, crossover trial of ferrous sulfate 325mg daily for 6weeks. Iron-related hematologic parameters, anthropometric data, sputum iron, Akron Pulmonary Exacerbation Score (PES), and the sputum microbiome were serially assessed. Fixed-effect models were used to describe how ferrous sulfate affected these variables. RESULTS Ferrous sulfate increased serum iron by 22.3% and transferrin saturation (TSAT) by 26.8% from baseline (p<0.05) but did not affect hemoglobin, sputum iron, Akron PES, and the sputum microbiome. CONCLUSIONS Low-dose ferrous sulfate improved hypoferremia without correcting anemia after 6weeks. We did not observe significant effects on sputum iron, Akron PES, and the sputum microbiome. Although we did not identify untoward health effects of iron supplementation, a larger blinded randomized controlled trial would be needed to fully demonstrate safety.

Optimising inhaled mannitol for cystic fibrosis in an adult population.

Abstract There has been remarkable progress in the treatment of cystic fibrosis (CF) patients over the past 20 years. However, limitations of standard therapies have highlighted the need for a convenient alternative treatment to effectively target the pathophysiologic basis of CF-related disease by improving mucociliary clearance of airway secretions and consequently improve lung function and reduce respiratory exacerbations. Mannitol is an osmotic agent available as a dry powder, dispensed in a convenient disposable inhaler device for the treatment of adult patients with CF. Inhalation of mannitol as a dry powder is thought to change the viscoelastic properties of airway secretions, increase the hydration of the airway surface liquid and contribute to increased mucociliary and cough clearance of retained secretions. In two large phase 3 studies [1, 2], long-term use of inhaled mannitol resulted in a significant and clinically meaningful improvement in lung function relative to control in adult CF subjects and had an acceptable safety profile. Clinical experience with inhaled mannitol confirms that it is safe and effective. A minority of patients are unable to tolerate the medication. However, through training in proper inhaler technique and setting clear expectations regarding therapeutic effects, both the tolerance and adherence necessary for long term efficacy can be positively influenced. Educational aims To discuss the importance of airway clearance treatments in the management of cystic fibrosis. To describe the clinical data that supports the use of mannitol in adult patients with cystic fibrosis. To highlight the role of mannitol tolerance testing in screening for hyperresponsiveness. To provide practical considerations for patient education in use of mannitol inhaler. Key points Inhaled mannitol is a safe and effective option in adult patients with cystic fibrosis. Mannitol tolerance testing effectively screens for hyperresponsiveness prior to initiation of therapy. Physiotherapists and respiratory therapists play an integral role in the introduction and maintenance of dry powder inhalation therapy. Patient training and follow-up is important for optimising longer term adherence.

Clinafloxacin for Treatment of Burkholderia cenocepacia Infection in a Cystic Fibrosis Patient

ABSTRACT Respiratory infection with Burkholderia cenocepacia is associated with accelerated decline in lung function and increased mortality in cystic fibrosis (CF) patients (A. M. Jones, M. E. Dodd, J. R. W. Govan, V. Barcus, C. J. Doherty, J. Morris, and A. K. Webb, Thorax 59:948–951, 2004, http://dx.doi.org/10.1136/thx.2003.017210). B. cenocepacia often possesses innate resistance to multiple antimicrobial classes, making eradication uncommon in established infection (P. B. Davis, Am J Respir Crit Care Med 173:475–482, 2006, http://dx.doi.org/10.1164/rccm.200505-840OE). We report the use of clinafloxacin in a CF patient with advanced B. cenocepacia infection, present pharmacokinetic (PK) data, and discuss the potential therapeutic role of clinafloxacin in patients with this condition.

Complications of long and intermediate term venous catheters in cystic fibrosis patients: A multicenter study

BACKGROUND Totally implantable venous access devices (TIVADs) or peripherally inserted central venous catheters (PICCs) are commonly used in the care of patients with cystic fibrosis (CF), but they are associated with various complications, including thrombosis, infection, and insertion site symptoms. METHODS We conducted a retrospective review of PICC and TIVAD use in adults and children with CF over an 8-year period at 3 accredited care centers. Patient attributes included CFTR genotype, comorbidities, lung function, body mass index, use of anticoagulation, and respiratory tract microbiology. Catheter data included line type, caliber, and lumen number. We assessed practice variation by surveying physicians. RESULTS In a population of 592 CF patients, 851 PICC and 61 TIVADs were placed between January 1, 2003 and July 1, 2011. Larger catheter caliber and increased lumen number were risk factors for PICC complications in adults. Patient-related risk factors for PICC complications included poor nutritional status, infection with Burkholderia cepacia spp., and having ≥5 lines inserted during the study period. The probability of a PICC complication varied across centers (2.6% to 14.1%, p=0.001) and remained significant after adjustment for patient-and line-related risk factors. The median complication-free survival of TIVADs, however, did not vary significantly by center (p=0.85). CONCLUSIONS This is the first longitudinal, multicenter assessment of complication rates for PICCs and TIVADs in a large cohort of adults and children with CF. Specific patient- and catheter-related characteristics were associated with increased risk of complications. Center effects on complication rates were observed for PICCs.