Nanotechnologies in delivery of mRNA therapeutics using nonviral vector-based delivery systems.

Because of its safe and effective protein expression profile, in vitro transcribed messenger RNA (IVT-mRNA) represents a promising candidate in the development of novel therapeutics for genetic diseases, vaccines or gene editing strategies, especially when its inherent shortcomings (for example, instability and immunogenicity) have been partially addressed via structural modifications. However, numerous unsolved technical difficulties in successful in vivo delivery of IVT-mRNA have greatly hindered the applications of IVT-mRNA in clinical development. Recent advances in nanotechnology and material science have yielded many promising nonviral delivery systems, some of which were able to efficiently facilitate targeted in vivo delivery of IVT-mRNA in safe and noninvasive manners. The diversity and flexibility of these delivery systems highlight the recent progress of IVT-mRNA-based therapy using nonviral vectors. In this review, we summarize recent advances of existing and emerging nonviral vector-based nanotechnologies for IVT-mRNA delivery and briefly summarize the interesting but rarely discussed applications on simultaneous delivery of IVT-mRNA with DNA.

Nonviral gene delivery to the lung with copolymer-protected and transferrin-modified polyethylenimine.

Polyethylenimine (PEI) has been shown to efficiently mediate topical gene transfer to the lungs after either direct intratracheal instillation or nebulisation. Recently, the protection of polyplexes with novel copolymers of poly(ethylene glycol) (PEG) via electrostatic interaction has been reported. In this study, such coated PEI polyplexes were investigated for their stability and interaction with human plasma and bronchoalveolar lavage fluid (BALF). Further, their potential for gene delivery to the mouse lungs in vivo was examined. Plasma protein and mucin adsorption was effectively inhibited when polyplexes were coated with the novel copolymers. Gene transfer efficiency of the coated PEI polyplexes decreased as compared with uncoated PEI polyplexes when administered intratracheally to the lung. The higher the molecular weight of the copolymerized PEG was, the stronger the observed gene transfer reduction. Gene transfer decreased presumably due to reduced interaction of the coated gene vectors with the cell surface. To circumvent this problem, transferrin was combined with PEI/DNA polyplexes for specific binding to the cell surface. In this case, gene transfer efficiency decreased. Gene transfer of the copolymer-protected and transferrin-modified gene vectors increased as compared with the copolymer-protected gene vectors alone but did not reach the level of uncoated gene vectors. These data show that copolymers could be used to effectively shield polyplexes from interaction with components of the airway surface liquid (ASL). Increased gene delivery was found upon transferrin modification of the coated PEI polyplexes suggesting a targeting effect.

Diabetes mellitus and cystic fibrosis: comparison of clinical parameters in patients treated with insulin versus oral glucose-lowering agents.

The prevalence of cystic fibrosis-related diabetes melltitus (CFRD) is increasing as patients with cystic fibrosis (CF) live longer. Because patients with CFRD are insulin-deficient, the standard medical treatment is exogenous insulin. Sulfonylureas enhance insulin secretion by acting on a specific islet beta cell receptor. No data are available about the outcome of sulfonylurea treatment vs. insulin treatment. In this retrospective study, data from 45 patients with CFRD were analyzed regarding their clinical outcome as it related to the treatment protocol. The duration of DM treatment was 7.6 +/- 4.6 years in the insulin-treated group and 3.5 +/- 2.0 years in the sulfonylurea group (n.s.). The age of CFRD diagnosis was significantly earlier in patients treated with insulin (n = 34) than in the patients treated with sulfonylurea (n = 11) (16.4 +/- 3.6 vs. 24.2 +/- 4.8 years, P < 0.001). No statistical differences were found between the two groups in the time of CF diagnosis, the most recent forced expired volume in 1 sec, forced vital capacity, Shwachman score, hemoglobin A(1C) levels, or weight for height index at the end of the study. Our data suggest that a subgroup of CFRD patients can be managed for a number of years with sulfonylurea, and that the clinical outcome was not different in this group compared with the insulin-treated patients.

Relations between the frequency of the DeltaF 508 mutation and the course of pulmonary disease in cystic fibrosis patients infected with Pseudomonas aeruginosa.

The course of pulmonary disease in cystic fibrosis is variable. There are controversial data on the impact of the type of mutation on the cystic fibrosis transmembrane conductance regulator (CFTR) gene on the course of pulmonary disease in CF. Since selected mutations of the CFTR gene appear to be associated with relatively mild disease, this study addressed the question whether the course of pulmonary disease in CF patients colonized with P. aeruginosa is influenced by the frequency of the DeltaF 508 mutation. For this study, we assessed FVC and FEV1 in 127 CF patients who attended regularly the Munich CF Center over a mean period of 43 +/- 16 (SD) months. Of these 127 patients, 69 (54.3%) were homozygous for the DeltaF 508 mutation, 42 (33.1%) were compound heterozygous for the DeltaF 508 mutation, and 16 (12.6%) did not carry the DeltaF 508 mutation on either chromosome. In homozygotes 59 (85.5%) out of 69 CF-patients were colonized with P. aeruginosa as compared with 27 (64.3%) out of 42 in heterozygotes (p <0.05). The mean age of onset of P. aeruginosa colonization was 11.0 years, and there was no difference between the three groups. The mean FVC and FEV1 values did not differ significantly between the three genotype groups when P. aeruginosa infection was disregarded. However, when only P. aeruginosa colonized patients were compared FVC and FEV1 values were lower in heterozygotes than in the other two groups both at the beginning and at the end of the study. These findings indicate that the course of pulmonary disease in CF patients is at least partially influenced by the frequency of the DeltaF 508 mutation.

Towards gene therapy of cystic fibrosis.

Numerous gene mutations associated with hereditary disorders have been identified. In cystic fibrosis the hereditary defect is attributed to mutations in one single gene, the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR). Conventional therapies of CF have dramatically increased the life expectancy of afflicted individuals. However, the ultimate incurability of this disease calls for novel and better therapeutic strategies. As cystic fibrosis is believed to be caused by mutations in one single gene, it has appeared to be the ideal candidate for one of the most tempting approaches in clinical therapy, namely gene therapy. Laboratory protocols for the introduction of genes into various tissues have been developed and applied over the last 15 years. The ease of gene transfer under laboratory conditions gave rise to the hope that rapid advances in gene transfer protocols under clinical settings could be achieved as well. 20 clinical trials of gene therapy for cystic fibrosis have been initiated using viral and non-viral vectors for gene transfer (Marcel and David Grausz 1997). The outcome of the CF gene therapy studies as well as of those for other diseases have clearly demonstrated that gene transfer and gene therapy in humans is a much more complex and challenging task than originally thought. Still, the encouraging results achieved in animal models and the rapid progress in vector technology justify the hope that the novel genetic therapies will be applied successfully to the benefit of patients suffering from cystic fibrosis.

Diabetes mellitus in patients with cystic fibrosis: the impact of diabetes mellitus on pulmonary function and clinical outcome.

In this multicenter study, the impact of CF-related diabetes mellitus (CFRD) on pulmonary function and clinical outcome has been investigated. To better characterize the relationship between insulin deficiency and clinical outcome we prospectively followed a group of 56 CF patients, 28 with CFRD (group 1) and 28 without diabetes (group 2) for 5 years. The clinical course of the patients was registered at each center. Data included were mortality, pulmonary function, body mass index, in-patient treatment, and CF-typical and diabetes typical complications. At the end of the study nearly twice the number of patients had died in group 1 as compared to group 2, however due to the low patient number this did not reach statistical significance. In patients with diabetes FEV1 and FVC declined significantly over the five year study period, whereas patients without diabetes did not show a significant decline during the study period. Retinopathy, nephropathy, and neuropathy were only observed in diabetic patients. In conclusion, the data presented in this prospective, multicenter study give evidence that insulin deficiency leads to a direct decline in pulmonary function suggesting a cause and effect relationship between insulin deficiency and lung disease.

Posaconazole for treatment of refractory invasive fungal disease.

Invasive fungal infections are usually associated with immunocompromised states About 40-60% of these patients are refractory to standard antifungal therapy We describe the effect of posaconazole in the treatment of a 12 years-old girl with uncontrolled diabetes mellitus with life-threatening cerebral mucor mycosis and a 4 year old girl boy with chronic granulomatous disease presenting with invasive Aspergillus nidulans infection.

Methodological optimization of polyethylenimine (PEI)-based gene delivery to the lungs of mice via aerosol application.

BACKGROUND: Polyethylenimine (PEI) has been successfully used for gene delivery to the lungs of mice via aerosol application using a whole body nebulization device. In this report we optimized the design of such an aerosol device. METHODS: Aerosol devices were constructed as either serial inhalation apparatus or as a whole body nebulization chamber connected to an aerosol spacer placed in a horizontal or vertical position. PEI-based gene vectors were nebulized using a standard jet nebulizer and luciferase gene expression of various tissues was examined. RESULTS: Using a whole body aerosol device resulted in luciferase gene expression in the lungs of mice at the same level as compared with a serial inhalation apparatus. Whereas gene expression was enhanced in the presence of 5% CO(2)-in-air, anesthesia of mice strongly decreased gene expression even when mice were intubated with an intravascular cannula. Reduction of the median mass aerodynamic diameter (MMAD) of the aerosol from 3.4 to 0.27 microm by interposition of an aerosol spacer increased gene expression significantly 3-fold. Drying of the aerosol by silica gel additionally increased gene delivery significantly 3-fold. Reporter gene expression mediated by branched PEI 25 kDa was 9- and 15-fold higher as compared with linear PEIs of 22 and 25 kDa, respectively, and was dependent on the DNA concentration. Gene expression was detectable as soon as 6 h after gene vector application and reached a maximum after 72 h but was still detectable after 14 days. The presence of Zn(2+) did not increase gene expression. CONCLUSION: We propose aerosol drying as a novel and simple method of optimizing PEI-based gene delivery to the lungs.

Jet nebulization of PEI/DNA polyplexes: physical stability and in vitro gene delivery efficiency.

BACKGROUND: Aerosol drug delivery currently represents the most acceptable and convenient delivery system for repeated drug application to the lungs. Although polyethyleneimine (PEI) has recently been demonstrated to mediate gene transfer successfully to mouse lungs via aerosol delivery, the effect of the jet nebulization process on the properties of PEI/DNA polyplexes has not yet been examined. METHODS: PEI/DNA polyplexes were generated in several commonly used solvents, such as distilled water, HEPES buffered saline (HBS), and 5% glucose. The complex parameters, such as particle size, zeta potential, DNA integrity, and transfection efficiency, were examined before and after jet nebulization. RESULTS: The complex parameters and the transfection efficiency were influenced by the solvent that was used for complex formulation and by the nebulization process itself. When polyplexes were formulated in HBS, the particle size, zeta potential, and DNA concentration were affected by the nebulization process and the transfection efficiency decreased dramatically. Polyplexes formulated in 5% glucose were less susceptible to the nebulization process, as indicated by only minor changes of the zeta potential and particle size when compared with HBS. The resulting transfecion efficiency was very low both before and after nebulization. Polyplexes formulated in distilled water had the most resistant behavior with the nebulization process. Zeta potential, particle size, and DNA integrity were influenced least of all by nebulization. CONCLUSION: As a result, the transfection efficiency of PEI/DNA complexes remained constant throughout the nebulization process only when formulated in distilled water. These data suggest that distilled water represents the most appropriate solvent for polyplex formulation when delivered by jet nebulization.

Genetic determination of diabetes mellitus in patients with cystic fibrosis. Multicenter Cystic Fibrosis Study Group.

Data from 1348 patients with cystic fibrosis in mid-Europe show that the prevalence of diabetes mellitus in these patients is 4.9%, that diabetes develops in more female than male patients with cystic fibrosis during childhood and adolescence and at a younger age, and that diabetes mellitus is more likely to affect delta F508 homozygous patients.

Specific and rapid detection by fluorescent in situ hybridization of bacteria in clinical samples obtained from cystic fibrosis patients.

We report on the rapid and specific detection of bacteria commonly isolated from clinical specimens from cystic fibrosis (CF) patients by fluorescent in situ hybridization (FISH). On the basis of comparative sequence analysis, we designed oligonucleotide probes complementary to species-specific 16S rRNA regions of these microorganisms and demonstrated the specificities of the probes by hybridization of different remotely related as well as closely related reference strains. Furthermore, in a pilot project we investigated 75 sputum samples and 10 throat swab specimens from CF patients by FISH and detected Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, Haemophilus influenzae, and Staphylococcus aureus within these specimens. The specificity of FISH was 100% in comparison to the results of conventional microbial culture. In contrast, the sensitivity of standard laboratory cultivation was moderately higher, since the limit for microscopic detection of bacteria within sputum samples by FISH was approximately 4 x 10(5) CFU/ml of sputum (resulting in a 90% sensitivity for FISH). Moreover, we demonstrated that FISH will be useful for the rapid detection of bacteria that cause acute pulmonary exacerbations in CF patients, as demonstrated in patients with H. influenzae, S. aureus, and P. aeruginosa exacerbations. Therefore, FISH is a valuable additional method for the rapid and specific detection of bacteria in clinical samples from CF patients, in particular, patients with pulmonary exacerbations.

In vivo gene delivery to the lung using polyethylenimine and fractured polyamidoamine dendrimers.

BACKGROUND: Gene transfer into the airways could be of importance for the treatment of chronic lung diseases such as cystic fibrosis. In the past few years several attempts have been made to effectively deliver DNA to the lung using different viral and non-viral vector systems. Viral vectors and cationic lipids have been tested intensively but the properties of cationic polymers such as polyethylenimine (PEI) 25 kDa and fractured polyamidoamine dendrimers to deliver DNA to the airways have not been studied. Surfactant preparations have been shown to influence pulmonary adenoviral and naked plasmid DNA mediated gene transfer in vivo. We investigated the gene delivery efficiency of branched PEI 25 kDa and fractured dendrimers to the murine lung in vivo and also examined the effect of surfactant on PEI 25 kDa mediated gene transfer to the lung. METHODS: Cationic polymer/DNA complexes were prepared in 25 mM HEPES buffer (pH = 7.4) or double distilled water and administered to the lungs of BALB/c mice via cannula intubation. The trachea, left and right lung, heart, liver and esophagus were examined for luciferase activity. Inflammation was assessed by performing standard histology. RESULTS: PEI/DNA complexes showed a high level of luciferase gene expression in the lung. Complexes formed in double distilled water exhibited higher gene expression than complexes formed in 25 mM HEPES buffer (pH 7.4). The optimal N/P ratio was found to be N/P = 10 in double distilled water. Luciferase activity was only detected in the lung and decreased rapidly in a time-dependent manner. The addition of a natural surfactant preparation, Alveofact, slightly reduced gene transfer of branched PEI 25 kDa. Luciferase gene expression obtained by using fractured dendrimers was very low. CONCLUSION: The present study demonstrates that PEI 25 kDa, but not polyamidoamine dendrimers, effectively mediates transient gene transfer to the murine lung after intratracheal intubation. In conclusion, branched PEI 25 kDa was found to be an effective vector for pulmonary gene delivery in vivo, being superior to fractured dendrimers.

Adenovirus infection in cystic fibrosis patients: implications for the use of adenoviral vectors for gene transfer.

Clinical trials using replication-deficient adenovirus as vectors for gene transfer into the airways of cystic fibrosis (CF) patients are in progress. However, little is known about the prevalence of wild-type adenovirus infections in patients with cystic fibrosis and their effect on lung function. To answer these questions, serum IgG and IgM antibody titers against adenovirus type 5 were prospectively measured by an indirect immunofluorescence assay in 199 CF outpatients and in a control group of 45 healthy children and young adults. In addition, we performed pulmonary function tests when the patients were in stable clinical condition. IgM antibodies against adenovirus were present in 104 of the 199 cystic fibrosis patients (52.3%). IgG antibodies against adenovirus were detected in 192 of the 199 cystic fibrosis patients (96.5%), and were significantly higher in cystic fibrosis patients older than 7 years than in younger patients and in age matched controls. IgG antibody titers measured a second time 11.8 months later in 143 of the 199 patients had increased in 48 (33.6%) patients. In 27 of these 48 patients, who had at least a 2-fold increase in antibody titer, FVC and FEV1 decreased by 9.8% (p < 0.05) and 8.3% (p = 0.05), respectively, over 45 months. In a comparison group matched for age, sex, and chronic Pseudomonas aeruginosa infection but no increase in antibody titers, FVC and FEV1 were unchanged. The results indicate that wild-type adenovirus infections are prevalent in cystic fibrosis patients and that wild-type adenovirus infections in cystic fibrosis patients seem to be associated with deterioration in lung function. These observations may have important implications for efficacy and safety considerations when using adenoviral vectors for gene therapy.

Increased liposome extravasation in selected tissues: effect of substance P.

We have used a pharmacologic mediator to open intercellular connections in selected vessels to allow liposomes to escape from the blood stream and to extravasate into tissues that have appropriate receptors. We have examined the effects of substance P (SP), a peptide known to increase vascular permeability in selected tissues, such as trachea, esophagus, and urinary bladder in rats. We used quantitative fluorescence analysis of tissues to measure two fluorescent markers, one attached to the lipid (rhodamine-phosphatidylethanolamine) and another, doxorubicin (an anti-tumor drug), encapsulated within the aqueous interior. We have also examined the deposition of liposomes microscopically by the use of encapsulated colloidal gold and silver enhancement. Analysis of the biochemical and morphological observations indicate the following: (i) Injection of SP produces a striking increase in both liposome labels, but only in tissues that possess receptors for SP in postcapillary venules; (ii) liposome material in these tissues has extravasated and is found extracellularly near a variety of cells beyond the endothelial layer over the first few hours; (iii) 24 h following injection of liposomes and SP, liposome material is found in these tissues, localized intracellularly in both endothelial cells and macrophages. We propose that appropriate application of tissue-specific mediators can result in liposome extravasation deep within tissues that normally do not take up significant amounts of liposomes from the blood. Such liposomes are able to carry a variety of pharmacological agents that can be released locally within selected target tissues for therapeutic purposes.

Mutation analysis in the diagnosis of cystic fibrosis.

Since the characterization of the gene encoding the cystic fibrosis transmembrane conductance regulator protein and identification of its main mutation, delta F508, causing cystic fibrosis (CF), more than 150 mutations in this gene have been reported, most of them only in a few or even single CF patients. Attempts to use mutation analysis in genetic counselling or for the diagnosis of CF depends on prevalence data of certain mutations in the respective population, and considerable ethnic differences have been reported. In this study we determined the prevalence of the mutations delta F508, G551D, R553X, and G542X and of genotypes defined by these mutations in 239 CF patients (444 independent CF chromosomes) seen in our clinic. The analysis for those four mutations alone now permits identification of approximately 75% of all mutations in our CF patients. The complete genotype can be resolved in approximately 63% of patients. This represents the diagnostic sensitivity which can be achieved by mutation analysis in patients without a family history of CF. We conclude that in situations where conventional diagnostic tests are not feasible or difficult to interpret, mutation analysis using a limited set of mutations can contribute significantly to an early and specific diagnosis of CF.

Interaction of liposomal and polycationic transfection complexes with pulmonary surfactant.

BACKGROUND: The delivery of genes to the airways holds promise for the treatment of lung diseases such as cystic fibrosis and asthma. Current non-viral gene delivery systems lack sufficient transfection efficiency. Pulmonary surfactant has been reported to be a barrier to gene transfer into the airways. Here we analyze the interaction of liposomal and polycationic transfection complexes with pulmonary surfactant. METHODS: The efficiency of non-viral transfection of cultured human airway epithelial cells (16HBE14o-), COS7 cells and porcine primary airway epithelial cells was studied in the presence of various surfactant preparations in order to model the conditions prevailing in the airways during transfection. RESULTS: The natural pulmonary surfactant, Alveofact, an extract from bovine lung lavage, was found to inhibit lipofection with lipofectAMINE for all cell lines investigated. Dendrimer meditated polyfection was unaffected for pulmonary cell lines and was weakly affected for COS7 cells. PEI-mediated polyfection was unaffected for all cell lines tested. The synthetic surfactant preparation Exosurf containing L-alpha-phosphatidylcholine-dipalmitoyl (DPPC) as the sole lipid ingredient had no statistically significant effect on polymer- and lipid-mediated transfection. The transfection efficiencies are related to structural changes in the DNA complexes as demonstrated by DNase-accessibility tests and fluorescence spectroscopy. In the presence of the phospholipid POPG, which is a constituent of Alveofact, DNA condensed in lipofectAMINE lipoplexes became accessible to DNaseI, while DNA condensed with PAMAM dendrimer or PEI was less accessible to DNase I as compared to lipoplexes. Consistently, the fluorescence of a DNA-intercalating dye increased after addition of Alveofact only in the case of lipoplexes. CONCLUSIONS: In contrast to lipofection, gene transfer with cationic polymers to airway epithelial cells is not inhibited by pulmonary surfactant in vitro. Depending on the surfactant concentration even an increase in polymermediated transfection can be seen. In conclusion, cationic polymers appear to be the more stable gene delivery systems for topical application into the airways.

Interaction of bronchoalveolar lavage fluid with polyplexes and lipoplexes: analysing the role of proteins and glycoproteins.

BACKGROUND: Plasmid DNA complexed with cationic lipids (lipoplexes) or cationic polymers (polyplexes) has been used for gene transfer into the lung. Topical gene administration of lipoplexes or polyplexes into the lung after intratracheal instillation or aerosolisation could cause interaction of the complexes with extracellular substances of the airway surface liquid (ASL). These extracellular interactions might be causal for the observed inefficient transfection rate in vivo after topical administration. Therefore, we studied the impact of bronchoalveolar lavage fluid (BALF) on reporter gene expression mediated by non-viral gene vectors. BALF was considered as a model system to mimic possible interactions of the gene vectors with the ASL. METHODS: BALF was taken from 15 patients who underwent diagnostic bronchoscopy. Lipoplexes and polyplexes were incubated with increasing concentrations of BALF and major components of the BALF such as albumin, mucin and alpha(1)-glycoprotein, as a representative of glycosylated proteins. As cationic polymers, we tested dendrimers (fractured PAMAM) and polyethylenimine 25 kDa (PEI) and, as cationic liposomes, we used Lipofect-AMINE. The effect of BALF on polyplexes and lipoplexes was analysed by transfection experiments, fluorescence-quenching assay, 2-D-gel electrophoresis, SDS-PAGE, DNAse protection assay, size and zeta-potential measurements. RESULTS: BALF inhibited polyplex- and lipoplex-mediated gene transfer. Analysing components of BALF, we found that dendrimer-mediated gene transfer was not inhibited by any specific component. PEI-mediated gene transfer was dose-dependently inhibited by alpha(1)-glycoprotein, slightly inhibited by mucin, but not inhibited in the presence of albumin. Lipoplex-mediated gene transfer was inhibited by mucin at higher concentrations and by albumin, but not by alpha(1)-glycoprotein. 2-D-gel electrophoresis revealed that proteins of the BALF were adsorbed more intensively to lipoplexes than to polyplexes. In addition, mucin and alpha(1)-glycoprotein also adsorbed more intensively to lipoplexes than to polyplexes. Adsorption of BALF components led to a decrease in the positive zeta-potential of lipoplexes and led to a negative zeta-potential of polyplexes. Complement cleavage fragment C3 beta, and in the case of lipoplexes also the C3 alpha fragment, were found among the proteins opsonised on gene vectors. CONCLUSIONS: Our study shows that BALF contains inhibitory components for non-viral gene transfer. We could not detect a specific inhibitory component, but inhibition was most likely due to the change in the surface charge of the gene vectors. Interestingly, there is evidence for complement activation when the route of pulmonary gene vector administration is chosen. Consequently, shielding of gene vectors to circumvent interaction with the ASL environment should be a focus for pulmonary administration in the future.