Composition polyethylenimine: dna for aerosol delivery
The invention relates to the field of molecular biology. The invention demonstrates that poly-composition/lipid:DNA are resistant to induced sputtering of degradation and are much more specific and effective than compositions based on lipids, transfection tissues of the respiratory tract in vivo. The invention is a composition polyethylenimine (PEI), they are approximately 10 times more efficient than previous compositions based on lipids, when delivered in vivo using a jet nebulizer. The technical result - aerosol compositions on the basis of polyethylenimine effective for gene therapy through the respiratory tract. 2 N. and 22 C.p. f-crystals, 17 ill.
The scope of the invention
This invention relates in General to the field of gene therapy and delivery of pharmaceutical drugs. More specifically, this invention relates to the delivery of therapeutic genes aerotolerant polyethylenimine.
Description of the prior art.
A serious problem associated with aerosol gene delivery to the lungs (in the form of “naked” DNA, viruses or liposomes), is a prominent Uchenye by transfection in vitro (Crook et al., 1996; Schwarz et al., 1996; Eastman et al., 1997a,b, 1998). In addition, it was shown that the lipids and proteins of pulmonary surfactant (surface-active substance that forms a monomolecular layer on the alveolar surface of the lungs) inhibit mediated by cationic liposomes transfection (Duncan et al., 1997; Tsan et al., 1997). These effects likely contribute to the message about the low level of gene transfer in vivo aerotolerance compositions based on lipids and slowed down the development of systems that efficiently deliver DNA into the lungs.
Recently it was reported that the composition containing one specific cationic lipid (bis-guanidine-trangression; BGTC), was more stable during spraying than some previously tested lipids. Despite this improvement in transfection in vivo via aerosol, clinical application of this therapy may require even more effective delivery vectors. In recent years it was reported that the polycation are effective in transfection of cells in vitro and in vivo. One derivative with notable activity is polyethylenimine (PEI) (Boussif et al., 1995, 1996). Here it is reported that the composition of PEI-DNA is not only resistant to induced spray reduction efficiency is ecchi lung tissue in vivo, so the composition based on polyethylenimine are approximately 10 times more efficient than previously optimized composition based on lipids, when delivered in vivo using a jet nebulizer. Such compositions based on polyethylenimine are more efficient in transfection light, but produce relatively low levels of transfection in the nasal passages of mice. Compositions based on polyethylenimine are, apparently, good candidates for aerosol gene delivery for the treatment of various genetic pulmonary disorders, including lung tumors, and can be non-invasive tool for genetic immunization.
In the prior art there is insufficient non-lipid carriers and compositions for aerosol delivery of DNA or therapeutic genes for use in targeted gene therapy through the respiratory tract. This invention satisfies this long existing need and desire in this field.
BRIEF description of the INVENTION
Aerosol delivery of plasmid DNA into the lungs provides a non-invasive treatment of various lung disorders. However, the sputtering process often leads to rapid loss of efficiency is x for DNA delivery, here it is reported that compositions using polyethylenimine and macromolecules such as DNA, lead to a high level of lung transfection and are stable during spraying. Composition RE-DNA also reveal a high degree of specificity in relation to the lungs and are non-toxic. The optimum ratio and concentration RE-DNA were determined for in vitro transfection and in vivo transfection. It turned out that the serum factor increases PEI mediated transfection, whereas surfactants possess the minimum inhibitory action.
One purpose of this invention is the representation of the cationic, non-lipid carriers and compositions for use in aerosolized delivery of DNA or therapeutic genes for targeted gene therapy through the respiratory tract.
In one embodiment of the present invention is provided a method of directed gene therapy through the respiratory tract, providing stage: delivery of aqueous dispersions through an aerosol of fine particles of a therapeutic gene, located in the complex with polyethylenimine, through the respiratory tract of an individual in need of such treatment.
In another embodiment, the present invention pre is Asa polyethylenimine and DNA encoding a therapeutic gene.
In another embodiment, the present invention presents a composition polyethylenimine-DNA for delivery of a therapeutic gene through an aerosol of fine particles obtained by the method comprising the stage of: dissolving DNA that encodes a therapeutic gene in a suitable solution; dissolving polyethylenimine in a suitable solution; complexation of dissolved DNA and dissolved polyethylenimine with obtaining the composition polyethylenimine-DNA; and placing the composition polyethylenimine-DNA in a device for spraying with obtaining the composition polyethylenimine-DNA for delivery of a therapeutic gene by means of an aerosol of small particles.
Other and further aspects, features and advantages of this invention will be apparent from the following description of the preferred at the present time variations of the present invention. These options are given for descriptive purposes.
BRIEF DESCRIPTION of DRAWINGS
The accompanying drawings have been included here so that visheopisannie features, advantages and objectives of this invention will be clear and can be understood in detail. These drawings form part of the description. However, it should be noted that the accompanying drawings illustraing of the invention.
Fig.1 shows cells A with RE-DNA at different ratios of N:P in the absence of serum. Composition PEI-pCMVand PEI-luciferase with an increasing ratio of N:P was received as described. Throughout the experiment used a constant concentration of DNA, except for the option “0”, which did not transicional, but which was subjected to otherwise identical environment changes. Cells I was transfusional for 6 h and was extracted to determine-galactosidase or luciferase activity 48 hours after the initiation of transfection. Fig. 1A illustrates levels-galactosidase, detected for each state, and Fig. 1B illustrates the levels of luciferase defined for each state. Fig. 1C illustrates the levels of protein (approximate measurement of the density of cells and, therefore, cytotoxicity), defined for cells transfected with PEI-pCMV(from the same experiment illustrated in Fig. 1A). N=3 for all groups. Vertical lines indicate standard error.
Fig. 2 shows the transfection of cells A increasing concentrations RE-DNA the concentrations at the indicated ratios of N:P. Cells I was transfusional for 6 h and was extracted for determination of luciferase activity at 24 h after the initiation of transfection. N=3 for all groups.
Fig. 3 shows the action of serum on transfection efficiency PEI-pCMVin vitro. Cells I was transfusional constant concentration of DNA (1 μg per well) and PEI (15 nmol per well) at a constant ratio of N:P 20 with these additions for 6 h and was extracted to determine-galactosidase after 48 h after the initiation of transfection. N=3 for all groups. Vertical lines indicate standard error.
Fig. 4 shows the action of the surfactant and BAL-fluid on the efficiency of transfection with PEI-pCMVin vitro. Cells I was transfusional constant concentration of DNA (1 μg per well) and PEI (15 nmol per well) at a constant ratio of N:P 20 with these additions for 6 h and then tested on-galactosidase after 48 h after the initiation of transfection. N=3 for all groups. Vertical lines indicate standard error.
Fig. 5 shows the effect of polyethylenimine and cationic lipid on stable transfection during raspy is the specified time periods before sampling from the reservoir for spraying and subjected to quantitative monitoring in the test transfection in vitro (CPRG) using cells A in culture, as described. N=3 for all groups.
Fig. 6 shows intranasal treatment in vitro BALB/c mice. In this experiment, mice were administered intranasally plasmid pCMVHi-CAT (containing the CAT gene), prepared or polyethylenimine, or with a cationic lipid BGTC:DOPE (figure 6A). Equal amounts of DNA (24 µg) was administered to all animals. Animals were first anestesiology and the DNA solution is the vector was administered intranasally. Animals were killed after 48 h after instillation, the lungs were removed and tissues were extracted and analyzed (using ELISA) on the expression of CAT activity. An identical experiment was performed using expression plasmids luciferase (pGL3), prepared or polyethylenimine or BGTC:DOPE (Fig. 6B). Animals were killed after 48 h after instillation, the lungs and nasal tissues were removed and were extracted and analyzed for the expression of luciferase, as indicated. Each processing group consisted of 6 animals. Vertical lines indicate standard error.
Fig. 7 shows the aerosol processing in vivo in BALB/c mice. In this experiment, animals were subjected to either RE-DNA (gene chloramphenicolchloramphenicol), lipid-DNA (the same plasmid, with the same concentration of DNA) or not exposed near the u, connected with one jet spray Puritan Bennett 1600, and 1-minute aerosol processing with subsequent 9-minute delay to allow the animals to inhale the aerosol before you start the next cycle. This is repeated until then, until it was exhausted fluid sprayer (40 ml) (approximately 16 hours). Animals were killed after 48 h after aerosol treatment, the lungs were removed and tissues were extracted and analyzed (using ELISA) on the expression of CAT activity. Each processing group consisted of 6 animals. Vertical lines indicate standard error.
Fig. 8 shows the induction of antibodies by genetic immunization PEI-pCMV-hGH. Groups of 5 mice were subjected to drug PEI-pCMV-hGH using aerosol or intranasal installation and the results were compared with the results obtained with naked DNA provided by intramuscular injection. Serum samples were obtained at 2-week intervals and the presence of anti-hGH antibodies were measured using ELISA. The average optical density and standard deviation for each group is shown at a dilution of 1:500. Background OD of sera from unimmunized mice was 0.1 in this analysis. Vertical che the effect of aerosol RE-DNA generated using air or air containing 5% CO2. 1 mg CAT plasmids used for the formation of a complex with PEI at a ratio of N:P 10:1 and the resulting complex was aerosolizable in mice within 30 minutes Lungs were removed after 24 h and CAT analysis was performed as described. Values represent mean ± SD (n=6 mice per group, p=0.001).
Fig. 10 shows that the gene expression in the lung by aerosol RE-DNA is dose-dependent. Increasing doses of CAT-plaidy was aerosolizable using 5% CO2in the air at a fixed ratio of N:P 10:1. There is an increase in the total number of delivered DNA, and the concentration delivered RE-DNA. Mice were killed after 24 h, the lungs were harvested and CAT protein were analyzed. Values represent mean ± SD (n=5 mice per group).
Fig. 11 shows the effect of the ratios of N:P in the transfer efficiency RE-DNA into the lungs by aerosol. Used different ratios RE-DNA (N:P) with a fixed number of CAT plasmid (2 mg). The complex was aerosolizable using 5% CO2in the air. Mice were killed after 24 h, the lungs were harvested and CAT protein were analyzed. Values represent mean ± SD (n=5 mice per group).
Fig. 12 aznoe plasmid (2 mg) were taken at various ratios of N:P. Complexes were aerosolizable using 5% CO2in the air. Mice were killed 24 h after aerosol delivery, the lungs were collected and determined the luciferase activity. Values represent mean ± SD (n=5 mice per group).
Fig. 13 shows the time course of transgene expression after a single exposure to the impact of aerosol PEI-DNA. Fig. 13A: mice were aerosolizable 2 mg CAT plasmids at a ratio of N:P 15:1 using 5% CO2in the air. Mice were killed after 24 h, the lungs were collected and immediately frozen. CAT analysis was carried out after the last time point. Values represent mean ± SD (n=5 mice at the time point). Fig. 13B: the stability of expression of CAT using two different ratios of N:P. Both groups of mice (n=5 mice at each time point per group) received 2 mg CAT plasmids at the ratios of N:P 15:1 or 10:1 using 5% CO2in the air. Time points for the ratio of 10:1 are 1, 2, 3 and 6 days after exposure to aerosol, and 15:1 are 1, 3, 7 and 10 days after exposure to the aerosol.
Fig. 14 shows the tissue distribution of transgene after a single exposure to the impact RE-DNA. Used the same groups of mice as in Fig. 1 the temporal point. Values represent mean ± SD (n=5 mice at the time point).
Fig. 15 shows a tumor index for aerosol gene therapy of melanoma B16-F10. Tumor cells were injected with day 0 and treatment was started on day 1. PEI-p53 and PEI-RB was delivered twice a week by aerosol for 3 weeks. At day 24 after injection of tumor cells, the mice were killed and lungs were weighed. The lung metastasis was evaluated on a scale of 1-4 points. The swelling index was calculated taking into account metastasis and light weight (p<0,01 versus control for both groups p53 and Rb group).
Fig. 16 shows the steps of aerosol Raman therapy for adenocarcinoma M109. Tumor cells were injected with day 0 and treatment was started on day 1. PEI-p53 was delivered twice a week and drug, 9NC, also delivered twice a week for 4 weeks. After 4 weeks, mice were killed, lungs were weighed and tumor foci were counted. The swelling index was calculated taking into account the weight of the lung, the number of tumor foci and tumor lesions (p<0,0001 versus control for both groups p53, and group p53+9NC).
Fig. 17 shows the steps compositions PEI and p53 on tumor growth in models naked mouse lung meta is th DNA in the lungs provides the possibility of direct application of drugs, genes on the lung surface as a means of treatment of various genetic lung disorders. However, the process of jet spray quickly destroys the “naked” DNA, viral vectors, and many compositions based on lipids. Although it has been shown that complexation of DNA with cationic lipids significantly stabilizes the plasmid DNA, loss of activity occur during spraying, seriously limiting aerosol delivery of many such complexes. Together with the development of systems for aerosol delivery, suitable for DNA delivery have been developed compositions using polyethylenimine (PEI, poly-polymer) and macromolecules, such as DNA, which lead to high levels of lung transfection (10-100 times higher than in the case of cationic lipids) and exhibit a high degree of stability during procedures spraying. In addition, these compositions are based on polyethylenimine show a high degree of specificity in respect of the lungs in comparison with DNA-liposomal complex and are non-toxic. The optimal composition of polyethylenimine and the ratio and concentration of complex polyethylenimine-DNA were determined for both in vitro and in vivo transfection. We have studied properties of compositions based on polyethylenimine that make them resistant to raspy the ke, apparently, increases mediated by polyethylenimine transfection, whereas surfactants have a minimal inhibitory effect. Potential applications of this method include the use of aerotolerance complex polyethylenimine-DNA for genetic immunization.
This invention relates to delivery through the nozzle. A number of inkjet nozzles are available to deliver a single dose or for the delivery of low-dose aqueous slurry. Composition polyethylenimine:DNA of the present invention can also be delivered by ultrasonic atomization. In addition, mixtures of polyethylenimine and DNA can also be issued by compressed air or gas in the inhaler with measured doses”.
Specifically, this invention relates to a method of targeting therapy, such as gene therapy through the respiratory tract to which the stages of delivery of aqueous dispersions of genetic macromolecule, such as a therapeutic gene, located in the complex with polyethylenimine, via an aerosol of small particles through the respiratory tract of an individual in need of such treatment. Representative examples of genetic macromolecules that can be delivered to fit the AK ribozymes, the antisense oligonucleotides, as well as other types of modified nucleic acids. This method of the present invention may be used for the treatment of an individual having a disease, such as cystic fibrosis, asthma, lung cancer, esophageal cancer, cancer of the colon, leukemia, breast cancer, sarcoma or melanoma. In addition, genetic macromolecule in complex with polyethylenimine in aqueous dispersions can be injected into an air mixture containing 10% of carbon dioxide gas to potenzirovti action polyethylenimine, as described in detail below.
This invention relates also to the composition of the polycation-genetic macromolecule for delivery of genetic macromolecule, such as a therapeutic gene, by means of an aerosol of fine particles containing polyethylenimine and genetic macromolecule. Representative examples of genetic macromolecules that may be contained in the compositions of this invention include DNA, RNA, catalytically active nucleic acids, such as ribozymes, antisense oligonucleotides, as well as other types of modified nucleic acids.
Further, this invention relates to compositions, steriade particles, and this song get method comprising the stages: dissolution of genetic macromolecules in a suitable solution; dissolving polyethylenimine in a suitable solution; complexation of dissolved genetic macromolecules and dissolved polyethylenimine with the formation of the composition polyethylenimine genetic macromolecule; and placing the composition polyethylenimine genetic macromolecule in a device for spraying with obtaining the composition polyethylenimine genetic macromolecule for delivery of genetic macromolecules through an aerosol of small particles. Representative examples of genetic macromolecules that may be contained in the compositions of this invention include DNA, RNA, catalytically active nucleic acids, such as ribozymes, antisense oligonucleotides, as well as other types of modified nucleic acids.
Methods of delivery for vysheopisanym methods and compositions include oral inhalation and nasal inhalation via face mask or tube for the mouth and using oxygen boxes for children who otherwise used to supply oxygen. Typically, the aerosol of fine particles create% the e, preferably, therapeutic gene contained functionally related elements necessary for expression, and representative therapeutic genes include regulatory gene transmembrane conductance of cystic fibrosis, the gene encoding a tumor suppressor, such as p53 or retinoblastoma gene, or engineered variants of these genes, cytokine genes, such as interleukin-2 or interleukin-12, and genes that can be used as DNA vaccines, which will stimulate direct the immune response against tumors or other infectious agents. Another representative of therapeutic gene is a gene timedancing HSV. Typically, the final concentration of therapeutic gene in polyethylenimine not more than about 10 μg DNA/50 nmol nitrogen polyethylenimine/ml and not less than about 0.1 μg DNA/50 nmol nitrogen polyethylenimine/ml in Addition to visheopisannie composition can be added serum. Next, cladoselache the ligand can be covalently anywhereman with polyethylenimine, and representative cladosporioides ligand is transferrin.
In accordance with this invention there can be used conventional methods of molecular biology, MAZ described in the literature. See, for example, Sambrook, Fritsch &Maniatis, "Molecular Cloning: A Laboratory Manual (1982); "DNA Cloning: A Practical Approach," Volumes I ana II (D. N. Glover ed. 1985); "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" [B. D. Hames &S. J. Higgins eds. (1985)]; "reduced and Translation" [B. D. Hames &S. J. Higgins eds. (1984)]; "Animal Cell Culture" [R. I. Freshney, ed. (1986)]; "Immobilized Cell And Enzymes [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984). Thus, in this description, the following terms will have the following values.
“DNA molecule” refers to a polymeric form of deoxyribonucleotides (adenine, guanine, thymine or cytosine) or in its single-stranded form, or in double-stranded helix. This term refers only to the primary and secondary structure of this molecule and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes.
“Genetic molecule” will include DNA, including therapeutic genes, RNA, catalytically active nucleic acids, such as ribozymes, antisense oligonucleotides, as well as other types of modified nucleic acids.
“Promoter sequence” is a regulatory owned the STI in the forward direction (3-direction). For the purposes of determining the present invention promoter sequence linked to the 3'-end of the site of transcription initiation and extends in the opposite direction of transcription (in 5'-direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. In this promoter sequence is the site of transcription initiation, as well as blocksgateway domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters are often, but not always, contain “TATA”boxes and “SAT”boxes. Prokaryotic promoters contain sequences Shine-Dalgarno, in addition to the -10 and -35 consensus sequences.
“The coding sequence of the DNA is double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of suitable regulatory sequences. The boundaries of the coding sequence are determined by the start-codon at the 5' (amino)-the end and the stop codon broadcast on 3' (carboxyl)-the end. The coding sequence can include, but are not limited to, prokaryotes apicauda) and even synthetic DNA sequences.
Regulatory sequences for transcription and translation are regulatory DNA sequences, such as promoters, enhancers, polyadenylation signals, terminators, etc., that provide for expression of the coding sequence in a cell host. “CIS-element” is a nucleotide sequence, also called the “consensus sequence” or “motif, which interacts with other proteins that regulate the expression of specific gene loci in the forward or reverse direction. In the coding sequence can also be enabled “signal sequence”. This sequence encodes a signal peptide located on the N-Terminus of this polypeptide which interacts with the host-cell and directs the polypeptide to the appropriate cellular location. The signal sequence can be detected in connection with various proteins, native to prokaryotes and eukaryotes. The polyadenylation signal and the sequence of the transcription termination will usually be localized with 3' hand coding sequence.
“Regulatory sequence expression is posledovatelya sequence “functionally associated with” transcriptional and translational regulatory sequences and is under the control” of transcriptional and translational regulatory sequences in the cell, when RNA polymerase Transcriber the coding sequence into mRNA, which is then translated into the protein encoded by this coding sequence.
“Gene” may include the intact gene in the form in which this gene occurs in nature, i.e., the coding sequence run by the promoter and/or additional sequences found in nature. Alternatively, the gene may include the coding sequence and the heterologous promoter (with heterologous transcriptional and/or translational regulatory sequences, or without them) to run the expression of that coding sequence. In the application here, “therapeutic gene” is usually genome, or with its native promoter or a heterologous promoter that initiates the expression of a coding for a protein that has a therapeutic effect, for example, the gene coding for the desired enzyme, otherwise absent in the cell.
Usually, expressing vectors containing promoter sequences which facilitate the efficient transcription and translation of the embedded DNA fragment used in conjunction with the owner. Usually expressing the vector is capable of providing phenotypic selection in transformed cells. Transformed hosts can be fermented and cultured according to methods known in this area, for optimum cell growth.
“Heterologous” in the construction of DNA is an identifiable segment of DNA within a larger DNA molecule that is not found in Association with this larger molecule in nature. Thus, when the heterologous plot encodes a gene of a mammal, the gene will usually flanked by DNA that does not flanks this genomic DNA of the mammal in the genome of an organism is the source of this DNA. In another example, the coding sequence is a construct where the coding sequence not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the codons of the native gene). Allelic variations of the naturally occurring mutational events do not lead to heterologous DNA defined here.
“Vector” is a replicon, such as plasmid, phage or cosmid, to which may be attached to another DNA segment in such a manner as to cause the replication priamo, virus) that functions as an Autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control.
“Start replication” refers to those DNA sequences that are involved in DNA synthesis.
In the application here, the term “aerosol” refers to the dispersion in air of solid or liquid particles of sufficiently small size and the low deposition rate, resulting in relative stability of being in the air (Knight, V., Viral and Mycoplasmal Infections of Respiratory Tract, 1973, Lea and Febigar, Phila. PA, pp.2).
In the application here, the term “dispersion” or “spraying” refers to the obtaining of fine aerosols from the fluid, i.e. the product back-flow producing a directed spray mist device (May, 1973).
In the application here, the term “jet spray” refers to a device in which compressed air is expanded in the nozzle, leading to a reduction in static pressure, which sucks the liquid from the reservoir to the atomizer through an attached tube. This fluid splits the air stream into the dispersion of droplets with a wide range of sizes. A large part of the drops (about 99,92%) hits the wall enclosing the bulb and return to cut the lump of air. He is atomized spray.
In the application here, “intranasal instillation” means the process by which liquid make a nose shot of the animal, which is an animal with artificial nose breathing, so this animal then inhales this liquid into the respiratory tract or lungs.
Specifically, the pharmaceutical composition (PEI):the DNA of this invention can be prepared for aerosol delivery of aqueous dispersions in the respiratory tract of an individual or animal. The person skilled in the art can easily determine, without undue experimentation, the appropriate dose of aerosol compositions. When applied in vivo for targeting gene therapy composition polyethylenimine:DNA of the present invention is administered to a patient or an animal in therapeutically effective amounts, i.e. amounts that effectively Express therapeutic gene and thereby helps eliminate or weaken the disease. The dose and regimen of drugs will depend on the nature of the disease, the properties of a particular composition polyethylenimine:DNA, such as its therapeutic index, the patient, the patient's medical history and other factors. Widemouth. Scheme applications will continue to optimize the efficiency ratio of the negative effects of the treatment. Cm. Remington's Pharmaceutical Science, 17thEd. (1990) Mack Publishing Co., Easton, Penn.; and Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8thEd (1990) Pergamon Press; which is incorporated herein by reference. Composition or finished form may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, such as buffers and preservatives. The composition of polyethylenimine:DNA is usually prepared at concentrations of about 0.1 μg DNA/20 nmol PEI nitrogen/ml - 10 μg DNA/150 nmol PEI nitrogen/ml
The following examples are provided to illustrate different variants of the present invention and are not intended to limit in any way the present invention.
PEI (25 KD) was obtained from Aldrich Chemical (Milwaukee, WI). The original solution of PEI was obtained at a concentration of 4.3 mg/ml (0.1 M for nitrogen) in phosphate buffered saline (SFR). DNA contains 3 nmol of phosphate on the MAG and the appropriate concentration was obtained in SFR. The desired amount of DNA in SPR were subjected to complexation with PEI by slow addition of DNA to PEI under intensive stirring vortex of this solution. The ZAT is E. the ratio of the reacting substances expressed as PEI nitrogen:phosphorus DNA (N:P).
The promoter of cytomegalovirus (CMV) with a reporter gene-galactosidase E. coli (pCMV; CLONTECH, Palo Alto, CA) and gene green fluorescent jellyfish protein (pEGFP; CLONTECH) was used to assess the expression of mammalian cells in vitro. Transfection in vivo was evaluated genome chloramphenicole-teletransfer (pCMVHiCAT) (a gift from Genzyme, Inc., Framingham, MA). Luciferase plasmid (pGL3, Promega, Madison, WI), modified by the incorporation of the promoter/CMV enhancer and polyadenylation sequence of the human growth hormone (Michael A. Barry from Baylor), was used as a reporter gene for transfection both in vitro and in vivo. The plasmid used for studies on immunization, pCMV-hGH, expresses the human growth hormone was obtained from Michael A. Barry. Bacterial cultures transformed by the above plasmid and received sufficient mass quantities of plasmid DNA was purified using reagents and columns not containing endotoxin DNA (Qiagen, Valencia, CA) and then was dissolved in does not contain endotoxins water to the desired concentration.
The synthesis of cationic lipids
Cationic lipids or synthesized, or recip is obam Gao and Huang (1991). Guanidinylation(bis-guanidinium-TREN-cholesterol; BGTC) was synthesized according to the methods Vigneron et al. (1996). N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradentate)-1-propanaminium (DMRIE) was obtained from Vical (San Diego, CA).
Obtaining complexes of cationic lipid: neutral colied-DNA
All forms of cationic lipid:DOPE was prepared first by mixing a cationic lipid (5 mg/ml in chloroform) with a suitable amount of dioleoylphosphatidylcholine (DOPE; Avanti Polar Lipids; 5 mg/ml in chloroform) or cholesterol (5 mg/ml in chloroform; Sigma) and drying in a nitrogen atmosphere. The mixture of lipids was dissolved in tertbutanol at 37°C, frozen at -80°C and liofilizirovanny for at least 24 hours Lipids were stored at -20°C to use, at this point, they were heated to room temperature and resuspendable in sterile water (Water for irrigation; water (for rinsing) WFI; Baxter, Deerfield, IL)) for at least 30 min before complexation with DNA. Lyophilized forms ready BGTC:DOPE, apparently, are stable at -20°C for a period of 6-8 months or more.
Cell line A lung cancer person bought from American type culture collection (Rockville, MD). To the serum (FCS), 2 mm L-glutamine and 50 μg/ml gentamicin (unless directed otherwise), or in medium RPMI 1640 (Gibco) with 10% FCS. Cells were cultured in the incubator for cell cultures at 37°C and in the presence of 5% CO2. Used only the first 8 passages of cells A.
Transfection of in vitro
For serum free of transpency cells were sown at 1.5×105cells per well in 12-hole tablets the day before transfection (unless specified otherwise). Cells were approximately 80% confluently during transpency. After complexation RE I-DNA or lipid-DNA forms ready RE-DNA or liposomal DNA brought in the medium Opti-mem I Reduced Serum Medium (Opti-mem; Gibco-BRL, Grand Island, NY) to a final DNA concentration of 1 μg/ml Immediately before transfection, the cells washed with Opti-mem and then put 1 ml solution for transfection per well (3 wells were usually transfusional at each time point or in each state). Complexes of plasmid-RE received at different ratios of nitrogen to phosphate (N:P) on the basis of PEI (mol. weight 25000; 581 moles of nitrogen per mole) and DNA (3 nmol of phosphate on ug). After 24 h (except where specified a shorter time of transfection) solution for transfection was removed, cells were washed and added 1 ml dnim phosphate saline solution (SFR) and literally Lisinym buffer (0.1 M Tris-HCl, 0,5% Triton X-100 (Sigma, St. Louis, MO) and analyzed for the expression-galactosidase or luciferase.
To determine the optimal proportions of nitrogen polyethylenimine to phosphate DNA (N:P) and the optimal concentration of nitrogen polyethylenimine for applications in vitro in the presence of serum was developed the following Protocol: 1) complexes of PEI-DNA were prepared in the range of ratios of N:P and gave them incubated at room temperature for 15-20 minutes before use; 2) cells were sown at 105cells per well in 24-hole format, and incubated at 37°C overnight in 1 ml of RPMI medium 1640, supplemented with 10% FCS; 3) then the volume of medium in each well was adjusted to obtain the desired final concentration of PEI nitrogen; 4) a constant volume of solution of PEI-DNA was added to each well for each specific ratio of N:P; 5) the plates were incubated at 37°C overnight and examined for expression of GFP or analyzed for the expression of luciferase.
The effect of serum surfactant and fluid bronchoalveolar lavage on the PEI transfection in vitro
Cells A were sown and transfusional, as described above, except that instead of dilution of the formed complexes coppernose-active substance or fluid bronchoalveolar lavage). Fetal calf serum (FCS) was obtained from HyClone Laboratories, Inc. (Logan, UT), mouse and human serum were obtained from Gibco-BRL and synthetic surfactant (Exosurf, containing mainly collateralised at a concentration of 81 mg/ml) was obtained from Burroughs Welcome Co. (Research Triangle, N. C.). Fluid bronchoalveolar lavage (BAL) were obtained from untreated mice, which were killed by penthrane anaesthesia and were bled through the abdominal aorta. The trachea was surgically exposed and Coulibaly tube RE (outer diameter 0,965 mm, Clay Adams). Using the total volume of 2.0 ml of physiological saline were lung lavage 5 times volume of approximately 1.0 ml (volume of 1.0 ml of the same liquid is pumped into the lungs and pumped from the lungs to 5 times using a syringe 3 ml, containing only 2 ml). The output was usually 85% from 2 ml of lavage fluid. United BAL fluid from multiple mice then liofilizirovanny and reconstituirea at various concentrations for analysis of transfection.
Quantitative analysis of the efficiency of transfection in vitro
After cell lysis, the protein concentration was determined using BSA-protein analysis from Pierce (Rockford, IL).-galactosidase activity is galactopyranoside (CPRG; Boehringer Mannheim, Indianapolis, IN) in the format of a 96-hole of the tablet and read the results on the tablet reader for microtiter tablets Dynatech MR5000 (Dynatech Laboratories, Inc., Chantilly, VA).
Tests luciferase was performed with commercial reagents according to the manufacturer's instructions (Promega, Madison, WI). For the expression of luciferase in cell cultures, the medium was removed and to each well was added 1 ml lisanova buffer (Promega, Madison, WI). After incubation tablet for 20 min at room temperature on a rotary shaker lysates were analyzed by mixing 1-10 ál of the lysate with 50 μl of the substrate and measuring light emission in 15 seconds in a luminometer TD-20C (Turner Designs, Sunnyvale, CA).
Functional stability of sputtered complexes of PEI-DNA and liposome-DNA
Prepared RE-DNA and lipid-DNA was obtained by dilution of DNA up to 80 μg/ml in 2.5 ml WFI, adding an appropriate amount of lipid in 2.5 ml WFI to DNA or an appropriate amount of DNA to PEI in SFR and inquira at room temperature for 15-30 minutes before spraying. At specified time intervals aliquots 50-75 μl was removed from the tank atomizer and prepared for transfection in vitro. pCMVused as a reporter gene in all of these Issac described above. It should be noted that in order jet nebulizers produce aerosols of fine particles, they must be continuously subjected to the fluid recirculation, since only a small fraction of the liquid spray (less than 1%) is released in the form of an aerosol with each passage through the nozzle of the sprayer. Large particles lipid-DNA (>400) is quickly processed to smaller sizes, so within a short period of time, the particle size of the lipid-DNA in the tank atomizer becomes very similar to the size of aerosol particles retrieved from the aerosol device All Glass Impinger (AGI-4, ACE Glass Co., Vineland, NJ)).
Dispenser with two nozzles Puritan Bennet 1600 twin jet nebulizer (Carlsbad, CA) used in this study was modified by removing one tube of one of the two injectors and was named sprayer with a single nozzle Puritan Bennet 1600 single jet (PB sj). PB sj worked at the speed of a current of 15 l/min, unless otherwise specified.
Transfection in vivo using complexes of PEI-DNA and liposome-DNA using instillation.
Composition PEI-DNA and lipid-DNA was obtained as described here, except that the DNA concentration was increased to 6 μg in 40 μl (after adding the appropriate concentration of liposomes or debapriya of random incomplete instillation (animals sometimes throw part instaliranog material) each animal was instillirovti a total of four times: once in the morning and once in the afternoon in each of two consecutive days using the same amount of DNA. Animals were slightly anestesiologi the penthrane just before the introduction of the composition. While holding the animal straight taking in the neck 40 µl was slowly introduced into one nostril, where the fluid ingaliovanyi. Animals were kept in a vertical position within a short period of time, to give instalirovany fluid to reach the lung. The effectiveness of pulmonary delivery using this approach was confirmed using radioactively labeled liposomal drugs, and most of this material is found in the lungs after a few minutes. Animals do not show visible signs of physiological deterioration or disturbance as a result of this treatment, and there was no obvious pathological consequences at the macroscopic examination of the lungs and other organs. Because previous work has shown that the optimal time for the expression of instalirovany reporter genes was 1-2 days after installation, the animals were killed at 48 h after the first installation.
An EXAMPLE IS TO receive, as described here, except that the DNA concentration was increased to 2 mcg per 20 ml (after adding the appropriate concentration of liposomes or polyethylenimine) for the single-dose dispenser. If there are no other indications, spray PB sj worked at the speed of a current of 8 l/min Coming out of the spray stream is passed through a sealed plastic cage(13×17×30 cm), which contained mice (usually 6-8). The air went out of the cell through the HEPA filter, and the entire device was placed in a laminar box, vented through additional filtration HEPA outwards. To minimize the use of aerosol spray worked under automatic control for only 1 min every 10 min After consumption of the first 20 ml of the suspension nebulizer has added an additional 20 ml of the same concentration. Exposure exposure required approximately 12 hours, during this time, mice were provided food and water and allowed to move freely in the cage.
Quantitative analysis of gene expression in vivo
It was found that the expression of CAT is more sensitive way of measuring the efficiency of transfection in vivo than expression-galactosidase, which dulali at appropriate time points (48 h, if there are no other indications) and the lungs and trachea were removed and frozen in liquid nitrogen. Later these tissues were crushed in Lisina buffer and homogenized using a homogenizer with beads Wig-L-Bug (Crescent Dental Mfg., Lyons, IL). To determine transfection nose nose section of the skull of the mouse cut out from the rest of the skull and frozen in liquid nitrogen. Frozen bow sections were crushed into powder and then subjected to homogenization in Lisina buffer using a homogenizer with beads Wig-L-Bug. The tissue extracts were analyzed on a content CAT using a commercially available ELISA kit for CAT (Boehringer Mannheim Grobh, Germany). The results obtained using ELISA, were equivalent to or better than the results obtained using conventional methods TLC and methods with separation of the organic phase. Due to the variability observed in the case of virtually all studies with aerosols in vivo, experimental and control groups usually consisted of 6-8 animals.
For analysis of in vivo expression of luciferase after instillation or aerosol delivery, animals were killed with a lethal dose of anesthesia, was eviscerated with a transverse incision of the abdominal aorta and collected light and La analysis of luciferase using a glass tissue grinder with conical base (Kontes Duall 23). An aliquot of 10 µl of the homogenate was added to 50 μl of the substrate and measured the emission of light.
Groups of 5 mice were subjected to pCMV-hGH combined with PEI (the ratio of N:P 10), by aerosol or the effects of intranasal plasmids. As a positive control group of five mice received empty plasmid (50 μg in 50 μl SFR) in the form of intramuscular injections in the front of the tibia (tibial) muscle. Serial serum samples were obtained at two-week intervals and were analyzed using ELISA for the total specific antibodies to human growth hormone (purified protein hGH was obtained from Cal-Biochem, San Diego, CA). A pool of sera from non-immunized mice served as negative control in each ELISA.
Optimization of in vitro ratios and concentrations of the reacting substances RE-DNA.
Due to the importance of the ratio of the reacting substances for the efficiency of transfection of most of the compositions of the polycation-DNA, the relationship between the ratio of PEI-DNA and transfection was investigated in vitro using cells A in the absence of serum. As shown in Fig. 1A, the ratio of nitrogen polyethylenimine to phosphate DNA (N:P) of approximately 5 is ressie-galactosidase. Transfection decreases rapidly when the ratio is below 4 and decreases as this ratio increases above 5. Very similar results were obtained with compositions PEI-luciferase (Fig. 1B). In the study of the action of these correlations on the levels of protein in the cell extracts from the same experiments (which roughly correlates with the density of cells) observed low toxicity or no toxicity at the ratios of less than 8 (Fig. 1C). Reduction of protein levels for groups treated with higher ratios may indicate cellular toxicity.
Because transfection in vivo must occur in the presence of proteins of the extracellular fluid and due to the above-mentioned toxicity, investigated the effect on the transfection efficiency as the concentration of polyethylenimine, and the ratio of N:P using cells A in culture medium containing 10% fetal calf serum. Fig. 2 shows that the ratio of N:P of 16 with a concentration of PEI 50-60 nmol/ml gives the highest degree of transfection, indicating that as the concentration of polyethylenimine, and the ratio of polyethylenimine to DNA strongly affects the efficiency of transfection in the presence of serum proteins. Persons who, the eat in its absence (Fig. 1B). The overall efficiency of transfection with optimal conditions in the presence of serum was indeed higher than in its absence. The same Protocol optimization used in other cells, which indicates that this is a relatively simple way, suitable for execution on the individual requirements of transfection with PEI for a particular application.
Action whey and surfactants on the efficiency of transfection in vitro
Although previous reports have shown that whey proteins tend to inhibit the transfection mediated by different cationic lipid and polycation, the enhancement observed here, in optimal conditions, the concentration of polyethylenimine and the ratio of N:P, led to the study of the action of serum on transfection efficiency using polyethylenimine. At test concentrations of fetal calf serum from 0 to 50%, it was obvious that the concentration of 5-20% increased transfection in vitro, whereas higher concentration (50%) led to reduced transfection (Fig. 3). Inhibition associated with high serum concentrations, could partially be overcome by increasing concentrations of PEI:DNA. Suede, both resulted in a similar increase transfection, whereas mouse serum did not show action. Rabbit serum also leads to increased PEI-mediated transfection. Action whey, it seems, is thermolabile with complete loss of reinforcing activity observed when heated to 95°C for 10 min, while heating for 30 min at 56°C in subsequent experiments did not reinforcing actions.
Also investigated the effect of pulmonary surfactant on PEI mediated transfection. This is performed bronchoalveolar lavage fluid of mice (dried and re-hydrated with increasing concentrations) and synthetic surfactant (Exosurf) in increasing concentrations. The cells were subjected to concentrations of BAL-fluid equivalent of 6.25-12.5% concentrations deliverydiovan BAL-fluid (concentrated BAL fluid was added to each well, 0.5-10% of the total volume of liquid in each well, respectively). The cells were subjected to concentrations of Exosurf equivalent of 4.05-81 µg/ml collaterality, the active agent in Exosurf. As shown in Fig. 4, higher concentrations of both BAL fluid and Exosurf premoderatsii surfactant, seems to have little effect.
The effect of sputtering on the efficiency of transfection in vitro complexes liposome-DNA and PEI-DNA
Preparations of complexes of PEI-DNA at ratios of N:P 5 and 10 were tested for transfection efficiency for 10-minute period of spraying. In one experiment, the composition PEI-DNA (N:P=10) resulted in an average of 96%, 94% and 89.6% of the holding activity transfection control (not subjected to sputtering) of the material in the test in vitro using cells A lung tumor person. Aliquots cyclically sprayed by the spray material is removed from a tank atomizer (with an initial volume of 5 ml) at intervals of 3 minutes For comparison, drugs PEI-DNA, drug DC-cholesterol:D:V(2:2:1) and the drug DMRIE:DOPE:pCMV(3:3:1) was sprayed to dryness (approximately 12 min) (Fig. 5). Compositions DC-cholesterol:D and DMRIE:DOPE used here were optimized for transfection in vitro cell A and used the ratio of DC-cholesterol:D similar to the ratio reported elsewhere for optimal transfection cell culture (Gao &Huang, 1991). Watched only a small loss of activitylost 60% of its activity for 3 min and was inactive after that. The activity of DMRIE:DOPE:pCMVdecreased by more than 70% in 3 min and approximately 90% after 6 minutes At higher speeds, power sprayer loss of efficiency of transfection with compositions containing other cationic lipids (Schwarz et al., 1996), is even higher. Interestingly, the drug with N:P=5 PEI-pCMVshowed a marked increase in the efficiency of transfection with increasing sputtering, which may reflect the degradation of DNA and thereby the effective increase in the ratio N:P.
Comparison of the efficiency of transfection in vivo complexes liposome-DNA and PEI-DNA after intranasal instillation
When the test compositions PEI-pCMV-HiCAT using intranasal instillation in vivo in BALB/c mice (Fig. 6A), it was found that the expression of CAT originating from PEI-DNA was 10-20 times higher in the lungs than those obtained with the use of BGTC:DOPE:pCMV-HiCAT, the most stable and effective compositions with cationic lipid for transfection in vitro up to the present time. Apparently mediated by PEI Express the ASU. In contrast, expression mediated installed compositions based on cationic lipids was higher in the nose than in the lungs. Similar data were obtained when animals when instaliranje complexes PEI-luciferase or BGTC:DOPE-luciferase (Fig. 6B).
Intranasal installation was also used to compare the efficiencies of transfection in vivo of various compositions PEI-pCMV-HiCAT. When tested ratios of N:P 5-20 it was found that the ratio of 10-20 resulted in comparable levels of CAT expression in vivo. However, when increasing the concentration of PEI-DNA (at a ratio of N:P 10) several times, thereby allowing delivery 3 times higher doses of DNA in the same intelliroom volume were observed an additional increase in the expression of the reporter gene.
Comparison of the efficiency of transfection in vivo complexes liposome-DNA and PEI-DNA after aerosol delivery.
Upon delivery of the compositions PEI-pCMV-HiCAT and BGTC:DOPE:pCMV-HiCAT (using identical concentrations of DNA) by sputtering polyethylenimine was 10 times better vector for expression in the lungs than the lipid (Fig. 7), which was described above confirmed the findings using the installation.
Also in agreement with the findings of the first transfection in lung, than in the nose, for compositions based on PEI, whereas aerotolerance composition BGTC:DOPE:pCMVresulted in comparable levels of expression in the nose and lungs. Aerosol delivery compositions luciferase-based PEI again led to similar ndings concerning their relative efficiencies in the lung and nose.
Genetic immunization and aerosol installationin the introduction of PEI-DNA vaccines
One potentially valuable applications for aerosol injection expressing vectors is the delivery of DNA vaccines. To determine, could in this way be achieved immunization, drug pCMV-hGH plasmid-PEI ratio (N:P 10) was administered via aerosol and nasal installation and, as positive control, were intramuscularly injected with naked DNA. As shown in Fig. 8, these treatments induced high levels of serum antibodies to human growth hormone in all groups. A significant humoral immune response (antibodies) was present at 2 weeks, and he went out on a plateau between 4 and 8 weeks. In Fig. 8 optical density is shown at a dilution of 1:500 serum in comparison with the background OD of 0.1 in unimmunized animals. Average titers Arup respectively. However, as shown in Fig. 8, all groups had clearly large standard deviation, similar to previous results with intramuscular injections. Thus, although the response of individual mice were vysokopribylnymi, persistence of antibodies within 8 weeks of a single injection of plasmid-RE suggests that this method of immunization may have significant application in the provision intranasal or by aerosol.
Efficient delivery of genetic material into the lungs and the respiratory tract is targeted and non-invasive approach to the treatment of a wide range of pulmonary disorders. However, transfection of pulmonary sites was usually ineffective when using non-viral vector for DNA delivery to minimize pathological and immune risks associated with viral delivery. It was caused, in part, by the inaccessibility of the lung environment for delivery of drugs or genes. In addition, the loss of activity associated with jet spray, limited the value of this methodology for DNA delivery to the lung targets (Crook et al., 1996; Schwarz et al., 1996). This study found that complexes of DNA with poly-polymer-polyethylenimine are beings who in gene delivery into the lung.
Other non-lipid poly-polymers that are used as vectors for DNA delivery include poly-L-lysine, which mediates only a low degree of transfection, but is significantly improved by conjugation with agents or activating agents to facilitate cellular uptake (Wu &Wu, 1988; Tang & Szoka, 1997), and dendrimers polyamidoamine (Haensler & Szoka, 1993). In addition, it was shown that modified forms PEI covalently conjugated with ligands, such as transferrin (Kircheis et al., 1997; Ogris et al., 1998; Ogris et al., 1999), target specific cell types with a high degree of specificity. There were reports of PEI mediated transfection in lung with the systemic administration of compositions (Goula et al., 1998), but to date little attention has been paid to the use of non-lipid polymers as DNA vectors for aerosol delivery. These discoveries show that compositions based on PEI give a significant degree of stability of plasmid DNA during the process of spray atomization. In the case of some kompozizii on the basis of PEI, transfection, apparently, even enhanced by the process of sputtering. Thus, the PEI is, apparently, a more effective mediator transfection in it is relatively non-toxic. The fact that polyethylenimine can be easily anywhereman with site-specific ligands increases the versatility delivered by aerosol compositions based on PEI.
Strengthening PEI mediated transfection thermo-labile serum factor (factors) may be a promising, although a mechanism (s) involved or the consequences for gene delivery in vivo are unknown at present. The fact that this effect is lost when heated to 100°C, is an argument against the possibility that it is associated with a small heat-stable factors, such as steroids. The fact that this strengthening is not valid temperature 56°C, is an argument against a role for the complement system. The identification of this increase transfection factor will be eventually useful in improving the overall efficiency of gene delivery in vivo.
Various regional specificity (lung or nose) polyethylenimine and cationic lipid (BGTC:DOPE), marked by the use of two individual reporter genes is unexpected discovery. Assuming that the size of the particles delivered from the same nozzle are similar, tion of the epithelium, or may be due to differences in cell response in natalina and pulmonary sigah. This discovery could be important in applications in which nasal expression of the delivered gene may have unintended consequences. On the other hand, these different delivery vectors may be suitable for different clinical applications. In the field of genetic immunization, discovery, reported here, suggest that persistent humoral responses (formation of antibodies) can be achieved by a single introduction of the plasmid using methods that deliver complex RE-DNA in lung tissue.
Because the drugs PEI-DNA can be effectively deposited in the periphery of the lung by aerosol (Weibel generations, 17-23), mediated RE transfection of respiratory epithelium in vivo is subjected to plasmid DNA and PEI effects of endogenous surfactants and proteins in the alveolar space. The actions of surfactants on PEI mediated transfection was investigated in vitro using both synthetic surfactant (Exosurf), and bronchoalveolar lavage fluid obtained from untreated mice. Although both of these substances really led to dependent on the concentration inhibition PEI mediated transfection, action surfactants may be relatively lower than the texts say, what tracheal insufflate (inhalation) plasmid DNA and complexes of plasmid DNA-cationic liposome resulted in similar amounts of gene expression in the lung. A much higher expression level in the lung is achieved with PEI-pCMV-HiCAT than only with DNA plasmid during installation that provides evidence that pulmonary surfactants do not offset the properties of gene transfer of polyethylenimine to the extent to which they do this cationic lipid compositions of cationic lipid:co-lipid. Thus, compositions based on PEI are a promising alternative is relatively weak cationic lipids as non-viral carriers for the treatment of various pulmonary disorders by aerosol. Results presented here also show perspective in relation to the delivery of genetic vaccines.
In the following experiments we used female mice BALB/c mice (aged 5-7 weeks).
Bacterial gene chloramphenicolchloramphenicol (R, reference 15) is used mainly as a reporter gene to measure the expression of the transgene. This CAT gene is under the control of the early promotiona embedding a promoter/enhancer element CMV and the polyadenylation sequence of the human growth hormone, was obtained from Dr. Michael Barry (Center for Cell and Gene Therapy, Baylor). All plasmids were purified on Qiagen columns (Qiagen, Valencia, CA), and they did not contain endotoxin. Plasmids were quantified by absorption in the UV at 260 nm. Analysis on agarose gel revealed that these plasmids are a mixture of mainly superspiritual plasmids with a small amount of broken plasmids.
Obtaining complexes of PEI-DNA
PEI (25 kDa branched) were purchased from Aldrich Chemical (Milwaukee, WI). The original solution polyethylenimine was prepared at a concentration of 4.3 mg/ml (0.1 M for nitrogen) in SFR, pH 7-7,5. PEI and DNA were mixed separately in 5 ml of water at required concentrations. The PEI solution was slowly stirred on the vortex and to it was added to the DNA solution to a final volume of 10 ml of the Mixture was allowed to stand at room temperature for about 15-20 minutes before spraying. The obtained ratio of the charge were expressed as the ratio of PEI nitrogen:phosphate DNA (N:P), which can be calculated by taking into account the fact that DNA has 3 nmol of phosphate at the mcg, and 1 μl of 0.1 M solution of PEI has 100 nmol amine. of nitrogen. A ratio of 10:1 N:P corresponds 1,29:1 PEI:DNA.
Aerosol delivery of PEI complexes-DNA
Mice were placed in plastic cages, which zakhaev the Oia spray the entire body. Complexes of PEI-DNA was aerosolizable using spray Aerotech II (AT-II) (CIS-US, Inc., Bedford, MA) at a speed of DC 10 l/min using air or air containing 5% CO2. AT-II is a high-performance, efficient dispenser for which retrieves aerosol in the optimal range of 1-2 microns mass median aerodynamic diameter (MMAD) for peripheral pulmonary delivery (17, 18). It was estimated that the size of aerosol particles PEI-DNA was 1.6 ám MMAD with a geometric standard deviation (GSD) of 2.9, with the use of device Andersen Cascade Impactor (Andersen Instruments, Atlanta, GA) according to previously described method (18). The source of dry air (Aridyne 3500, Timeter, Lancaster, PA) was delivered into the gas mixer Bird 3M (Palm Springs, CA) connected to a gas compressor and container CO2. The mixture of air and CO2delivered in a spray bottle. The final concentration of 5% CO2in the air was determined using solution Fyrite (Bacharach, Pittsburgh, PA). Spraying 10 ml of solution was taken approximately 30 minutes
Mice were anestesiology and squashed after each time point and the lungs and other tissues were collected and immediately frozen. The test kit CAT ELISA kit (Boehringer Mannheim Gmbh, GermT using a homogenizer with beads Wig-L-Bug (Crescent Dental Mfg., Lyons, IL). After centrifugation of the homogenate 200 µl of the extract was used for analysis of CAT ELISA performed in the format of a 96-well plate. The absorption was read using a tablet reader for microtiter tablets (Molecular Devices, Sunnyvale, CA). He exposed mice were used as controls. The CAT activity was expressed in ng SAT/ml of tissue extract using the standard curve obtained with purified CAT enzyme. The sensitivity of this test was further strengthened in accordance with the suggestions of the manufacturer in such a way as to detect such low levels of CAT protein as 0.1 to 0.3 PG per well.
Analysis of luciferase
Mice were anestesiology and were killed and lungs were collected. Set for analysis of luciferase (Promega, Madison, WI) was used to measure the expression of luciferase. Light homogenized in 1 ml lisanova buffer for analysis of luciferase using a homogenizer with beads Wig-L-Bug. After centrifugation of the homogenate of 10 μl of extract was added to 50 μl of luciferase substrate and the luminescence was read within 10 s at 96-hole tablet on a luminometer (Microlumat LB 96 P, EG&G Berthold, Germany). He exposed mice were used as controls. Acts when using purified luciferase from Promega.
Histological analysis of tissue slices
Mice were anestesiologi with isoflurane and killed by exsanguination through the abdominal aorta. Lungs were isolated, were Coulibaly and recorded by filling in 10% neutral buffered formalin, embedded in paraffin and processed for histological analysis. Did small sections at 4 μm and observed under a microscope for observing any signs of inflammation or toxicity using staining with hematoxylin and eosin. After conducting a univariate analysis of variance (ANOVA) for comparison of these methods produced processing using t-test, Student (two-tailed unpaired Student's t-test). The differences were considered significant if p0,05.
Spraying complexes RE-DNA with 5% CO2
Spraying complexes RE-DNA with 5% CO2enhances transgene expression in the lung with comparison with normal air: inhalation of 5% CO2in the air was associated with an increase in respiratory air volume and respiratory rate in mice and humans (19, 20, 21). Inhalation of aerosols containing 5% CO2could lead to a higher inhalation aerosol particles and, consequently, a higher expression with cf who It was delivered to BALB/c mice by aerosol using either normal air, or air containing CO2. A fixed number of CAT plasmid (1 mg/10 ml solution) at a ratio of N:P 10:1 was aerosolizable for 30 min as indicated. Lungs were collected after 24 hours and SAT-analysis was performed to determine the extent of transfection. 5% CO2in the air leads to 3-fold increase (p=0.001) detectable levels of CAT, in comparison with the aerosol spray only air (Fig. 9).
The DNA transfer using PEI is dependent on dose
For additional optimization of transgene expression ratio of N:P was maintained constant at 10:1, and the amount of DNA varied from 250 μg to 4 mg per 10 ml aerotolerance solution. This leads to an increase in the concentration in the tank and the amount of total DNA, spray produced by the spray. Complexes were aerosolizable using 5% CO2in the air with 2 mg of DNA, providing the highest level of CAT expression in the lung (Fig. 10). The CAT levels measured with 250 µg DNA were not statistically different from control lungs (p=0.34). In addition, by dissolving 4 mg of DNA in 10 ml at a ratio of N:P 10:1 was observed by a visual precipitation of DNA, which may be responsible for the lack of further increase in the level of CAT, delenia reacting substances that would be perfect for aerosol delivery in vivo, different ratios of PEI-DNA (N:P) were evaluated for their ability to transliterate easy. The amount of DNA was maintained constant at 2 mg, and the concentration of polyethylenimine varied with obtaining ratios 5:1, 10:1, 12,5:1, 15:1, 17,5:1 and 20:1. These ratios were chosen based on preliminary studies in vitro and in vivo (with introduction by instillation). These complexes were aerosolizable using 5% CO2in the air. The ratio of N:P 15:1 gave the highest level of CAT expression in the lungs, while 5:1 resulted in very low levels of CAT expression (Fig. 11). There were no statistically differences between the ratios 10:1, 12,5:1, 17,5:1 and 20:1 (p>0,1), but there was a significant difference between 15:1 and 20:1 (p=0.05) and between 10:1 and 15:1 (p=0.014).
To determine the optimum ratio for plasmids other than the CAT, different ratios of N:P was tested for the expression of the luciferase gene in the lung. The estimated ratios were 5:1, 10:1, 15:1, 20:1, 30:1 and 40:1. The optimal curve for luciferase shifted to the right compared to the CAT, with the highest expression at 20:1 (p<0,05 in comparison with other relations) (Fig. 12). This implies that different plasmids may require different ratios N:P.
Temporary hut after a single aerosol delivery. This is important information for planning the treatment regimen for therapeutic research. Two mg CAT plasmids were aerosolizable using 5% CO2in the air in mice at two different ratios of N:P 15:1 and 10:1. Different time points investigated for a group of 10:1 were 1, 2, 3 and 6 days after exposure to the action of the spray. The lungs and other tissues were collected at different time points and immediately frozen. All tissues were analyzed at the same time after the last time point (6 days). For a group of 15:1 mice were killed on days 1, 3, 7 and 10 after aerosol treatment. Lungs were collected and frozen after each time point and CAT protein was analyzed after the last time point (day 10).
For both ratios of N:P CAT expression is highest at 24 hours and remains constant (statistically there is no difference between day 1 and day 3, p=0.4 for the ratio of 15:1 and p=0,12 for the ratio 10:1) for 3 days (figure 13A and 13B). The level of CAT is reduced to approximately 50% of maximal levels after a week, and significant levels are detected even after 10 days (p=0.001 compared with control).
Tissue distribution of transgene
Intravenous or intraperitoneal delivery victoriathe lead to systemic delivery of genes various tissues were collected from the same group of mice, which was used in the above experiment (group 10:1), and SAT-analysis was carried out after the last time point. The analyzed tissues were lung, liver, spleen, kidney, thymus, brain and blood. The level of CAT, detektirovanii in nulegacy tissues was very low and was not significantly different (p>0,1 for all tissues from control tissues (Fig. 14), which indicates that aerosol delivery complexes RE-DNA leads to highly specific pulmonary gene expression without the presence of systemic delivery.
Histological analysis did not show signs of inflammation
To determine whether aerosol delivery complexes RE-DNA to any type of toxicity or acute inflammation in this system, 2 mg CAT plasmids were combined in the form of a complex with PEI at a ratio of N:P 15:1 and the mice were subjected to aerosol for 30 min using 5% CO2in the air. Mice were killed after 24 h and the lungs were fixed in formalin and stained with hematoxylin and eosin. Light has not found any histological evidence of deviation from the norm, as shown in Fig. 15. The use of 5% CO2in and highly specific for lung.
Action of the composition PEI-anticancer gene in studies with animals
Fig. 15 and 16 show the steps compositions PEI-anticancer gene in studies with animals. Fig. 15 shows that the composition PEI and p53 PEI and the retinoblastoma gene significantly reduce tumor growth in models of melanoma animal with the introduction of spray twice a week for 4 weeks. Fig. 16 shows that the composition PEI and p53 PEI, p53 anti-cancer drugs 9-nitrocamptothecin (9-NC) significantly reduce tumor growth in a model of adenocarcinoma of the animal with the introduction of spray twice a week for 4 weeks. Fig. 17 shows that the composition PEI and p53 significantly reduce tumor growth in a model of pulmonary metastasis of human osteosarcoma naked mouse (SAOS-LM6) when aerosol delivery composition twice a week for 4 weeks. Aerosol therapy was started 8 weeks after implantation of animal tumor cells. In the same experiment, PEI separately and PEI in combination with genes CAT or IL-12 (as non-specific controls) did not lead to inhibition, significantly lower than the untreated control.
In this study, the use of 5% CO2mi, achievable using conventional air during spraying. This could be caused by increased respiratory minute volume in the presence of 5% CO2. The use of 5% CO2in the air in the aerosol leads to a 4-5-fold deposition of particles of the drug - liposome in the lungs of rodents. When using 5% CO2in the air for delivery of the aerosol PEI-DNA could be observed visually that the mouse was breathing deeper and faster, suggesting that increased expression of the transgene in the lung could be due to increased deposition of aerosol particles. However, it is possible that elevated CO2influenced the efficiency of transfection complexes PEI-DNA by changing some other physiological parameters. Based on these observations, 5% CO2in the air could be used for aerosol delivery complexes other polymer-DNA or cationic lipid-DNA and with similar results. 5% CO2was well tolerated by the people, and shows that it increases minute volume, so this strategy could potentially be used for patients with lung diseases.
Other parameters for aerosol gene delivery using PEI in quality is Ino charged DNA is an important factor determining the transfection efficiency of the complex. Shipping genes aerosol could require different conditions. The ratio of N:P 15:1 gave the highest levels of CAT expression in this system, with a 5:1 ratio led to a very low level of expression in comparison with other ratios.
In contrast, higher expression for luciferase was obtained when the ratio of N:P 20:1. This could be due to the different size of luciferase plasmid, resulting in structurally characterized complex with PEI in comparison with SAT-plasmid. This could be due to differences in purity of the plasmid and share superspiritual patterns. Still, there is considerable overlap in the optimal ratios of N:P of these two plasmids. The optimum ratio for a variety of plasmids may be different. With regard to experimental variability ratio between 10:1 and 20:1 should work properly. A ratio below 10:1 did not give a very high transfection in lung.
Curve dose-response expression of CAT shows that 2 mg of DNA in complex with PEI, in 10 ml aerotolerance solution gives the best expression of this CO2-enhanced delivery system. Ek is akademiese in complex with PEI (with numerous different ratios), cause some precipitation of the DNA, and this, perhaps, is the reason for the low transfection. It should be noted that there is an increase both in concentration and in the number of delivered DNA.
It was estimated that sprayed the exit stream from the spray Aerotech II was approximately 80%. About 72% of the tank's DNA was delivered in an inhalation chamber, as determined using an all-glass instrument AGI, as described above. The remainder was located in the T-connector and tubing. Taking into account the mouse artificial nasal breathing, pulmonary physiology (cardiac output and deposited fraction) and the output concentration of the aerosol (4,8 mg/l) has been calculated that the amount of DNA otlichayushiesya in the lungs of mice, approximately 4-5 mcg during the 30 min exposure to the impact of aerosol (for the initial concentration of the tank 2 mg DNA/10 ml solution). These calculations are based on the inhalation of normal air, and the deposition may be higher in the presence of 5% CO2in the air due to the increase in tidal volume and breathing frequency.
The expression of CAT was also used to monitor the time course of gene expression. After a single exposure wodnych levels after one week. Was a very significant amount of expression (30% of maximum levels) even after 10 days. This suggests that the stability of gene expression after aerosol delivery using polyethylenimine may be more than adequate for various clinical applications. Stability of gene expression up to day 10 in this study is similar to or higher than reported in other studies using cationic lipids for instillation or aerosol gene delivery.
Information about the tissue distribution indicates that gene expression after aerosol delivery in this system is limited by light, which indicates the minimum system delivery. In contrast to the lung, tissues such as liver, spleen and kidney, which usually discover the detected levels of expression when delivered by intravenous or intraperitoneal administration, showed low expression or absence of detectable expression of the CAT when delivered by aerosol PEI-DNA. It is important, if the expression of the gene of interest is limited to the lungs. Aerosol delivery can distribute these particles are non-invasive and uniformly on all light.
P is bluedale infiltration of inflammatory cells or lung damage when examining thin slices. Although high levels of expression are detected even after a week, some treatments may require repeated and frequent gene delivery.
5% CO2in the air can increase the levels of gene expression when the gene delivery by aerosol. The expression is predominant in a lightweight, and highly significant levels detected even a week after aerosol delivery. PEI is, apparently, also non-toxic at concentrations optimal for expression in vivo. Aerosol delivery complexes extensive polyethylenimine-DNA is a low-cost alternative delivery of genes mediated by viral vectors and vector-based cationic lipids, and should be useful for diseases such as cystic fibrosis and lung cancer.
In the description cited the following links:
Boussif, O., et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7297-7301.
Boussif, O., et al. (1996) Gene Therapy 3, 1074-1080.
Crook K., et al. (1996) Gene Therapy 3, 834-839.
Duncan, J. E., et al. (1997) Human Gene Therapy 8, 431-438.
Eastman, S. J., et al. (1997) Human Gene Therapy 8, 765-773.
Eastman, S. J., et al. (1998) HUMAN Gene Therapy 9, 43-52.
Eastman J, et al. (1997) Human Gene Therapy 8, 313-322.
Gao, X., &Huang, L. (1991) Biochem. Biophys. Res. Comm. 179, 280-285.
Goula, D., et al. (1998) Gene Therapy 5, 1291-1295.
Haensler, J. & Szoka, F. C. (1993) Bioconj Chem. 4:372-379.
Kircheis, R., et al. (1997) Gene Therapy 4, 409-418.
Ogris, (1997) Gene Therapy 4, 823-832.
Tsan, M.-F., et al. (1997) Human Gene Therapy 8, 817-825.
Vigneron, et al. (1996) Proc. Natl. Acad. Sci. 93, 9682-9686.
Any patents or publications mentioned in this description correspond to the level of the person skilled in the art to which this invention. In addition, these patents and publications is incorporated herein as references to the same extent as if it had been specified that each individual publication is incorporated herein by reference.
The person skilled in the art will understand that the invention is well adapted to achieve the above goals and obtain the above results and benefits, as well as the objectives, results and advantages inherent in this invention. These examples together with the described methods, procedures, treatments, molecules, and specific compounds are representative of presently preferred variant, introduced as examples and are not intended to restrict the scope of the present invention. Changes in these and other applications, are obvious to experts in the area covered by the idea of the present invention defined by the framework of the attached claims.
1. Sposobem, in need of such treatment, comprising the stage of inhalation spray of small particles of specified genetic macromolecules, located in the complex with polyethylenimine in aqueous dispersions, in these Airways.
2. The method according to p. 1, characterized in that the inhalation is an oral inhalation, nasal inhalation, or both oral and nasal inhalation.
3. The method according to p. 1, characterized in that the aerosol of small particles created by the process of sputtering.
4. The method according to p. 3, characterized in that the spraying is carried out by means of the jet of spray.
5. The method according to p. 1, characterized in that the specified genetic macromolecule selected from the group consisting of DNA, therapeutic gene, RNA, catalytically active nucleic acids, ribozymes, antisense oligonucleotides and modified nucleic acids.
6. The method according to p. 5, characterized in that therapeutic gene contains functionally related elements necessary for expression of the indicated gene.
7. The method according to p. 5, characterized in that therapeutic gene is selected from the group consisting of a regulatory gene transmem is on, encoding a cytokine, a gene encoding a tumor-specific antigen, the gene coding for specific infectious agent antigen, and the gene encoding the target antibody.
8. The method according to p. 5, wherein the final concentration of the specified therapeutic gene in the specified polyethylenimine no more than approximately 10 μg DNA/50 nmol nitrogen polyethylenimine/ml, or less than about 0.1 μg DNA/50 nmol nitrogen polyethylenimine/ml
9. The method according to p. 1, characterized in that the individual suffers from a disease selected from the group consisting of cystic fibrosis, asthma, lung cancer, esophageal cancer, colon cancer, leukemia, breast cancer, sarcoma and melanoma.
10. The method according to p. 1, characterized in that therapeutic gene, located in the complex with polyethylenimine, further comprises serum.
11. The method according to p. 1, characterized in that the specified polyethylenimine covalently anywhereman with cleocinonline ligand.
12. The method according to p. 11, characterized in that the glue-technopolitical ligand is transferrin.
13. The method according to p. 1, characterized in that the aerosol of small particles contains a mixture of up to 10% gas astiz, containing polyethylenimine and genetic macromolecule, located in the complex with the specified polyethylenimine.
15. The composition according to p. 14, characterized in that the aerosol of small particles created by sputtering using a jet spray.
16. The composition according to p. 14, characterized in that the specified genetic macromolecule selected from the group consisting of DNA, therapeutic gene, RNA, catalytically active nucleic acids, ribozymes, antisense oligonucleotides and modified nucleic acids.
17. The composition according to p. 14, characterized in that the specified genetic macromolecule is a DNA encoding a therapeutic gene.
18. The composition according to p. 17, characterized in that therapeutic gene contains functionally related elements necessary for expression.
19. The composition according to p. 17, characterized in that therapeutic gene is selected from the group consisting of a regulatory gene transmembrane conductance of cystic fibrosis, the gene encoding timedancing HSV, the gene encoding a tumor suppressor, a gene encoding a cytokine, a gene encoding a tumor-specific antigen, the gene coding for specific infecciones ecna concentration specified therapeutic gene in the specified polyethylenimine no more than approximately 10 μg DNA/50 nmol nitrogen polyethylenimine/ml, or less than about 0.1 μg DNA/50 nmol nitrogen polyethylenimine/ml
21. The composition according to p. 17, characterized in that the composition prepared in concentrations of approximately 0.1 μg DNA/20 nmol nitrogen polyethylenimine/ml - 10 μg DNA/150 nmol nitrogen polyethylenimine/ml
22. The composition according to p. 14, optionally containing serum.
23. The composition according to p. 22, characterized in that the specified polyethylenimine covalently anywhereman with cleocinonline ligand.
24. The composition according to p. 23, characterized in that the specified cleocinonline ligand is transferrin.