A DRY POWDER FORMULATION FOR GENE THERAPY
BACKGROUND OF THE INVENTION
Gene therapy is rapidly becoming a new therapeutic modality in experimental and clinical medicine. It is desirable that a pharmaceutical formulation, containing DNA or RNA with the transgene sequence, be prepared in large quantity with pharmaceutical quality. The currently used non-viral vectors for gene therapy are mostly two-vial formulations which are mixed at the bedside prior to administration. Such an admixture formulation is acceptable but not ideal. Furthermore, the two-vial formulations are usually dilute in their concentrations of DNA, RNA, oligonucelotide or protein, necessitating the administration of solutions of relatively large volume. A highly concentrated, stable, one-vial formulation is thus more desirable than the current formulations. We describe here the composition and the method of preparation of a dry powder formulation suitable for topical and airway administration for gene therapy and other purposes. When reconstituted in an aqueous medium, it can also be administered in liquid form.
1. Field of the Invention The present invention relates to methods and compositions suitable for gene transfer or
delivery of gene therapy. More particularly, it relates to pharmaceutical compositions in which DNA, RNA, oligonucleotides and proteins can be complexed with lipid and delivered in a concentrated and convenient form, either as a lyophilized powder or in liquid form after reconstitution in a suitable aqueous medium.
2. Background Information
The field of gene therapy/gene transfer is currently advancing along a number of fronts, both experimentally and clinically. Genetic material has been transferred using viral vectors, for example adenoviruses and retroviruses, as well as nonviral methods such as microinjection, calcium phosphate coprecipitation, and transfer via liposomes. (For a comprehensive review, see Morgan and Anderson, 1993.) A number of diseases and disorders are potential candidates for some type of gene therapy, if appropriate techniques can be found to deliver genetic material to target tissues and cells. In a few cases, (e.g. Cystic Fibrosis, Familial Hypercholesterolemia and Severe Combined Immunodeficiency) gene therapy has advanced to the point of clinical trials. Cationic liposomes have been shown to be an efficient means of transfecting a wide variety of mammalian cells. Their advantages include simplicity of preparation, ability to complex a very high percentage of DNA, versatility of use with virtually any type of naked DNA or RNA (with no theoretical size limitation) , the ability to transfect many different cell types, superior
transfection efficiency, and lack of immunogenicity or biohazardous activity.
Cationic liposomes composed of a mixture of DC-Choi and DOPE have been used to transfect the airway epithelial cells with CFTR cDNA in CF knock-out mice (Hyde et al. , 1993/ Alton et al., 1993) . The same formulation has also been used in two phase I/II clinical trials for melanoma (Nabel et al., 1992) and for CF (Caplen et al., 1994). This formulation is generally regarded as effective and non-toxic and will become commercially available in the near future.
To date, however, DNA/liposome and RNA/liposome complexes have been administered in liquid form and consequently suffer from several disadvantages. Many are two-vial formulations that must be mixed shortly before administration, resulting in inconvenience in manufacture, shipping, storage and administration. In addition, it is difficult to obtain highly concentrated solutions of DNA and RNA using this technique. This may result in a need to administer large volumes of solution to achieve an optimal therapeutic effect.
SUMMARY OF THE INVENTION This invention provides a dry powder formulation which contains a lyophilized DNA/liposome complex, suitable for gene transfer or gene therapy. Lyophilized RNA/liposome, oligonucleotide/liposome and protein/liposome complexes are also provided. The dry powder can be easily reconstituted with water and is active in gene transfer i vi tro and in vivo. It is suitable
for airway delivery as an aerosol or for topical administration as a spray. The potency of gene transfer of the dry powder was at least 50-fold higher than that of a liquid formulation of similar composition. In addition, the powder can be reconstituted for delivery in liquid form.
This invention further provides a method of preparing DNA, RNA, oligonucleotides or protein in powder form suitable for therapeutic use or gene transfer.
This invention also provides an effective and efficient method of delivering DNA, RNA, oligonucleotides or protein to tissues or cells.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. A dry powder formulation of the
DNA/liposome complex. 500 μg pUCCMVCAT DNA and 500 nmol (300 μg) of DC-Choi liposomes were mixed and allowed to complex. Lactose was added to a final concentration of 0.25 M. This solution was placed in a -80°C freezer for several hours, further frozen in liquid nitrogen, and freeze-dried for 24 hours. The resulting powder was triturated to reduce the particle size.
Figure 2. Gene transfer activity and toxicity of DNA/liposome complex reconstituted from a dry powder formulation. 2 mg pRSV-LUC DNA and 2 μmol (1.2 mg) of DC-Choi liposomes were mixed and allowed to complex. Dextrose was added to make a 0.25 M solution with a volume of 4 ml. This mixture was freeze-dried and reconstituted with 1 ml of distilled water. This reconstituted complex was
added to CHO cells and allowed to transfect for 44 hours. Luciferase activity (□) and protein content (♦) of the treated cells were assayed. The reconstituted DNA/liposome complex was active in gene transfer at concentrations which showed minimal toxicity, as indicated by the high protein content of the treated cells.
Figure 3. CAT activity in the treated mice. Balb/C mice weighing 20 grams were anesthetized with i.m. injections of ketamine. The dry powder formulation of the DNA-liposome complex (as shown in FIG. 1) was delivered via intra-tracheal administration. The mice were sacrificed after 40 hours and their lungs and trachea were assayed for CAT activity. Each number (la, lb) represents a single mouse. Lung samples (for mice 1-8) are noted as "a", while trachea samples are noted as "b" . Mice 1 and 2 were instilled with 500 μg of DNA as a fluid reconstituted from dry powder. Mice 3-7 were insufflated with maximally lOμg of DNA as a dry powder. Mouse 8 was an untreated control. Lanes 9 and 10 are a positive control (1 unit CAT) and a negative control (no enzyme) for the CAT reaction, respectively.
DETAILED DESCRIPTION OF THE INVENTION
MATERIALS AND METHODS
The following abbreviations are used in the specification: DC-Choi: 3β[N- (N' ,N' -dimethylaminoethane) -carbamoyl] cholesterol
DOPE: dioleoylphosphatidylethanolamine CAT: chloramphenicol acetyl transferase CF: cystic fibrosis
CFTR: cystic fibrosis transmembrane conductance regulator protein
CHO cells: Chinese Hamster ovary cells
Plasmid DNA
Plasmid DNA was prepared by a CsCl/EtBr gradient method (Sambrook et al. , 1989) . It was run on an agarose gel to test for purity and quantitated by using the A260/A2β0 ratio. Plasmid pUCCMVCAT was constructed in this lab by Dr. Hassan Farhood (see Farhood et al. , 1995) . It contains the E. coli chloramphenicol acetyl transferase (CAT) gene driven by the human cytomegalovirus early promoter and cloned into a pUC18 background. Plasmid pRSV-LUC contains a luciferase gene driven by the Rouse Sarcoma Virus promoter (De Wet et al. , 1987) .
Liposomes DC-Chol was synthesized by the method of
Gao and Huang (1991) . DOPE was purchased from Avanti Polar Lipids, Inc. DC-Chol and DOPE were mixed in a 3:2 molar ratio, evaporated in a stream of nitrogen to form a thin film, and vacuum desiccated to remove any residual chloroform. The lipid film was rehydrated in distilled deionized water which had been autoclaved. The liposomes were allowed to hydrate overnight. DC-Chol liposomes were prepared by microfluidization by using an M-110S microfluidizer (Microfluidics Corp.) to an average diameter below 200 nm. The liposomes were filtered
through a 0.2 μm filter to provide sterilization. The liposomes were diluted to a final concentration of 2 μmol/ml ( 1.2 mg/ml of total lipid). Other lipids known in the art will also be effective for these purposes, with cationic lipids being particularly suitable for complexing with DNA, RNA and other negatively charged compounds.
Examples in addition to DC-Chol include DOTMA, DOSPA, DMRIE, DOTAP, DOGS, and DDAB, which may be used alone or in combination with DOPE or other compounds.
Preparation of DNA/Liposome Complex and Dry Powder
500 μg of DNA and 500 nmol (300 μg) of DC-Chol/DOPE liposomes were mixed and allowed to complex. By complex we mean an association of molecules by noncovalent bonds, such as electrostatic, ionic, hydrophobic, hydrogen bonding and others. Complexation is generally complete in a relatively short time (95% within 10 minutes using the method described here) , but will occur at a slower rate in more dilute solutions. This parameter can be determined by routine experimentation by one of ordinary skill in the art, e.g. by changes in light scattering or other methods.
Lactose (or another sugar) was added to a final concentration of 0.25 M. This solution was placed in a -80°C freezer for several hours, further frozen in liquid nitrogen, and allowed to freeze-dry for 24 hours. The resulting powder was triturated to reduce the particle size. The terms "oligo- nucleotide/lipid complex", "DNA/lipid complex",
"RNA/lipid complex" , and "protein/lipid complex" refer to the respective compounds that have undergone the steps of complexing and freeze-drying, with or without the addition of a cryoprotective agent. A DNA/lipid complex, for example is a lyophilized DNA/liposome complex.
As will be evident, use of the present method is contemplated for DNA, RNA (both sense and antisense) , oligonucleotides, proteins and similar compounds which have all been shown to be deliverable by cationic liposomes (see, for example, Felger and Ringold, 1989 (for DNA); Malone et al. , 1989 (for RNA); Debs et al. , 1990 (for protein); Bennett et al., 1992, (for oligonucleotides). Any cryoprotective agent which can be freeze-dried into powder form (e.g. amino acids, urea) could also be used for the preparation of the powder.
Measurement of Particle Size
Freeze-dried DNA:DC-Chol liposomes were reconstituted to their original volume by addition of deionized water and shaking. The particle size was measured by using a Coulter N4 SD sub-micron particle analyzer, using a uni-modal analysis. The sample was counted for 200 seconds. The particle size of the DNA:DC-Chol liposome complex was between 150-200 nm, both prior to and after the freeze-drying process.
EXAMPLE 1
Transfection of Mammalian Cells In Vitro 2 mg of pRSV-LUC and 2 μmol (1.2 mg) of
DC-Chol liposomes were mixed and allowed to complex.
Dextrose was added to make a 0.25 M solution in a volume of 4 ml. This mixture was freeze-dried and reconstituted with 1 ml of distilled water.
The transfection was carried out in a 48 well plate in 0.5 ml as follows. An appropriate amount of the reconstituted complex was diluted into 0.5 ml of serum-free media (composed of 0.25 ml of Hanks Balanced Salt Solution and 0.25 ml of CHO-S- SFM media, both obtained from Gibco BRL) , added to CHO cells (obtained from the American Type Culture Collection) and allowed to remain on the cells for 6-7 hours. The serum-free medium was then removed and replaced with 0.5 ml per well of F-12 media (also from Gibco BRL) supplemented with 10% fetal bovine serum (from Hyclone Labs) , and the incubation was continued to a total time of 44 hours. For protein measurement, cells were harvested and extracted for protein which was quantitated by using a Coomassie Plus Protein Reagent (Pierce) assay.
Luciferase Assay
CHO cells were allowed to transfect for a total time of 44 hours. The media was aspirated and the cells were washed once with a 0.9% sodium chloride solution. The cells were lysed with 100 μl of Lysis Buffer (Promega) per well. The cell lysate was collected, briefly centrifuged, and 4 μl was used for the luciferase assay. Luciferase Assay Buffer (Promega) was thawed and 10 ml was used to reconstitute the Luciferase Assay Substrate (Promega) . The samples were loaded into an
AutoLumat LB953 (Berthold) luminometer, 100 μl of the luciferase substrate was added to the cell
lysate, and the relative light units of each sample were counted for 20 seconds. The raw data from each sample was normalized for protein content.
EXAMPLE 2 Airway Delivery of Dry Powder and Reconstituted DNA/Liposome Complex
Balb/C mice weighing 20 grams were anesthetized with i.m. injections of ketamine. A small incision was made into the trachea and a 1 ml Tuberculin syringe with a 21 gauge needle was inserted. The dry powder formulation of DNA:DC-Chol liposome complex was loaded into the syringe. A 1 ml pipet tip with a rubber bulb attached to the top of the pipet tip was attached to the top of the syringe and used to blow the powder into the trachea and lungs. This procedure was repeated several times and a rise in the chest of the mouse indicated a successful insufflation.
In the case of the reconstituted DNA/liposome complex, a small volume (75-100 μl) of distilled water was used to reconstitute 500 μg of the dry powder formulation. A small incision was made in the neck of the mouse to expose the trachea. The entire volume (encompassing 500 μg of the complex) was instilled over a period of one minute.
CAT Assay
The mice were sacrificed 40 hours after insufflation. The lungs and trachea from each mouse were removed, placed in an extraction buffer and homogenized. The sample was briefly centrifuged and
a sample of the supernatant was assayed for protein content.
The CAT assay using [1C] chloramphenicol as a substrate was done according to a modified method of Gorman et al. (1982) . A sample of the supernatant was mixed with the assay reaction mixture and incubated for several hours. Ethyl acetate (1 ml) was added to the reaction mixture, vortexed briefly, and 800 μl of the upper layer was taken and dried in a Speedvac for 1 hour. The sample was resuspended with 30 μl of ethyl acetate and then spotted onto a TLC plate. The plate was developed in a chloroform: methanol (95:5) solution and quantified by a phosphorimager.
Effect of Crvoprotectant on the Particle Size and Activity of DNA/Liposome Complex
It is important in some instances, particularly for storage purposes, to include in the formulation of the invention a sufficient amount of a cryoprotectant to permit the formulation to be freeze-dried without loss of activity. Four different sugars (dextrose, sucrose, lactose, and mannitol) were tested for their potential to serve as cryoprotectants. Their ability to maintain the original size of the liposomes and the appearance of the powder mass were used as criteria to select the most appropriate sugar. As shown in Table 1, the original size of the liposomes was 177 nm. Lactose, dextrose, and sucrose were able to maintain the original size of the liposomes, while mannitol was not. Lactose produced a fine white powder upon freeze-drying, while dextrose and sucrose produced a
crystalline substance. Lactose was chosen as the model sugar for the dry powder formulation, although it will be appreciated that other sugars could be usefully employed.
Table 1: Size of Reconstituted Liposomes3
Mean Diameter (nm)
Cryoprotectant Cryoprotectant Concentration (M)
0 0.1 0.25 0.5 0.75 1.0
Lactose 1450 252 158 128 Sucrose 171 205 181 156 244 Dextrose 168 273 180 301 397 Mannitol 765 875 985
a Liposomes consist of a 3:2 molar ratio of DC-Chol and DOPE in a concentration of 2 μmol/ml. They were prepared in water and microfluidized to produce liposomes. The mean diameter of freshly prepared liposomes was 177 nm.
b The mean diameter was measured by using a Coulter N4SD submicron particle size analyzer using a unimodal analysis. The liposomes were in a volume of 2 ml prior to lyophilization and were subsequently reconstituted with water back to the original volume of 2 ml.
The amount of cryoprotection used in the present formulation can be varied depending on other factors . A suitable amount of the sugar or other cryoprotectant should usually be used to protect the DNA and liposomes during lyophilization. The amount is generally sufficient if the average size of the vesicles is increased or decreased by less than about 50%, and ideally by less than about 20%, as measured by the described method. A cryoprotective substance is considered to be particularly suited to these purposes if, in addition, it produces a powder with bulk and flow properties such that it can be administered as a dry aerosol. Identification of such compounds and suitable amounts or concentrations can be done by routine experimentation by those of ordinary skill in the art.
A Dry Powder Formulation
A dry powder formulation prepared by lyophilizing a 1 ml solution containing 500 μg of DNA, 300 μg of DC-Chol liposomes and 0.25 M lactose is shown in FIG. 1. The powder was fine and flowed very well. It was stored in a desiccator at room temperature until use. Upon reconstitution by adding water, the DNA/liposome complex continued to be 150-200 nm in mean diameter and there was no flocculates or precipitates in the reconstituted solution. The transfection activity of the reconstituted complex was assayed with CHO cells by using complexes containing pRSVLUC DNA. The treated cells were assayed for luciferase activity two days after the
transfection. As shown in FIG. 2, cells could be effectively transfected to express the luciferase activity by the reconstituted complex in a dose dependent manner. Lack of cytotoxicity of the complex was evident because the treated cells contained nearly the same amount of protein as the untreated control (FIG. 2) . Thus, we conclude the dry powder formulation, after reconstitution with water, is stable and active in gene transfer for the mammalian cells.
Airway Delivery of the Dry Powder
Dry powder containing pUCCMVCAT plasmid DNA and DC-Chol liposomes was insufflated intra-tracheally into mice. Up to 10 μg of DNA was delivered into each animal. 100 μl of reconstituted complex (500 μg of DNA) from the same dry powder was also intra-tracheally injected as a comparison. The mice were sacrificed two days later and the major organs were removed. CAT activities of protein extracts from trachea and lung tissues of the mice were assayed by using a TLC method. As can be seen in FIG. 3, no CAT activity could be found in either the trachea or lung of the control mouse which had not been injected with any complex (lane 8) . Mice treated with dry powders (lanes 3-7) had expressed CAT activity in both trachea and lung tissues. Mice injected with the reconstituted complex also expressed CAT activity in both tissues (lanes 1 and 2) . It is important to note that mice treated with the dry powder received only 10 μg of DNA maximally, yet the level of transgene expression
was similar to those treated with 500 μg of DNA as a reconstituted fluid from the same dry powder (FIG. 3) . This remarkable and surprising result indicated that the dry powder formulation was at least 50-fold more potent in activity than the reconstituted fluid.
EXAMPLE 3
Treatment of Cystic Fibrosis
DNA which encodes human CFTR (prepared, e.g., as described by Caplen et al. , 1994 or
Caplen et al . , 1995) will be complexed to DC-Chol liposomes, combined with lactose, and lyophilized, as described. The resulting powder will be delivered to the airway of a CF patient using a conventional type of dry powder inhaler (see, e.g., Atkins et al. , 1992, Byron, 1990 and Hickey, 1992) . In addition to CF, patients with lung cancer and other pulmonary diseases could also be treated using this method with other forms of DNA, RNA, oligonucleotides or proteins. The DNA could contain a tumor suppressor gene for cancer treatment or an antiinflamatory gene for asthma, for example. Since the dry powder is stable it lends itself to being produced in a conventional dosage form. This dosage form is preferred because it would allow patients to self-medicate as each actuation of the inhaler could deliver a predetermined dose. A patient could be treated at home as needed, eliminating the need for hospitalization or medical visits.
DISCUSSION
Since the pioneering work of Feigner et al. (1987), a number of different cationic liposome/micelle formulations have been developed, some of which are commercially available. Our work in this area started with a study of structure-activity relationships in which about 20 different cholesterol derivatives were synthesized and screened for activity and toxicity in a 1:1 mixture with dioleoyl phosphatidylethanolamine
(DOPE) . A set of empirical rules on the structure of cationic cholesterol was derived for the best transfection activity, best shelf stability, and least cytotoxicity (Farhood et al. , 1992). DC-Chol was synthesized according to these rules and determined to be one of the best performing cationic lipids for transfection (Gao and Huang, 1991) . It is known that the best transfection activity of cationic liposomes in a serum-free medium occurs at the ratio of positive/negative charge slightly greater than 1 (Gao and Huang, 1991) . This is because the complex (with slightly excess positive charge) can readily bind to the negatively charged cell surface. However, most of the optimal DNA/liposome complexes lose a substantial amount of activity when serum proteins are present in the transfection medium. This is the primary reason why the cationic liposomes do not work well if injected i.v., despite some isolated reports of success (Zhu et al., 1993; Brigham et al. , 1991). The ratio of DNA to liposome for airway administration varies a great deal among different investigators and ranges from
8:1 (Logan et al. , 1995) to 1:6 (Alton et al. , 1993) by weight. The role of proteins and other negatively charged molecules in the lining fluid of airway epithelium must play a crucial role in determining the optimal ratio of DNA/liposome for transfection. In the case of solid tumor, we found that 1:0.6 (w/w) ratio of DNA/liposome was optimal if the complex is directly injected into the tumor for transfection (unpublished data) . No significant aggregation or precipitation of the
DNA/liposome complex occurs at this ratio even if the concentration is greater than 1 mg of DNA per ml. A lyophilized dry powder of the complex could be prepared by using 0.25 M lactose as a cryoprotectant (see FIG. 1) . Upon reconstitution, there is no change in particle size (150-200 nm in diameter) and the transfection activity is restored (FIG. 2) . In vivo gene transfer activity of this powder formulation was shown by intra-tracheal administration into mice. The activity was much greater than that of the reconstituted fluid from the same dry powder.
The dry powder will preferably be administered as an aerosol in a metered dose inhaler. The powder will be suspended in Freon or another suitable agent and packaged into a sealed vial mounted onto an inhaler. Other agents that might be suitable (for example, dichlorotetrafluoroethane and dichlorodifluoro- methane) are known in the art. A unit volume of the Freon suspension will be delivered to the airway by inhalation when the inhaler is triggered to dispense the suspension. Alternatively, the
19 powder can be loaded into a capsule which is then punctured by an inhaler to release the powder and blown into the airway. Such devices are well known in the medical arts (see, for example, Pharmaceutical Inhalation Aerosol Technology, J. Swarbrick, ed., Marcel Decker, New York, 1992, particularly at pp. 155-185 and 255-288; and Respira tory Drug Delivery, P.R. Byron, ed., CRC Press, Inc., Boca Raton, Florida, 1990, particularly at pp. 167-206; the entire contents of each are incorporated herein by reference) . The dry powder can also be administered topically to skin or mucosal tissues by a similar delivery device. The DNA/liposome complex can also be reconstituted from the dry powder and used as a liquid in cases where that would be a preferred route of administration. As the powder provides a stable product until reconstitution, any current route of drug administration can be used (oral, intravenous, intramuscular, intraperitoneal, subcutaneous, topical, aerosol, etc.)
While the invention has been described most fully in terms of formulations and uses for gene transfer or therapy, it is not intended to be limited to those uses. It will be clear to the skilled artisan that formulations can also be made from a variety of suitable proteins that might be advantageous to administer diagnostically, therapeutically or experimentally in the manner described. Advantages would include increased stability of the protein/lipid powder over other available forms of the protein, increased efficacy
of delivering either systemic or targeted therapy to the lungs. The invention could be used, for example, in such diverse applications as delivery of chemotherapeutic agents for treatment of lung cancer, or for systemic delivery of insulin in diabetics.
The references earlier mentioned are more fully identified hereafter, and are hereby incorporated by reference and relied upon:
Alton, E.W.F.W., Middleton, P.G., Caplen, N.J.,
Smith, S.N., Steel, D.M. , Munkonge F.M., Jeffrey, P.K., Geddes, D.M., Hart, S.L., Williamson, R., Fasold, K.I., Miller, A.D., Dickson, P., Stevenson, B.J., McLachlan, G., Dorin, J.R., and Porteous, D. (1993) Nature Genetics 5:135-142.
Atkins, P.J., Barker, Ν.P. and Mathisen, D. (1992) In: Pharmaceutical Inhalation Aerosol Technology (Swarbrick, J. , ed.) pp. 155-185, Marcel Decker, New York. Bennett, C.F., Chiang, M.Y. , Chan, H. , Shoemaker, J.E., and Mirabelli, C.K. (1992) Molec . Pharmacol . 41: 1023-1033.
Brigham, K.L., Meyrick, B., Christman, B., Magnuson, M. , King, G., and Berry, L.C., Jr. (1989) Am . J. Med. Sci . 298:278-281.
Byron, P.R. (1990) In: Respiratory Drug Delivery (Byron, P.R., ed.) , pp. 167-206, CRC Press, Inc., Boca Raton, Florida.
Caplen, N.J., Alton, E.W.F.W., Middleton, P.G., Dorin, J.R. , Stevenson, B.J., Gao, X., Durham,
S.R., Jeffery, P.K. , Hodson, M.F., Coutelle, C, Huang, L. , Porteous, D.J., Williamson, R., and Geddes, D.M. (1995) Nature Medicine 1:39-46.
Caplen, Ν.J., Gao, X., Hayes, P., Elaswarapu, R., Fisher, G., Kinrade, E., Chakera, A., Schorr, J. , Hughes, B., Dorin, J.R., Porteous, D.J., Alton, E.W.F.W., Geddes, D.M., Coutelle, C. , Williamson,
R. , Huang, L. , and Gilchrist, C. (1994) Gene Therapy 1:139-147.
Debs, R.J., Freedman, L.P., Edmunds, S., Gaensler, K.L., Duzgunes, N. and Yamamoto, K.R. (1990) J. Biol . Chem . 265:10189-10192.
De Wet, J.R., Wood, K.V., DeLuca, M. , Helinski, D.R. , and Subramani, S. (1987) Mol . Cell . Biol . 7:725-737.
Farhood, H., Bottega, R, Epand, R.M., and Huang, L. (1992) Biochim. Biophys . Acta 1111:239-246.
Farhood, H., Serbina, N. , and Huang, L. (1995) Biochim. Biophys . Acta, In Press.
Feigner, P.L., and Ringold, G.M. (1989) Nature 337:387-388. Feigner, P.L., Gadek, T.R., Holm, M., Roman, R. , Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., and Danielsen, M. (1987) Proc. Natl . Acad. Sci . USA 84:7413-7417
Gao, X., and Huang, L. (1991) Biochem. Biophys . Res . Comm. 179:280-285.
Gorman, CM., Moffat, L.F., and Howard, B.H. (1982) Mol . Cell . Biol . 2:1044-1051.
Hickey, A.J. (1992) In: Pharmaceutical Inhalation Aerosol Technology (Swarbrick, J. , ed.) pp. 167- 206, Marcel Decker, New York.
Hyde, S., Gill, D., Higgins, C.F., Trezise, A.E., MacVinish, L.J., Cuthbert, A.W., Ratcliff, R, Evans, M.J., and Colledge, W.H. (1993) Na ture 362:250-255. Logan, J.J., Bebok, Z., Walker, L.C., Peng, S.,
Feigner, P.L., Siegal, G.P., Frizzell, R.A. , Dong, J. , Howard, M. Matalon, S. Lindsey, J.R., DuVall, M., and Sorscher, E.J. (1995) Gene Therapy 2:38- 49. Malone, R.W. , Feigner, P.L., and Verma, I.M.
(1989) Proc. Natl . Acad. Sci . USA 86:6077-6081.
Morgan, R.A. , and Anderson, W.F. (1993) Ann. Rev. Biochem. 62:191-217.
Nabel, G.J., Chang, E.G., Nabel, E.G., Plautz, G., Fox, B.A., Huang, L., and Shu, S. (1992). Hum. Gene Ther. 3:399-410.
Sambrook, J., Fritsch, E.F., and Maniatis, T. In Molecular Cloning: A Laboratory Manual , Vol . 1 . , Cold Springs Harbor Laboratory Press, 1989.
Zhu, N., Ligitt, D., Liu. Y.. and Debs. R. (1993). Science 261:209-211.
While the invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not to be limited to the disclosed embodiment, but is intended to cover various modifications included within the spirit and scope of the appended claims.