Title:  Sonic nebulized nucleic acid/cationic liposome complexes and methods for pulmonary gene delivery

United States Patent:  6,271,206

Assignee:  Valentis, Inc. (The Woodlands, TX)

Filed:  September 11, 1997

Compositions and methods are provided for gene delivery to the respiratory tract. In particular, compositions comprising a nebulized nucleic acid/stabilizing agent complex, and methods employing such complexes for pulmonary gene delivery, are provided. Such complexes are preferably sonic nebulized.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention is generally directed to compositions and methods for pulmonary gene delivery. It has been found, within the context of the present invention, that a gene complexed with a stabilizing agent (such as a cationic lipid carrier) may be efficiently formulated into an aerosol and delivered to a patient through the use of sonic nebulization. Accordingly, the present invention is particularly directed to compositions comprising a sonic nebulized nucleic acid/stabilizing agent complex, to sonic nebulizers containing such compositions, and to the use of such compositions for the delivery of therapeutic nucleic acid drugs to the respiratory tract of a patient.

It has also been found, within the context of the present invention, that the charge of a nucleic acid/stabilizing agent complex is an important factor influencing gene transfer efficiency via nebulization or intratracheal instillation. More specifically, positively charged complexes result in higher levels of transgene expression than negatively charged complexes, and the efficiency improves with an increase in the positive charge. Accordingly, the present invention is also directed to compositions comprising a nucleic acid/stabilizing agent complex having a charge ratio selected to enhance gene transfer (typically a ratio (-:+) of at least 1:2). Preferably, such complexes are nebulized prior to administration to a patient.

As used herein, the term "nebulized" refers to a complex that has been treated by jet or ultrasonic nebulization to form an aerosol, as described herein. The term "sonic nebulized" refers to a complex that has been treated by ultrasonic nebulization. Any commercially available nebulizer may be used to prepare nebulized complexes. For the preparation of sonic nebulized complexes, an ultrasonic nebulizer such as the Omron Model NE-U07 (Omron Health Care, Inc., Lake View, Ill.) may be employed. An ultrasonic nebulizer uses a piezoelectric transducer that vibrates at very high frequency to induce waves in the reservoir solution. Interference of these waves at the surface of the liquid leads to the production of droplets as an aerosol (i.e., a suspension of colloidal particles), which is then transported by an airstream. This method produces aerosols containing particles in the respirable range (typically 1-5 .mu.m). Without wishing to be bound by any particular theory or mechanism of the present invention, it is believed that the high delivery efficiency of sonic nebulized nucleic acid/stabilizer complexes can be attributed to the fact that the sonic nebulizer operates in a passive mode and delivers aerosols as a bolus, only during inhalation, through a one way valve. This minimizes aerosol loss during exhalation.

While any nucleic acid molecule may be used in the preparation of nebulized nucleic acid/stabilizing agent complexes of the present invention, plasmid DNA and RNA molecules are preferred. Preferably, the size of the complex is less than 2 .mu.m, to permit effective aerosol formation by the ultrasonic nebulizer. Preferred nucleic acid molecules are plasmid expression vectors containing genes of interest. Such plasmids may be prepared using techniques well known to those of ordinary skill in the art, such as the techniques found in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y., 1989. Preferred plasmids are those that encode a cytokine (such as, but not limited to, IL-12), the protease inhibitor alpha-1 antitrypsin, cystic fibrosis transmembrane regulator and other such proteins.

A "stabilizing agent" within the context of the present invention may be any compound or material that, when complexed to a nucleic acid molecule, permits ultrasonic nebulization of the complex without significant loss of transfection efficiency of the nucleic acid. Loss of the supercoiled form (the most potent and fragile of plasmid physical forms) upon nebulization should be less than 20%. Suitable stabilizing agents include one or more lipids, peptides and/or polymers. For example, a peptide/DNA complex containing a condensing peptide (GM208) and a lytic peptide (GM225.1) formulated at a charge ratio (-:+:-) of 1:3:1 is stable after nebulization, as indicated by particle size and zeta potential measurements. Lipids are preferred stabilizing agents, and cationic lipids, such as N-[1-(2,3-dioleyloxy)propyl]-N-N-N-trimethylammonium chloride (DOTMA), are particularly preferred. Cationic lipids are typically employed with helper lipids (or co-lipids) that facilitate the release of DNA from the endosomes following endocytic uptake of the nucleic acid/lipid complex by fusing with endosomal membranes and modulating their physical state. Suitable co-lipids include dioleylphosphatidylethanolamine (DOPE) and/or cholesterol (Chol) (see Brigham et al., Am. J. Med. Sci. 298:278-281, 1989; Canonica et al., Am. J. Resp. Cell. Mol. Biol. 10:24-29, 1994; Bennett et al., Biosci. Rep. 15:47-53, 1995). Lipids for use in the present invention may generally be obtained from commercial sources, such as Avanti Polar Lipids Inc. (Alabaster, Ala.).

In particularly preferred embodiments, the amounts and types of lipids are selected so as to generate a complex that results in enhanced gene transfer to respiratory epithelial cells following intratracheal instillation and/or aerosol inhalation. It has been found, within the context of the present invention, that particle size and charge are important factors that influence pulmonary transgene expression. These properties are determined by the cationic lipid, the co-lipid and methods of formulation. The size and charge ratio of nucleic acid/cationic lipid complexes can be varied by altering the stoichiometry of lipids and nucleic acid (see Tomlinson and Rolland, J. Control. Rel., in press, 1996). The mean size of such complexes varies as a function of the relative level of cationic lipid (for instance, from 100 nm for a 1:0.5 (-:+) charge ratio to 220 nm for a 1:3 charge ratio). In addition, for a fixed DNA/lipid ratio, the increase in DNA concentration has been found to result in an increase in the mean diameter of the complex.

Complex size and surface charge may generally be evaluated using methods well known to those of ordinary skill in the art. For example, the charge ratio may be determined by Doppler electrophoretic light scattering. The mean diameter and zeta potential of the complexes may be characterized by dynamic light scattering and Doppler electrophoretic light scattering (see Tomlinson and Rolland, J. of Cont. Rel., 39:357-372, 1996). Doppler electrophoretic light scattering may generally be performed using any suitable equipment, such as the Coulter DELSA 440 (Coulter Corp., Hialeah, Fla.). Such analyses may be performed by collecting the scattered light front four different angles. The frequency of operation of the instrument may be about 500 Hz with an amplitude of current equal to or lower than the conductivity of the sample. Any equipment suitable for performing dynamic light scattering analyses may be employed, including the Coulter N4 MD Sub-Micron Particle Size Analyzer (Coulter Corp., Hialeah, Fla.).

In general, positively charged complexes give higher levels of expression than negatively charged complexes. In addition, the level of transgene expression increases as the particle size of the complex increases. Accordingly, nucleic acid/stabilizing agent complexes typically have a charge ratio (-:+) of at least 1:2, preferably ranging from 1:2 to 1:6, and more preferably about 1:3. Particle size of the nucleic acid/stabilizing agent complex should be greater than 200 nm for efficient uptake.

Nucleic acid and stabilizing agents may be formulated into a complex using any technique appropriate for the particular components. Such techniques are well known to those of ordinary skill in the art. In complexes containing one or more lipid stabilizing agents, large multilamellar vesicles (MLVs) or extruded liposomes (i.e., unilamellar vesicles) may be prepared prior to the addition of nucleic acid. For example, cationic liposomes composed of DOTMA, along with co-lipids DOPE or cholesterol at a 1:1 mole ratio, may be extruded through polycarbonate filters with different pore diameters (such as 200, 400 and 800 nm) to prepare unilamellar vesicles of different size. Small unilamellar vesicles (SUVs), which have diameters less than 100 nm, may also be prepared by extrusion through appropriately sized filters. Plasmid/lipid complexes may then be prepared by controlled mixing of the cationic liposomes with plasmid DNA at a pre-determined ratio (the stoichiometry of DNA to cationic lipids depends on charge and concentration) using a continuous infusion apparatus. Preferably, the complexes are prepared in 10% lactose so that the formulation is isotonic. Liposomes and plasmids may be delivered to the mixing chamber at a precise rate using, for example, a variable flow self-priming peristaltic pump (e.g., VWR Model Number 54856 (VWR, Houston, Tex.). Steady state conditions may be maintained in the mixing chamber such that the input flow rate of the components into the mixing chamber is equal to the output rate of the formulated complex from the mixing chamber. The complexation efficiency (ie., the fraction of plasmid condensed onto the cationic lipid) may be determined by agarose gel electrophoresis. Plasmid integrity may also be determined using agarose gel electrophoresis, by stripping the DNA from the complex with Triton X, and comparing the stripped DNA bands with that of a naked DNA control.

The colloidal stability of nucleic acid/stabilizing agent complexes is important for clinical applications. Studies have shown that formulated complexes aggregate over time (see Rolland et al., Proc. Intern Symp. Contl. Rel. Bioact. Mat. 21:240-241, 1994), resulting in decreased transfection efficiency. It has been found, within the context of the present invention, that the stability of complexes in an isotonic medium may be increased by lyophilizing the formulated complex in the presence of cryoprotectants (e.g., lactose, mannitol, sucrose and/or trehalose). For example, complexes formulated in lactose may be lyophilized at -30.degree. C. using a freeze dryer (e.g., Model TDS2C0T500, FTS Systems Inc., Stone Ridge, N.Y.). The cooling down, primary cooling, secondary cooling and vacuum may be controlled using a microcomputer. Following freeze drying under controlled conditions and rehydration to isotonicity, the stability characteristics of the complex (size and zeta potential) are maintained.

The lyophilized complex may be jet milled to produce particles in the 1-3 .mu.m range for preparation of a dry powder inhalation dosage form. Lyophilized and jet milled complexes that are rehydrated and instilled maintain their ability to transfect cells; moreover the transfection efficiency is comparable to plasmid/lipid complexes that are not lyophilized. Jet milling may be performed using, for example, Micro-Jet Model 00, Fluid Energy Aljet, Plumsteadville, Pa., with a 60 psig grinding pressure, a 50 psig feed pressure and a manual feed. Prior to administration to a patient, lyophilized and jet milled complexes should be rehydrated to isotonicity.

Nucleic acid/stabilizing agent complexes may be aerosolized using a ultrasonic nebulizer, such as the Omron Model NE-U07. This device produces aerosols by vibration of a piezoelectric crystal at high frequency (2.4 Hz). While other nebulizers may be employed, three parameters are of particular importance in nebulizer selection: the mass median aerodynamic diameter (MMAD), respirable dose (RD) and delivery efficiency, which may be determined using standard techniques. Preferably, the MMAD is less than about 5 .mu.m, the RD is greater than about 60% and the delivery efficiency (ie., the ratio of the mass of drug deposited in the lower airways to the mass of drug aerosolized) is greater than about 10% (see Pillai et al., J. Aerosol Med. 9:227-240, 1996).

An aerosolized nucleic acid/stabilizing agent complex may be collected using, for example, a modified test tube impaction apparatus. Aerosols may be fed into a flexible tygon tubing and through a narrow glass pipet such that the aerosol particles that exit the pipet impact on an ice-cooled test tube and condense. In this manner, aerosols may be collected at predetermined time intervals.

Nucleic acid/stabilizing agent complexes prepared as described herein may generally be used for gene delivery to the respiratory tract of a patient. Within the context of the present invention, a patient may be a human or other mammal, and may be afflicted with one or more diseases or may be free of detectable disease. Accordingly, treatment of a patient with one or more nucleic acid/stabilizing agent complexes may be for preventive purposes or for treatment of an existing disease. For example, a complex containing nucleic acid encoding IL-12 could be administered by aerosol inhalation to modify the immune response to an allergen-induced asthma attack. The nucleic acid/stabilizing agent complexes described herein are suitable for administration by inhalation, which may be oral and/or nasal. Preferably, the complex is sonic nebulized, but jet nebulization may also be employed for complexes having a net positive charge, as described above.

For administration to a patient, one or more nucleic acid/stabilizing agent complexes are generally formulated as a pharmaceutical composition. A pharmaceutical composition comprises a nucleic acid/stabilizing agent complex in combination with a physiologically acceptable carrier (i.e., a non-toxic material that does not interfere with the activity of the active ingredient). Any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of the present invention. Representative carriers include synthetic particles carriers such as peptides and biodegradable polymers or a combination of such materials. Optionally, a pharmaceutical composition may additionally contain other additives such as, for example, preservatives, antimicrobial agents, anti-oxidants, chelating agents, inert gases, and/or other active ingredients.

The frequency of administration and dosage will vary from patient to patient, and depending on the particular nucleic acid administered, the bioavailability of the formulated plasmids to airway epithelial cells and the level and duration of gene expression. Because the protein encoded by the transgene is expressed at the target site, the dose required for prophylactic effect is orders of magnitude lower than that required for drug delivery by conventional routes, with concomitantly fewer side effects. In general, 1-2 doses may be administered every week, depending on the level of therapeutic protein required at the target site for prophylactic or therapeutic effect. A suitable dose is an amount of sonic nebulized/stabilzing agent complex that is sufficient to show improvement in the symptoms of a patient afflicted with a disease. Such improvement may be detected based on fewer systemic side effects, improved patient compliance and/or an improvement in clinical symptoms associated with the disease state. In general, the amount of sonic nebulized/stabilizing agent complex present in a dose ranges from about 500 .mu.g to 4 mg. The emitted dose from the ultrasonic nebulizer for formulations detailed herein ranges from about 10 to 15 .mu.g/L. The exposure time, estimated from the output concentration of aerosols, the ventilatory parameters for human beings under normal breathing conditions and, the deposition efficiency based on the mass median aerodynamic diameter (MMAD) of aerosols, may vary from about 15 minutes to about 2 hours.

Claim 1 of 22 Claims

What is claimed is:

1. A method for preparing a sonic nebulized nucleic acid/stabilizing agent complex, comprising the steps of:

(a) admixing a nucleic acid and a stabilizing agent comprising cationic liposomes at a negative to positive charge ratio ranging from about 1:2 to about 1:6 to form a positively charged nucleic acid/stabilizing agent complex;

(b) formulating the positively charged nucleic acid/stabilizing agent complex in a physiologically acceptable carrier to form a stabilized nucleic acid formulation; and

(c) aerosolizing said formulation with a sonic nebulizer.

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