Title:  Method for preparing a viral aerosol and its use in gene therapy treatment

United States Patent:  6,344,194

Assignee:  Transgene S.A. (Strasbourg, FR)

Filed:  July 21, 1999

Foreign Application Priority Data:  Oct 26, 1993[FR] (93 12743)

A method of preparing a viral aerosol from a dilute viral suspension prepared by dissolving a virus in an aqueous solution containing 6-12 g/l of a monovalent cation salt, or 50-100 g/l of a hexose, which is then nebulised with a gas pressure of 0.5-3.5 bars or an ultrasonic frequency of 2-5 MHZ. The resulting aerosol composition is also disclosed.

Description of the Invention

The present invention relates to a method for preparing an aerosol comprising a virus.

Aerosol formulation of various substances, in particular medicinal substances, has been known for a very long time. Stability and negligible sedimentation rate constitute the essential features of an aerosol. For this reason, on the one hand, in order to prevent the particles aggregating with one another or displaying too high a sedimentation rate, properties incompatible with good efficiency, the proportions of constituents must satisfy a number of specific constraints due, in particular, to different environmental parameters such as the degree of humidity and the temperature, and on the other hand the formulation must take account of a number of difficulties mainly associated with the efficiency of the defence of the respiratory tract against aerial contaminants (clearance mechanisms), which tends to degrade inhaled active principles rapidly.

In particular, in the case where the active principle consists of viruses, the integrity of the viral particle is necessary for infection and penetration of the cells of the pulmonary epithelium, thereby imposing very specific conditions for obtaining the aerosol.

Generally speaking, during an aerolization, a virus may be inactivated as a result of three major causes. In the first place, loss of infectious power may result from the damage undergone by the virus during the nebulization process (spraying into the airways), it may also be inactivated in the aerosol by dehydration, and lastly it may be degraded in the potentially hostile environment represented by the mucus which coats the whole of the respiratory tract and which contains a large number of (proteolytic and other) enzymes.

It has now been demonstrated that viral inactivation can be limited and maintained within reasonable limits by applying, in order to obtain the aerosol, the method according to the invention employing particular conditions of nebulization and of packaging. In addition, this method enables the virus to be delivered effectively and in suitable amounts to the lung, in particular in the tracheobronchial passage.

Accordingly, the subject of the present invention is a method for obtaining a viral aerosol permitting, in particular, its administration via the airways of a mammal, characterized in that:

(a) a dilute viral suspension is prepared corresponding to the dilution of a viral suspension containing 10⁴ to 10¹³ plague forming units (pfu) of a virus in an aqueous solution containing at least 6 to 12 g/l of a salt of a monovalent cation or 50 to 100 g/l of a hexose; and

(b) the dilute viral suspension is nebulized with a gas pressure of 0.5 to 3.5 bars or an ultra-sound frequency of 2 to 5 MHz.

The method according to the present invention is applicable to a very large number of viruses which can be administered by aerosol, in particular viruses chosen from the group consisting of poxviruses, retroviruses, herpesviruses, adeno-associated viruses, rhinoviruses, influenza viruses and adenoviruses.

In the context of the present invention, the term "virus" denotes both a natural virus as found in nature, and a modified virus whose genome contains modifications relative to that of the parent virus from which it originates. It can be an attenuated virus which has lost all or part of its pathogenic power relative to the natural virus from which it is derived. Its genome is modified in vivo in the course of successive passages in cell culture or in a living organism.

The term "virus" can also refer to a recombinant virus whose genome is modified in vitro by genetic engineering techniques. The modification can, for example, enable at least one gene essential to the viral replication to be inactivated (rendering the virus defective for replication), and/or a DNA fragment coding for a heterologous protein (normally not encoded by the natural virus) to be inserted. Insertion takes place in a suitable region of the viral genome, so as to permit the expression of the heterologous DNA fragment in a host cell. A host cell consists of any eukaryotic cell which can be infected by said virus, advantageously a human cell and preferably an epithelial cell of the tracheo-bronchial passage.

For the purposes of the present invention, the heterologous DNA fragment can originate from a eukaryotic organism or from a virus other than the one into which it is inserted. It may be isolated by any conventional technique in the field of the art, for example by cloning., PCR (Polymerase Chain Reaction) or chemical synthesis. It can be a fragment of genomic type DNA (containing all or part of the set of introns of the natural gene), of the complementary DNA type (cDNA; lacking introns) or of the minigene, that is to say mixed, type (containing at least all or part of an intron).

In accordance with the objectives pursued by the present invention, the heterologous DNA fragment may be placed under the control of the elements needed for its expression. "Elements needed" denotes the set of elements permitting transcription of said DNA fragment into messenger RNA (mRNA) or translation of the mRNA into protein.

The heterologous DNA fragment can code for (i) an intracellular protein, (ii) a membrane protein present at the surface of the host cell, or (iii) a protein secreted into the external medium. It can hence contain a signal sequence permitting secretion of the protein towards the membrane or out of the host cell.

Many recombinant viruses capable of benefitting from an administration by aerosol are described in the prior art. Their construction and propagation are within the capacity of a person skilled in the art. As examples, there may be mentioned Ad-.alpha.-1AT (Rosenfeld et al., 1991, Science, 252, 431-434), into the genome of which the human gene coding for al-antitrypsin (.alpha.1AT) is inserted, and Ad-CFTR (Rosenfeld et al., 1992, Cell, 68, 143-155), into the genome of which the human gene coding for the CFTR (for Cystic Fibrosis Transmembrane Conductance Regulator, in English) protein is inserted.

The method according to the present invention involves the preparation of a dilute viral suspension. This dilute viral suspension corresponds to the dilution of a viral suspension containing 10⁴ to 10¹³ pfu of virus in an aqueous solution containing at least 6 to 12 g/l of a salt of a monovalent cation or 50 to 100 g/l of a hexose.

In this step, the suspension to be diluted is generally composed of viruses placed in a buffered medium optionally containing a bivalent cation such as magnesium, calcium or manganese. This type of suspension also being usable for storage. For storage in frozen form, the suspension should be supplemented with a stabilizing agent such as glycerol at a concentration of at least 10%, or sucrose at a concentration of approximately 1 M.

The suspension of viral particles to be diluted can optionally comprise other substances, in particular human serum albumin (HSA), urea, sodium glutamate, glycine and inositol.

According to the method of the present invention, it is necessary for the viral suspension to comprise 10⁴ to 10¹³, advantageously 10⁶ to 10¹² and preferably 10⁸ to 10¹¹ pfu of a virus; the activity of this suspension will be able to depend, in particular, on the virus used.

This suspension to be diluted is then diluted with the aqueous solution according to a suspension/aqueous solution ratio by volume of 1:5 to 1:20, advantageously of 1:10 to 1:20, preferably of 1:12 to 1:18, and as an absolute preference approximately 1:16.

The aqueous solution preferably comprises 6 to 12 g/l of a salt of a monovalent cation, preferably a sodium salt or a potassium salt, and as an absolute preference potassium chloride, sodium lactate and/or sodium chloride. The concentration of salt of a monovalent cation is preferably 6 to 10 g/l, and as an absolute preference approximately 9 g/l.

When the aqueous solution comprises 50 to 100 g/l of a hexose, and preferably approximately 50 g/l, possible hexoses are, in particular, glucose and mannose.

In addition, the aqueous solution can comprise other compounds, such as a calcium salt, for example calcium chloride.

The suspension thus diluted enables an aerosol to be generated whose particular stability in the respiratory tract is sufficient to enable the virus to pass through it. Naturally, it is possible to obtain a corresponding viral suspension by another manipulation sequence.

The nebulization of the dilute suspension may be achieved by a gas pressure or by ultrasound. The conditions of this nebulization also constitute critical parameters of implementation of the method. According to a preferred embodiment of the method according to the present invention, the dilute suspension is subjected to a gas pressure of 0.5 to 3.5 bars, and as an absolute preference of 2 to 3.5 bars. Alternatively, it may also be subjected to an ultrasound frequency of 2 to 5 MHz, and preferably approximately 2 to 3 MHz. The gas pressure and also the ultrasound frequency may be applied by means of a nebulizer.

Generally speaking, a nebulizer is an apparatus permitting the administration of aerosols. The nebulizers may be of any type and their structures are known to a person skilled in the art, and these devices are commercially available.

When the method used entails the application of a gas pressure, a nebulizer of the pneumatic type, connected either to a source of compressed gas such as air or medical oxygen or to a pneumatic pump, is preferably employed. As regards the nebulizing of a dilute viral suspension with an ultrasound frequency, it is preferable to use an ultrasonic type nebulizer, which is provided with a quartz crystal vibrating at high frequency.

The method according to the invention is most especially well suited to the preparation of an aerosol intended for therapeutic purposes, to deliver an optimum amount of viral particles into the tracheobronchial passages of the respiratory tract.

Generally speaking, in man, the respiratory tract is composed of three distinct regions:

the upper part which extends from the nose to the top of the trachea;

The tracheobronchial passages which extend from the top of the trachea to the terminal bronchioles; and

the alveolar region extending from the bronchioles to the alveolar sacs.

Among the diseases capable of being treated by employing the method according to the invention, lung diseases effecting the tracheobronchial passages may be mentioned, and in particular cystic fibrosis, pulmonary emphysema, asthma and lung cancer.

In this context, the viruses which it would be advantageous to deliver by carrying out the method according to the invention are preferably recombinant viruses whose genome comprises a DNA fragment coding for a heterologous protein capable, in particular, of inhibiting or slowing down the progression of a lung disease or preventing it becoming established. Among heterologous proteins, there may be mentioned those which are capable of:

participating directly or indirectly in the transport of ions through cell membranes, and more specifically in the transport of chloride (CI^- or sodium (Na⁺) ions, such as the CFTR protein (Riordan et al., Science, 245, 1066-1073);

reducing the activity of the proteases present in the lungs, in particular in inflammatory conditions, such as native .alpha.1AT (Long et al., 1984, Biochemistry, 23, 4828-4837) or modified .alpha.1AT (Jallat et al., 1986, Protein Engineering, 1, 29-35); and

inhibiting the growth of tumor cells by strengthening cellular immunity, such as interleukins (IL), interferons (IFN) or tumor necrosis factors (TNF), and by having a tunor-suppressive activity, such as the protein p53 (Baker et al., 1989, Science, 244, 217-221) or Rb (Friend et al., 1986, Nature, 323, 643646).

This list is not limiting. DNA fragments coding for other proteins described in the literature for their antitumor effect or their inhibitory effect with respect to the destruction of lung tissues may be used.

Lastly, the present invention also relates to a method of treatment of diseases of the airways, in particular of lung diseases in man, according to which an aerosol prepared according to the method according to which:

(a) a dilute viral suspension is prepared corresponding to the dilution of a viral suspension containing 10⁴ to 10¹³ pfu of virus in an aqueous solution containing at least 6 to 12 g/l of a salt of a monovalent cation or 50 to 100 g/l of a hexose;

(b) the dilute viral suspension is nebulized with a gas pressure of 0.5 to 3.5 bars or an ultrasound frequency of 2 to 5 MHz, is administered by inhalation, in the nasal or buccal cavity of a patient requiring such a treatment.

The inhalation can take place in a single dose or a dose repeated one or several times after a certain time interval.

The invention is described more completely by means of the following examples:

EXAMPLE 1

Preparation of an Aerosol from a Viral Suspension Stored in the Presence of 10% Glycerol and Diluted in a Solution of Monovalent Cation

A viral suspension is prepared from a recombinant adenovirus into the genome of which the human gene coding for the CFTR protein (Ad-CFTR) is inserted, as described in Rosenfeld et al. (1992, supra).

Briefly, it is derived from adenovirus type 5, the genome of which lacks on the one hand the E1A gene coding for a trans-activating protein essential to the replication of the adenovirus, and on the other hand the nonessential E3 gene. The CFTR gene is inserted in place of the E1A gene.

Ad-CFTR is propagated by means of human embryonic kidney cell line 293 (Graham et al., 1977, J. Gen. Virol., 36, 59-72), which expresses the E1 function constitutively. This line is available at the ATCC (CRL 1573). The 293 cells are cultured according to the supplier's recommendations.

Cells in culture are infected before confluence with an initial inoculum of Ad-CFTR virus and according to a multiplicity of infection (moi) of 2 to 10. They are incubated at 37^oC. and, as soon as cytopathic effects are observed, normally after 4 to 10 days of culture, the viruses are harvested according to the following protocol.

The cell suspension is centrifuged at low speed for 10 minutes. The cell pellet is resuspended in approximately 10 ml of GMEM medium (Glasgow Modified Eagle Medium, Gibco BRL, Cergy-Pontoise) supplemented with 2% of fetal calf serum. The viruses are separated from the cells by several successive freezing/thawing cycles in an ethanol-dry ice bath/water bath at 37^oC. Following the last cycle, cell debris is removed by centrifugation at low speed for 5 to 10 minutes.

The viruses are purified from the centrifugation supernatant by fractionation on a cesium chloride gradient with two density layers, 1.40 and 1.25 g/ml, respectively. Centrifugation is carried out at 100,000 g for a few hours at room temperature. The viruses appear in the form of a white band located at the interface of the two layers, and are recovered using a syringe. They are subjected to EL second purification on a self-generated cesium chloride gradient prepared using a solution containing 1.33 g/ml. The viruses are likewise recovered by puncturing the tube with a syringe, and glycerol equivalent to 1/10th of the volume is added.

The cesium chloride is removed by dialysis against a buffer containing 10 mM Tris-HCl, pH 7.4, 1 mM MgCl₂ and 10% glycerol.

The titer of the viral suspension thereby obtained is determined accurately according to the titration method in agar or at 30 h (Graham and Prevec, 1991, Methods in Molecular Biology, 7, 109-128, ed. E. J. Murray, The Human Press Inc. Clinton, N.J.). The suspension is distributed in tubes (Nunc or Nalgene) in 100 .mu.l aliquots so as to contain 5x10⁹ to 5x10¹⁰ pfu each. These tubes, which constitute the stock viral suspension, are stored at -60^oC. until used.

At the time of use, 1.5 ml of sodium chloride solution containing 9 g/l (Meram, Melun) are added to 100 .mu.l of the Ad-CFTR suspension (at a concentration of 10⁷, 10⁸ or 10⁹ pfu). Controls performed show that the titer of the dilute viral suspension thereby obtained is stable for several hours at room temperature.

An accelerated stability study was also under-taken at 370^oC. After dilution of the viral suspension to different concentrations (10⁹, 10⁷, 10⁵ pfu/ml) in the above sodium chloride solution, a sample of each dilution is taken at regular intervals (t=0 h, 2 h, 4h and 24 h), and the viral infectivity is titrated by the agar or 30 h technique. Stability at 37^oC. of the dilute viral suspension for more than 3 h is observed, especially at high concentration (10⁹ and 10⁷ pfu/ml).

An aerosol is generated by placing the dilute viral suspension in the reservoir of an Optineb 709 nebulizer (Air Liquide, Paris). The latter is a pneumatic generating apparatus operating at high pressure and affording a choice of the carrier gas. It is connected to a source of compressed air, and a variable pressure of medical air may be applied. Aerosolization tests are performed in order to evaluate the optimum conditions to be applied to patients suffering from cystic fibrosis. In particular, the effect of pressure (from 1 to 3.5 bars) and of the respiratory rate are studied. The latter defines a number of breaths per minute, and may be predetermined by setting the apparatus. In this connection, setting to position 7 corresponds to the respiratory rate of a child, while position 9 corresponds to that of an adult.

Aerosolization is carried out in 3 steps (viral suspension diluted, followed by two rinses of the cup with the sodium chloride diluent solution). The Optineb is set to self-triggering and connected to a trap or collector, for example a collectron MD8 (Sartorius, Palaiseau, FRANCE), to the end of which is fitted a gelatin filtering membrane enabling the viral particles to be recovered after nebulization. This membrane is then dissolved at 37^oC. in 10 mM Tris-HCl buffer, pH 7.4, 1 mM MgCl₂ and 10% glycerol, and the infectivity of the viruses recovered is titrated by the 30 h and agar technique. This measurement enables the amount of active virus capable of entering a patient's airways to be evaluated. Under the different test conditions and relatively repeatedly, the trapped infectious viruses represent 15 to 40% of the initial number of viruses. Although some fluctuations are observed, best recovery yields are obtained at high pressure (3.5 bars), this being the case in both setting positions. As a guide, the recovery yield of medicinal products applied by aerosolization is usually approximately 10%. These results make it possible to envisage the use of the aerosolization of Ad-CFTR recombinant adenoviruses for the treatment of patients suffering from cystic fibrosis.

Claim 1 of 11 Claims

What is claimed is:

1. An aerosol obtained by a method comprising:

a) preparing a dilute viral suspension corresponding to the dilution of a viral suspension containing 10⁴ to 10¹³ pfu of recombinant adenovirus particles in an aqueous solution, wherein the dilute viral suspension limits inactivation of the virus and wherein said dilution is performed according to a viral suspension/aqueous solution ratio by volume of 1:5 to 1:20 and wherein said aqueous solution contains at least 6 to 12 g/l of a salt or a monovalent cation of 50 to 100 g/l of a hexose: and

b) nebulizing the dilute viral suspension under conditions sufficient to produce a viral aerosol composition.

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