Therapeutic delivery compositions and methods of use thereof

IS 2 189 808 T3

DESCRIPTION

Compositions of therapeutic contribution and methods of use of the same

Technical field

The present invention relates to therapeutic delivery compositions and compositions comprising therapeutic delivery compounds that destroy or suppress the growth of bacteria, viruses, fungi and protozoa, and methods of using the same. The compounds, compositions and methods are effective for the delivery of drugs and other compounds to the interior of cells and for controlling intracellular organisms.

Background of the invention

Many new and potentially useful technologies are being developed that may form the basis for future medical cures and therapies. Examples of such technologies include gene substitution, antisense gene therapy, triplex gene therapy, and ribozyme-based therapy. However, to be successful, these technologies require efficient means for delivery of the therapeutic agent through cell, nuclear and microorganism membranes.

The recent advent of technology and advances in the ability to understand the structure and function of many genes make it possible to cut or modify the activity of a given gene. The alteration of gene activity can be effected in many ways. For example, oligonucleotides that are complementary to certain gene messages or viral sequences, known as "antisense" compounds, have been found to have an inhibitory effect against viruses. By creating an antisense compound that hybridizes to the targeted cell or virus RNA message, translation of the message into protein can be interrupted or prevented. In this way, gene activity can be modulated.

The ability to turn off specific genes provides great therapeutic benefits. For example, theoretically, it is possible to combat viral diseases with antisense DNA and RNA molecules that seek out and destroy viral gene products. In tissue culture, antisense oligonucleotides have inhibited infections with herpesviruses, influenza viruses, and the human immunodeficiency virus that causes AIDS. It is also possible to target antisense oligonucleotides against mutated oncogenes. Antisense technology also holds the potential to regulate growth and development. However, for gene therapy to work, antisense therapeutic compounds must be delivered across cell plasma membranes into the cytosol.

Gene activity is also modified using sense DNA with a technique known as gene therapy. Defective genes are replaced or complemented by the administration of "good" or normal genes that are not subject to the defect. Administered normal genes that insert on a chromosome, or may be present on extracellular DNA, produce normal RNA, which in turn leads to a normal gene product. In this way, genetic defects and deficiencies in the production of the gene product can be corrected. Furthermore, gene therapy has the potential to increase the normal genetic complement of a cell. For example, it has been proposed that one way to combat HIV is to introduce into the T cells of an infected person a gene that makes the cells resistant to HIV infection. This form of gene therapy is sometimes called "intracellular immunization." Genetic material such as polynucleotides can be administered to a mammal to elicit an immune response against the gene product of the administered nucleic acid sequence. Such gene vaccines elicit an immune response in the following manner. First, the nucleic acid sequence is administered to a human or an animal. The administered sequence is then expressed to form gene product within the human or animal. The gene product within the human or animal is recognized as foreign material and the immune system of the animal or human mounts an immunological response against the gene product. However, this system is currently not feasible due to the lack of effective gene delivery systems that facilitate the delivery of genetic material through both cellular and nuclear membranes.

Finally, gene therapy can be used as a method of delivering drugs in vivo. For example, if genes encoding therapeutic compounds could be delivered to endothelial cells, the gene products would have facilitated access to the blood stream. Currently, genes are delivered to cells ex vivo and then reintroduced into the animal.

Retroviral vectors can be used to deliver genes ex vivo to isolated cells, which are then infused back into the patient. However, retroviral vectors have some disadvantages, such as

ES 2 189 808 T3 such as being able to deliver genes only to dividing cells, a random integration of the gene to be delivered, potentially causing unwanted genetic alterations, and possibly reversing to an infectious wild-type retroviral form. Another disadvantage of antisense gene therapy is that it is effective at the messenger RNA domain, meaning that antisense oligonucleotides must be introduced in an amount to interact with all or a substantial number of the mRNA in the cytosol, and that such treatment alone it is effective during active mRNA synthesis. In addition, oligonucleotides must be maintained at this high level throughout mRNA synthesis to be effective over time.

The recently developed "DNA triplex" technology represents an improvement in gene regulation. DNA triplex technology uses oligonucleotides and compounds that specifically bind to particular regions of duplex DNA, thereby inactivating the targeted gene. An advantage of triplex DNA technology is that only a single copy of the oligonucleotide or compound is required to alter gene expression because the binding is at the DNA site, not at the mRNA site. A disadvantage of triplex DNA technology, however, is that the oligonucleotide or compound must pass through not only the cell membrane but also the microbial membrane in the case of treating microbial infections, or the nuclear membrane in the case of altering the eukaryotic gene function or expression of foreign DNA integrated into chromosomal DNA.

Another emerging technology concerns the therapeutic use of ribozymes for the treatment of genetic disorders. Ribozymes are catalytic RNA molecules that consist of a hybridizing region and an enzymatic region. Ribozymes can be manipulated in the future to specifically bind to a targeted region of the nucleic acid sequence and to cut or otherwise enzymatically modify the sequence to alter its expression or translation into a gene product.

Therefore, there is a great need for improved delivery systems for genetic material, such as genes, polynucleotides, and antisense oligonucleotides, that can be used in gene therapy. More specifically, there is a need for non-toxic compositions that have surfactant properties that can facilitate the transport of genetic and other drugs and therapeutic compounds across cell membranes.

There is a particular urgent need for an effective treatment for Acquired Immune Deficiency Syndrome, or AIDS, a disease believed to be caused by a human retrovirus, Human T Lymphotropic Virus III (HTLV-III) which is also called immunodeficiency virus. human or HIV. Like other retroviruses, HIV has ribonucleic acid, or RNA, as its genetic material. When the virus enters the host cell, a viral enzyme called reverse transcriptase exploits the viral RNA as a template to assemble a corresponding DNA molecule. DNA travels through the nucleus of the cell and inserts itself between the host's chromosomes, where it provides the basis for viral replication. In the case of HIV, the host cell is often a T4 lymphocyte, a white blood cell that plays a major regulatory role in the immune system. Once inside a T4 cell, the virus can remain latent until the lymphocyte is immunologically stimulated by a secondary infection. The virus then reproduces rapidly, destroying or rendering the host cell ineffective. The resulting depletion of T4 cells and loss of activity makes the patient vulnerable to "opportunistic" infections by an agent that would not normally harm a healthy person. In addition, the virus damages the host through many other mechanisms.

Many therapies against AIDS infection are currently being investigated. Several of these therapies under investigation are based on disrupting reverse transcriptase as it assembles viral DNA destined to become the virus. The drugs used for this purpose are chemical analogs of the nucleic acids that make up the DNA subunits. When the analog is delivered to an infected cell, reverse transcriptase will incorporate it into a growing DNA strand. However, because the analog lacks the correct point of attachment for the next subunit, the chain is terminated. The truncated DNA cannot integrate into the host chromosomes or provide the basis for viral replication, and thus the extent of infection is interrupted. One of the compounds believed to act by mimicking a nucleotide is azidothymidine, or AZT. However, AZT is known to have serious side effects and its effectiveness in mitigating the disease AIDS has been questioned. The efficacy of AZT and other antiviral and antimicrobial drugs could be increased if improved means and methods were available to deliver therapeutic agents to the site of infection.

WO-93/08845 describes the local application of antisense oligonucleotides that are effective in inhibiting the expression and translation of a variety of genes. Antisense oligonucleotides can be applied

ES 2 189 808 T3 to the tissue surface at the location within a biocompatible matrix or carrier. The matrix or carrier can be a hydrogel material such as a poly (propylene oxide-ethylene oxide) gel. Pluronic mentioned^TM 127 (Poloxamer 407) as a suitable poly (propylene oxide-ethylene oxide). Poloxamer 407 consists of 73% polyoxyethylene and 27% polyoxypropylene.

Summary of the invention

The present invention includes a method of delivering therapeutic drugs to a human or animal to treat disease states such as, but not limited to, bacterial infection and infections caused by HIV and other DNA and RNA viruses.

The present invention relates particularly to compositions and methods for treating infectious diseases and genetic disorders through genome therapy and intracellular delivery of antisense oligonucleootides or other nucleic acid sequences.

The present invention comprises a therapeutic delivery composition effective for treating a disease state, comprising an administrable mixture of an effective amount of a therapeutic compound capable of altering the function of nucleic acid sequences and an effective amount of a non-block copolymer. ionic surface active having the following general formula:

HO (C2H4O) b (C3H6O) a (C2H4O) bH in which a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight of between 750 and 15,000, preferably between 2,250 and 15,000, more preferably between 3,250 and 15,000, and b is an integer number such that the hydrophilic portion represented by (C2H4O) constitutes 1% to 50% by weight of the compound, preferably 5% to 20%.

A particularly useful composition is a mixture of a compound capable of altering the gene expression and / or the translation of proteones, such as an antisense oligonucleootide, a DNA troplex compound, a ribozyme or another compound capable of altering the function of acid sequences. nucleic, and the previously described non-ionic block copolymer.

The composition of the present invention can be administered by a number of routes including, but not limited to, topical, transdermal, oral, transmucosal, subcutaneous injection, intravenous injection, intraperitoneal injection, and intramuscular injection.

Accordingly, an objective of the invention is to provide a therapeutic drug delivery vehicle.

Another objective of the present invention is to provide compositions that facilitate the delivery of one or more therapeutic agents that alter the function of nucleic acid sequences within a cell, such as a phagocotic cell, when mixed with a therapeutic agent.

Another objective of the present invention is to provide compositions that act synergistically with an agent delivered once within a cellula.

Yet another object of the invention is to provide non-ionic block copolymers that have surfactant properties that facilitate the transmission and introduction through cellular plasmaotic membranes of nucleic acid sequences and compounds capable of altering the function of nucleic acid sequences.

A further objective of the present invention is to provide compositions and a method for treating physiological genetic disorders using nucleic acid and antisense oligonucleotide sequences in combination with non-ionic block copolymers.

Another objective of the present invention is to provide useful compositions and method for manipulating gene expression using troplex DNA compounds.

Yet another objective of the invention is to provide DNA vaccines.

An objective of the present invention is to provide compositions that can be used to treat people with infectious diseases.

IS 2 189 808 T3

Yet another object of the present invention is to provide a method for treating viral infections in humans or animals.

Another object of the present invention is a compound and a method that is effective in inhibiting virus replication in both animals and humans.

Another object of the present invention is to provide a compound and method that is effective in inhibiting the replication of HIV and other RNA and DNA viruses.

Yet another object of the present invention is to provide a method for treating microbial infections in humans or animals.

A further objective of the present invention is to inactivate a virus in a blood product prior to infusion into a person or an animal.

These and other objectives, features, and advantages of the present invention will become apparent upon review of the following detailed description of the disclosed embodiment and the appended claims.

Brief description of the drawings

Figure 1 is a grid illustrating block copolymers by hydrophobic molecular weight and hydrophilic percentage.

Figure 2 is a grid illustrating therapeutic delivery block copolymers by hydrophobic molecular weight and hydrophilic percentage.

Figure 3 is a grid illustrating more preferred therapeutic delivery block copolymers by hydrophobic molecular weight and hydrophilic percentage.

Detailed description

The present invention includes gene therapy compositions that are mixtures of a non-iogenic block copolymer and nucleic acid sequences or compounds capable of altering the function of nucleic acid sequences and methods to deliver these compositions to a human or animal in need. of the same for the intracellular alteration of the gene expression and / or the translation of proteins. High molecular weight non-ionic surface active polyoxyethylene-polyoxypropylene block copolymers having a low percentage of polyoxyethylene have been unexpectedly found to facilitate the transport of DNA and other compounds within cells and to be aseíuteile for intracellular delivery of therapeutic agents in vivo. for the treatment of a disease. Block copolymers are believed to be particularly useful in helping to reclose membranes and thus increase the percentage of survival of cells into which nucleic acid sequences or other compounds have been introduced intracellularly. Surprisingly, it has also been found that compositions comprising the non-ionic block copolymers of the present invention and nucleic acid sequences are less susceptible to the degrading effects of DNAase than nucleic acid sequences alone.

The present invention also encompasses therapeutic compositions and methods that destroy or inhibit the growth of microorganisms and alter the expression or function of nucleic acid sequences. An example of bacteria against which the present invention is effective are species of mycobacteria, such as Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium leprae. Other microorganisms against which the invention is effective include, but are not limited to, Chlamydia trachomatis, Chlamydia pneumoniae, Listeria monocytogenes, Candida albicans, Cryptococcus neoformans, Toxoplasma gondii, Pneumocystis carinii, herpes simplex virus, Cytomegalovirus, influenza viruses type A and B and respiratory syncytial virus.

The present invention includes therapeutic compositions and methods for treating DNA viruses and RNA viruses, and infections and infectious diseases caused by such viruses in a human or animal, including infections caused by HIV or herpes or antigenically related strains thereof. Antigenically related strains are strains that cross-react with antibodies specific for HIV. One skilled in the art can easily determine viral strains that are antigenically related to HIV by performing standard immunoassay tests using anti-HIV antibodies and the viral strain to be tested, and looking for positive cross-reactivity. Therapeutic compositions comprising the surface active polymers described herein are effective in inhibiting or suppressing the replication of such viruses in cells.

IS 2 189 808 T3

The present invention includes a therapeutic composition useful for delivering antimicrobial drugs and treating disease states, comprising a mixture of a surface-active non-ionic block copolymer, a compound capable of altering the function of nucleic acid sequences, and an antibiotic or therapeutic drug. Examples of such compounds capable of altering nucleic acid sequence function include genes, oligonucleotides, antisense oligonucleotides, DNA troplex compounds, and ribozymes. Drugs that can be used with the non-ionic copolymers of the present invention include, but are not limited to, rifampin, isoniazid, ethambutol, gentamicin, tetracycline, erythromycin, pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinloxacin, and of sparphloxacin, such as of sparphloxacin. , clarithromycin, dapsone, doxycycline, ciprofloxacin, ampicillin, amphotericin B, fluconazole, ketoconazole, pyrimethamine, sulfadiazine, clindamycin, azithromycin, paromycin, diclazaril, clarithromycin, atovaquone, pentamidine, acyclovir, trifluorouridine, AZT, DDI, DDC and other antiviral anaologs, foscarnet, ganciclovir, viral protease inhibitors, antisense and other modified oligonucleootides, and ribavirin.

Preferred drugs to use for various infectious microorganisms are listed in Table I.

Table I

<td>Organism Bacteria</td><td>Drugs</td>

<td>Mycobacterium tuberculosis</td><td>Isoniazid, rifampin, ethambutol, pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones such as ofloxacin and sparfloxacin</td>

<td>Mycobacterium avium</td><td>Rifabutin, rifampin, azithromycin, clarithromycin, fluoroquinolones</td>

<td>Mycobacterium leprae</td><td>Dapsone</td>

<td>Chlamydia trachomatis</td><td>Tetracycline, doxycycline, erythromycin, ciprofloxacin</td>

<td>Chlamydia pneumoniae</td><td>Doxycycline, erythromycin</td>

<td>Listeria monocytogenes Mushrooms</td><td>Ampicillin</td>

<td>Candida albicans</td><td>Amphotericin B, ketoconazole, fluconazole</td>

<td>Cryptococcus neoformans Protozoa</td><td>Amphotericin B, ketoconazole, fluconazole</td>

<td>Toxoplasma gondii</td><td>Pyrimethamine, sulfadiazine, clindamycin, azithromycin, clarithromycin, atovaquone</td>

<td>Pneumocystis carinii</td><td>Pentamidine, atovaquone</td>

<td>Cryptosporidium sp. Virus</td><td>Paromomycin, diclazaril</td>

<td>Herpes simplex virus type 1</td><td>Acyclovir, trifluorouridine, and other nucleoside analogs</td>

<td>and type 2</td><td>antivirals, foscamet, antisense oligonucleotides and triplex-specific DNA sequences</td>

<td>Cytomegalovirus</td><td>Foscamet, ganciclovir</td>

<td>HIV</td><td>AZT, DDI, DDC, foscamet, viral protease inhibitors, peptides, antisense oligonucleotides, triplex sequences, and other nucleic acid sequences</td>

<td>Influenza virus type A and B</td><td>Ribavirin</td>

<td>Respiratory syncytial virus</td><td>Ribavirin</td>

<td>Varicella zoster virus</td><td>Acyclovir</td>

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Optionally, surfactants and low molecular weight alcohols are added to the therapeutic mixture of antimicrobial drug and nonionic block copolymer. Examples of useful surfactants in the present invention include Tween 80 and emulsions with fatty acids such as phospholipids, cholate, and amino acids. The preferred surfactant is Tween 80. Surfactants are added to the mixture at a concentration ranging from about 0.1% to about 5% w / w. The preferred surfactant concentration is about 2%. The term "approximately", as it applies to concentrations expressed herein, means the indicated concentration plus or minus ten percent. The term "low molecular weight alcohols" means alcohols having two to eight carbons. An example of useful low molecular weight alcohols in the present invention is ethanol, which is the preferred low molecular weight alcohol. Low molecular weight alcohols are added to the mixture in a concentration ranging from about 0.5% to about 5% v / v. The preferred low molecular weight alcohol concentration is between about 1% and about 3% v / v.

The present invention also includes compositions and methods for immunizing animals or humans, otherwise referred to as DNA vaccination. Immunization is carried out by administering a composition comprising the gene that codes for the gene product to be immunized against contained in an expression vector, in combination with a block copolymer that promotes and facilitates the uptake of genetic material through membranes. cell phones. The introduced gene is expressed, resulting in the production of an antigenic gene product.

In addition, compositions comprising nonionic block copolymers and genes encoding compounds effective to destroy, reduce or retard cancer, such as lymphokines, can be administered to humans or animals for the treatment of cancer.

The present invention comprises a surface active copolymer which is preferably an ethylene oxide-propylene oxide condensation product with the following general formula:

HO (C2H4O) b (C3H6O) a (C2H4O) bH in which a is an integer such that the hydrophobe represented by (C3H6O) has a molecular weight between 750 and 15,000, and b is an integer such that the hydrophilic percentage represented by (C2H4O) constitutes 1% to 50% by weight of the compound.

The present invention also comprises a therapeutic delivery composition useful for altering gene expression and / or protein translation, comprising a administrable mixture of an effective amount of an antisense oligonucleotide or other nucleic acid sequence and an effective amount of a copolymer. of non-iogenic blocks that have the following general formula:

HO (C2H4O) b (C3H6O) a (C2H4O) bH in which a is an integer such that the hydrophobic represented by (C3H6O) has a molecular weight of between 750 and 15,000, preferably between 2,250 and 15,000, more preferably between 3,250 and 15,000, and b is an integer such that the hydrophilic portion represented by (C2H4O) constitutes 1% to 50% by weight of the compound, preferably 5% to 20%. The term "mixture", as used herein, means any combination of therapeutic drug and non-ionic block copolymer, including solutions, suspensions or encapsulations of drug in copolymer micelles. An "effective amount" is an amount sufficient to alter the activity and / or the amount of gene product produced by the gene or genes that are sought to be modulated in a human or animal.

The present invention also comprises a therapeutic delivery composition useful for immunizing an animal or a human against a particular gene product, comprising a administrable mixture of an effective amount of an expression vector, the gene that codes for the gene product against the one to be immunized contained in the expression vector and an effective amount of a non-iogenic block copolymer having the following general formula:

HO (C2H4O) b (C3H6O) a (C2H4O) bH where a is an integer such that the hydrophobic represented by (C3H6O) has a molecular weight of between 750 and 15,000, preferably between 2,250 and 15,000, more preferably between 3,250 and 15,000, and b is an integer such that the hydrophilic portion represented by (C2H4O) constitutes 1% to 50% by weight of the compound, preferably 5% to 20%. An effective amount is an amount sufficient to elicit an immunological response against the gene product of the nucleic acid sequence administered to the human or animal.

It should be understood that the molecular weight and percentage ranges that are described for the block copolymer are to be considered external ranges and that any populations of molecules that fall within

ES 2 189 808 T3 of the indicated ranges is considered an embodiment of the present invention.

The entire block copolymer molecule is poorly soluble in water and is substantially non-ionic. It is believed that the steric configurations and the physicochemical properties of the molecule, rather than the chemical nature of the constituent parts, are largely responsible for the anti-infective activity and the therapeutic delivery activity. The compositions of the present invention include, but are not limited to, aqueous solutions, suspensions, or emulsions, such as oil-in-water emulsions.

The polymer blocks are formed by the condensation of ethylene oxide and propylene oxide, at elevated temperature and pressure, in the presence of a catalyst. There is some statistical variation in the number of monomer units that combine to form a polymer chain in each copolymer. The molecular weights given are approximations of the average weight of the copolymer molecule in each preparation and depend on the test methodology and calibration standards used. It should be understood that the propylene oxide and ethylene oxide blocks do not have to be pure. Small amounts of other materials can be mixed as long as the overall physicochemical properties are not substantially changed. A detailed discussion of the preparation of these products is found in US Patent No. 2,674,619, which is incorporated herein by reference in its entirety.

Ethylene ioxide-propylene ioxide condensation products that can be used in the present invention are summarized in Table II. It should be understood that these compounds are merely representative of the compounds that can be used to implement the present invention and do not include all possible compounds that could be used to implement the present invention. The high molecular weight copolymers listed in Table II that do not have a BASF trade name are new compositions that have never been synthesized before.

Table Π

<td>CRLN °</td><td>Poloxamer</td><td>BASF Business Name</td><td>Molecular Weight of POP</td><td>% of POE</td>

<td></td><td> 122</td><td>L42</td><td> »1200</td><td> »20%</td>

<td>CRL-85171</td><td> 181</td><td>L61</td><td> «1750</td><td> «10%</td>

<td>CRL-85172</td><td> 182</td><td>L62</td><td> »-1750</td><td> «20%</td>

<td>CRL-85173</td><td> 183</td><td>L63</td><td> «1750</td><td> «30%</td>

<td>CRL-85174</td><td> 184</td><td>L64</td><td> »1750</td><td> «40%</td>

<td>CRL-85175</td><td> 185</td><td>Q65</td><td> «1750</td><td> «50%</td>

<td>CRL-85178</td><td> 188</td><td>F68</td><td> «1750</td><td> «80%</td>

<td>CRL-85202</td><td> 212</td><td>L72</td><td> «2050</td><td> «20%</td>

<td>CRL-85221</td><td> 231</td><td>L81</td><td> «2250</td><td> «10%</td>

<td>CRL-8122</td><td> 282</td><td>L92</td><td> «2750</td><td> «20%</td>

<td>CRL-8131</td><td> 331</td><td>L101</td><td> «3250</td><td> «10%</td>

<td>CRL-8133</td><td> 333</td><td>P103</td><td> «3250</td><td> »30%</td>

<td>CRL-8135</td><td> 335</td><td>P105</td><td> «3250</td><td> »50%</td>

<td>CRL-9038</td><td> 338</td><td>F108</td><td> «3250</td><td> «80%</td>

<td>CRL-8141</td><td> 401</td><td>L121</td><td> »4000</td><td> «10%</td>

<td>CRL-8142</td><td> 402</td><td>L122</td><td> «4000</td><td> «20%</td>

<td>CRL-8143</td><td> 403</td><td>P123</td><td> »4000</td><td> «30%</td>

<td>CRL-8941</td><td> 441</td><td>L141</td><td> «4400</td><td> «10%</td>

<td>CRL-8950</td><td> —</td><td> —</td><td> «6000</td><td> «5%</td>

<td>CRL-1235</td><td> —</td><td> —</td><td> «7500</td><td> «5%</td>

<td>CRL-1190</td><td> —</td><td> —</td><td> «10.000</td><td> «5%</td>

<td>CRL-336</td><td> —</td><td> —</td><td> »14.000</td><td> «5%</td>

<td>CRL-1183</td><td> —</td><td> —</td><td> »3750</td><td> «10%</td>

<td>CRL-1122</td><td> - -</td><td> —</td><td> «5900</td><td> «12%</td>

<td>CRL-3362</td><td> —</td><td> —</td><td> «3900</td><td> «11%</td>

<td>CRL-3632</td><td> —</td><td> —</td><td> »4740</td><td> »11%</td>

<td>CRL-9352</td><td> —</td><td> —</td><td> «7750</td><td> «15%</td>

<td>CRL-1187</td><td> —</td><td> —</td><td> «750</td><td> «25%</td>

IS 2 189 808 T3

A grid illustrating the copolymer range encompassed by the present invention based on the molecular weight of the hydrophobic portion and the percent hydrophilicity, and showing selected non-ionic block copolymers, appears as Figure 1. Polymer blocks are formed by condensation, at high temperature and pressure, of ethylene oxide and propylene oxide in the presence of a catalyst.

There is some statistical variation in the number of monomer units that combine to form a polymer chain in each copolymer. The molecular weights given are approximations of the average size of copolymer molecules in each preparation. A further description of the preparation of these block copolymers is found in US Patent No. 2,674,619. (See also "A Review of Block Polymer Surfactants", Schmolka IR, J. Am. Oil Chemist Copolymerization, Volume 2, edited by RJ Ceresa, John Wiley and Sons, New York, 1976.

It has been discovered that copolymers particularly effective as therapeutic delivery agents are shown in Figures 2 and 3. As is evident from Figures 2 and 3, the most effective copolymers as therapeutic delivery agents are of high molecular weight and have low POE percentages generally less than 20% POE.

Non-ionic block copolymers form micelles above their chromic micellar concentration. Non-ionic copolymers have negative solubility toothermic coefficients. In cold, the kinetic energy of the water molecules is reduced and they form weak hydrogen bonds with the oxygen of the POP blocks. This hydration of the hydrophobic promotes solubility at low temperatures. As the temperature rises, the "cloud point" is reached; the increased kinetic energy of the water breaks the hydrogen bonds, the polymer becomes insoluble and micelles are formed.

Thus, the copolymers, which are therapeutic in their own right, can form phosphoric structures that can be combined or loaded with an additional different therapeutic agent. Accordingly, the non-ionic block copolymers of the present invention can be used as therapeutic drug delivery vehicles. Mixtures of therapeutic drugs with non-ionic block copolymers have the advantage of the synergistic activity of two therapeutic agents. In addition, copolymers having specific characteristics can be selected for use with particular therapeutic drugs. For example, CRL-8131, which is hydrophobic, is an excellent carrier for hydrophobic antibiotics such as rifampin. However, other agents that are not clearly hydrophobic can be used in accordance with the present invention.

A therapeutic agent delivery vehicle is prepared using any of the surface active nonionic block copolymers of the present invention in combination with any of a variety of antimicrobial agents. In one embodiment, CRL-8131 is used at a concentration of from about 3% to about 5% to construct a therapeutic agent delivery vehicle. Therapeutic delivery vehicles made using copolymers that are more hydrophilic than CRL-8131 typically require a higher concentration (from about 5% to about 10%) of the copolymer.

Using copolymer-based micelles as a therapeutic drug delivery vehicle is particularly desirable because micelles readily accumulate and are present for a prolonged period of time, in macrophages, the site of HIV and other viral infections and a primary target for viral therapy. Examples of such therapeutic compositions based on therapeutic copolymers include CRL-8131 combined with 2% Tween 80 and 1% ethanol, and CRL-8142 combined with 1% Tween 80 and 5% ethanol.

Nucleic acid sequences or other compounds capable of altering the function of nucleic acid sequences are administered to a human or animal to alter gene expression and / or modify the amount or activity of the gene product. For example, antisense oligonucleootides mixed with previously described non-ionic block copolymers give useful compositions for the supply of antisense oligonucleootides with the purpose of altering or regulating gone expression and / or proteon translation. In addition, nucleic acid sequences such as genes can be delivered, which are incorporated into the chromosome replacing or augmenting the defective gene. Alternatively, the intracellularly administered gene may reside in the cocell and be expressed on an extrachromosomal element.

The present invention also provides novel compositions and methods for immunizing an animal or a human. The compositions comprise an expression vector, a gene encoding the gene product to be immunized against contained in the expression vector, and a copolymer of

ES 2 189 808 T3 blocks effective in transferring genetic material, such as expression vectors, through the cell membrane. The method of immunizing an animal or human comprises administering the vector-containing copolymer composition expressed to the animal or human. A preferred mode of administration is by intraperitoneal injection. This embodiment of the invention provides means for the delivery of gene sequences capable of expressing an antigenic gene product directly to human or animal cells, in vivo or ex vivo, with subsequent reintroduction into the human or animal. Once introduced into cells, antigenic gene product production induces and maintains an immune response by the human or animal against the introduced gene product.

The following specific examples illustrate various aspects of the invention, such as compositions and methods of the invention useful for gene therapy, and compositions and methods of the invention useful for gene-mediated immunization. It should be appreciated that other embodiments and uses will be apparent to those skilled in the art and that the invention is not limited to these specific illustrative examples. Example I

A therapeutic agent delivery vehicle is prepared by combining any of the surface active non-ionic block copolymers, such as CRL-8131, with any of a variety of compounds capable of altering the function of nucleic acid sequences. For CRL-8131, a concentration of three to five percent by weight by volume is desirable to construct the therapeutic vehicle. For a more hydrophilic copolymer, five to ten percent by weight by volume is used.

300 milligrams of CRL-8131 are added to 10 ml of 0.9% NaCl and the mixture is solubilized by storage at 2-4 ° C until a clear solution is formed. An appropriate amount of a compound capable of altering the gene function of a nucleic acid is added to the mixture and micelles are formed that associate the copolymer and the compound, raising the temperature above 5^°C and allowing the micelle suspension to equilibrate. The balanced suspension is adequate for administration.

For example, an antisense oligonucleotide sequence, such as one described by Matsukara, M. et al., Proc. Natl. Acad. Sci. USA 84: 7706-7710 (1987), which is expressly incorporated herein in its entirety by reference, is combined with the copolymer to form a micellar composition.

Briefly, phosphorothioate or methylphosphonate derivatives of a sequence complementary to regions of HIV art / trs genes having the sequence 5'-TCGTCGCTGTCTCG-3 'are prepared according to the method of Matsukura et al. 300 milligrams of CRL-8131 are added to 10 ml of 0.9% NaCl and the mixture is solubilized by storage at temperatures of 2-4^°C until a clear solution is formed. The desired antisense oligonucleotide is subsequently mixed with the copolymer solution to provide a concentration effective to inhibit antiviral activity when administered to a patient infected with the HIV virus. Generally, the effective amount of antisense compound will be such that the final concentration in the blood is in the range of 1 µΜ to 100 µΜ, although other effective amounts of antisense compounds outside of this range may be found for specific antisense compounds. One of ordinary skill in the art can easily test the relative efficacy of any particular antisense oligonucleetide according to the in vivo test of Matsukura et al.

An average person has approximately 6.25 liters of blood. Thus, oligonucleotide concentrations of about 6 mM to 600 mM are required in the composition when 1 ml injections are to be administered. Lower oligonucleotide compositions with higher delivery volumes can be used.

Example II

The antisense oligonucleotide anti-infective composition of Example I is administered to HIV patients by any route effective to reduce viral activity. The preferred route of administration is by intravenous injection. The antisense composition can be administered multiple times per day to ensure that an effective amount of the antisense oligonucleotide is maintained.

Example III

A gene therapy composition is made to treat an animal or human suffering from the effects of a defective or missing gene by combining a copolymer, such as CRL-8131, with a normal copy of the defective gene. For example, for patients suffering from adenosine deaminase deficiency

ES 2 189 808 T3 (ADA), a gene therapy composition is made that contains a normal copy of the adenosine deaminase gene. The gene therapy composition is made by mixing a copolymer prepared as previously described in Example I with the desired gene, withdrawing blood from the human or animal, transferring blood cells with the composition containing ADA gene, and reintroducing the blood cells. transfected into human or animal. The introduced gene is expressed in vivo, alleviating the effects of the original gene deficiency.

Example IV

Similarly, the gene therapy composition of Example III combines with isolated T lymphocytes to form T lymphocytes containing the ADA gene. T lymphocytes containing the ADA gene are subsequently administered, for example by injection, to the patient suffering from adenosine deaminase deficiency. The administered cells express ADA and produce adenosine deaminase, thus increasing the supply of the enzyme in the patient and correcting the deficiency.

Example V

DNA vaccination is carried out essentially as described for gene therapy in Examples III or IV, except that the gene that is introduced into the host expresses an antigenic gene product that is recognized as foreign by the host animal, thereby causing an immune response.

Example VI

A composition comprising CRL-8131 copolymer and an expression vector containing the herpes simplex virus type 1 gD gene was used in a transfection experiment. DNA transfection is normally performed using standard techniques of precipitation with calcium chloride and DEAE-dextran. DEAE-dextran is used to roughen the cell membrane and calcium is used to precipitate DNA on the cell surface, facilitating the uptake of DNA in cells. However, this procedure is generally toxic to cells and causes substantial cell mortality.

A new transfection system was discovered using the block copolymer of the present invention in place of caloric chloride. In fact, it was surprisingly found that copolymer-assisted transfection occurs even in the absence of DEAE-dextran.

Vero cells were incubated in DEAE-dextran for 30 seconds. A mixture of copolymer and an expression vector containing herpes simplex virus type 1 glycoproleia gD DNA was added to Vero cells immediately after removal of DEAE-dextran. Up to 40% of the cells were found to be efficiently transfected with the gD gene.

Surprisingly, in two out of four experiments, the copolymers were able to transfect Vero cells with less than 40% efficiency even when the DEAE-dextran incubation step was omitted. Example VII

Other studies have shown that block copolymers are effective in transferring genetic material across cell membranes in vivo. DNA vaccine-induced immunization was successful when an expression vector containing the herpes simplex virus type 1 gD gene combined with copolymer was injected intraperitoneally into rabbits every two weeks. Sera were collected and tested for the presence of anti-gD antibody. Low levels of anti-gD antibody were detected after 4 weeks of inoculation in this way. These results demonstrate that genetic material administered intraperitoneally with a copolymer delivery vehicle is collected by cells in vivo and expressed to give the gene product in amounts sufficient to elicit an immune response.

Example VIII

DNAse protection experiments

Five different compounds (CRL 1122, 3362, 3632, 9352 and 8131) were used in experiments to test the degree of protection. DNA was mixed with the compounds at 4 ° C and, after 15 minutes at 37 ° C, DNAse I (1 µl of 10 mg / ml solution) was added. After 30 minutes of incubation at 37 ° C,

ES 2 189 808 T3 removed DNAse I by treatment with proteinase K (3 µl of 10 mg / ml solution). Controls were: DNAse I in the absence of non-ionic block copolymer and DNA alone without any DNAse I treatment.

DNA was protected from degradation by DNAse I in all samples in which non-ionic block copolymers were present. The best DNA protection was achieved with CRL-3362 and 8131. The DNA-copolymer compositions did not migrate on horizontal agarose electrophoresis and remained within the wells (stained with ethidium bromide). An effective protection against the action of DNAse I was achieved in solutions of one volume of DNA solution (1 pg / ml) to 5 volumes of non-ionic block copolymer (30 pg / ml). The estimated amount of protection varied from experiment to experiment and was estimated to be within 15-40% of total DNA.

Additional experiments showed that the DNA-copolymer compounds did not transform competent E. coli cells through the calcium method. The phenol also did not dissolve the nonionic block copolymer separately from the DNA. DNA bound to NBC can be precipitated by adding 5 volumes of isopropyl alcohol.

Example IX

Transfection experiments

Topical transfection experiments for transient expression of herpes viral glycoprotein genes and other genes of interest involved the following procedure. Cells such as COS (African monkey kidney cells; CV1) are seeded in 6-well plates. Transfection is performed when the cells are 50-80% confluent (still in logarotmic growth phase). The cells are first washed with PBS buffer, incubated with 0.5 ml of DEAE-dextran solution (500 mg / ml) for 1-2 minutes, this solution is aspirated and DNA precipitate is added to the cells. . The DNA to be transfected is mixed for 30 minutes at room temperature with CaCl2 under controlled pH conditions to form a fine precipitate. This solution is mixed with 1 ml of growth medium (DMEM) and placed on the cells for 4 hours at 37 ° C. At this time, the cells are shocked with 15% glycerol and subsequently washed with PBS. This osmootic shock facilitates the collection of the CaCl2-DNA precipitate in the cells. Cells are then washed again with PBS and incubated with growth medium at 37^°C for 48 hours.

Genic expression is detected in most cases using specific monoclonal antibodies directed against the expressed proteones using indirect immunofluorescence. The expressed proteones can also be labeled with radioactive tracers and immunoprecipitated or detected on Western blots.

25 µl of DNA (7 µg) and 25 µl of non-ionic block copolymer (30 µg / ml) were used. Additionally, mixing the non-ionic block copolymer with DNA on ice and adding the mixture to the cells produced similar results as when they were added separately (DNA added first and non-ionic block copolymer second).

Copolymers 1183, 1187, 8131, 1235, 8950AQ and 1190AQ (where AQ indicates that the nonionic block copolymers were diluted 1:10 and 25 µl was used). Topical results are as follows. Transfection with DNA alone, dextran alone, copolymer alone, and DNA plus dextran had negligible transfection of less than 0.2%. In contrast, the positive control of DNA plus dextran plus glycerol has a transfection of 2% while various copolymers plus DNA were satisfactory to transfect DNA in cells up to 2.5 times better than the control, as shown in Table III:

IS 2 189 808 T3

Table ΙΠ

<td>Copolymer</td><td>Percentage of Transfection</td>

<td> 1183</td><td> 2%</td>

<td> 1187</td><td> 5%</td>

<td> 8131</td><td> 2%</td>

<td> 1235</td><td> 3%</td>

<td>8950AQ</td><td> 4%</td>

<td>1190AQ</td><td> 5%</td>

There was no toxicity associated with the copolymer, except mild toxicity with 1187. The others were toxic especially after glycerol treatment.

It should be understood that the foregoing refers only to preferred embodiments of the present invention and that numerous modifications and alterations can be made without departing from the spirit and scope of the invention as set forth in the appended claims.