WO2005123142A1

WO2005123142A1 - A method using inorganic nanoparticles as non-viral vectors for gene therapy

Info

Publication number: WO2005123142A1
Application number: PCT/US2005/020986
Authority: WO
Inventors: Dalia Chattopadhyay; Susmita Mitra; Amaranath Maitra
Original assignee: Abraxis Bioscience, Inc.
Priority date: 2004-06-10
Filing date: 2005-06-10
Publication date: 2005-12-29

Abstract

A method of treating a disease using gene therapy comprising: administering a non-viral vector to a subject, where in the non-viral vector comprises genetic material contained in an artificial plasmid that is encapsulated in a CaPi NP, MgPi NP, or MnPi NP of less than 100 nm in diameter.

Description

A METHOD USING INORGANIC NANOP ARTICLES AS NON-VIRAL VECTORS FOR GENE THERAPY

BACKGROUND OF THE INVENTION

[0001] Gene therapy is a technique that introduces genetic material into a cell so that it may be inserted into a cell's genetic pool, either to correct an underlying defect or to modify the characteristics of a cell via expression of the newly inserted gene. With the use of gene therapy the potential exists to treat, and in some instances cure, a variety of diseases and conditions. Vectors are required for intracellular delivery of genetic material (ie., RNA, DNA, antisense) and both viral and non-viral vectors have been utilized. While viral vectors generally have very high transfection rates, they suffer significant drawbacks, which include inherent difficulties in pharmaceutical processing, immunogenicity, difficulty in targeting specific cell types, and the possibility of reversion of an engineered virus to a wild-type virus. [0002] Due to the problems associated with viral vectors, there has been a growing interest in the development of non- viral vectors. Non- viral vectors are attractive as the methods of gene transfer utilizing non-viral vectors require only a small number of the gene of interest, have virtually infinite capacity, are non infectious, non-mutagenic, and have large scale production capabilities. Further, non-viral vectors may be adapted for use with pharmaceutical techniques. One drawback of non- viral vectors, however, is their low transfection efficiency due to exposure of the genetic material, such as DNA, to the hostile DNAse environment. This is due to electrostatic compaction of DNA with the polymeric materials typically used to produce non-viral vectors. While there are non-viral vectors that are suitably used for laboratory in vitro transfection, these vectors and their methods of use are not effective for in vivo applications.

[0003] What is needed is a method for introducing genetic material into a cell utilizing a non-viral vector that has high transfection efficiency and may be utilized to treat a variety of diseases and conditions. BRIEF SUMMARY OF THE INVENTION

[0004] The invention provides a method of treating a disease or condition using gene therapy comprising: administering a non- viral vector to a subject, where in the non- viral vector comprises genetic material contained in an artificial plasmid that is encapsulated in a CaPi NP, MgPi NP, or MnPi NP of less than 100 nm in diameter. [0005] Also provided is a method of suppressing gene expression comprising: administering a non- viral vector to a subject, where in the non- viral vector comprises genetic material contained in an artificial plasmid that is encapsulated in a CaPi NP, MgPi NP, or MnPi NP of less than 100 nm in diameter and wherein the genetic material is selected from the group consisting of genomic DNA, cDNA, RNA, siRNA, antisense DNA, antisense RNA, ribozyme, complimentary RNA DNA package with or without expression elements, a mini-gene, gene fragments, regulatory elements, promoters, and combinations thereof, wherein the genetic material administered to the subject has the effect of suppressing the expression of the gene of interest.

[0006] The invention further provides a method of augmenting gene expression comprising: administering a non- iral vector to a subject, where in the non- viral vector comprises genetic material contained in an artificial plasmid that is encapsulated in a CaPi NP, MgPi NP, or MnPi NP of less than 100 nm in diameter and wherein the genetic material is selected from the group consisting of genomic DNA, cDNA, RNA, siRNA, antisense DNA, antisense RNA, ribozyme, complimentary RNA/DNA package with or without expression elements, a mini-gene, gene fragments, regulatory elements, promoters, and combinations thereof, wherein the genetic material administered to the subject has the effect of augmenting the expression of the gene of interest.

[0007] The invention also provides a method of treating a disease or condition comprising: administering a non- viral vector to a subject, where in the non- viral vector comprises genetic material contained in an artificial plasmid that is encapsulated in a CaPi NP, MgPi NP, or MnPi NP of less than 100 nm in diameter and wherein the genetic material is selected from the group consisting of genomic DNA, cDNA, RNA, siRNA, antisense DNA, antisense RNA, ribozyme, complimentary RNA/DNA package with or without expression elements, a mini-gene, gene fragments, regulatory elements, promoters, and combinations thereof, wherein the genetic material administered to the subject has the effect of modulating expression of the gene of interest such that one or more symptoms of the disease or condition is ameliorated.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0008] Figure 1 is a graph showing the ELISA index of anti-liver specific protein for control, disease, and two treatment groups,

[0009] Figure 2 is a graph showing anti-liver specific protein counts for normal, disease, and treatment groups.

DETAILED DESCRIPTION OF THE INVENTION

[0010] The invention provides a method of treating a disease or condition using gene therapy comprising: administering a non- viral vector to a subject, where in the non- viral vector comprises genetic material contained in an artificial plasmid that is encapsulated in a CaPi NP, MgPi NP, or MnPi NP of less than 100 nm in diameter. [0011] The non-viral vector utilized in the inventive method must exhibit high transfection efficiency. Although other non-viral vectors may be suitable for use with the present invention, the preferred non-viral vector that exhibits high transfection efficiency while maintaining the integrity of the genetic material being introduced into the cell is disclosed in U.S. Patent No. 6,555,376, incorporated herein by reference. The inorganic nanoparticles of calcium, manganese, and magnesium, described in the '376 patent are suitable for use in vitro and in vivo and the process for their production is fully described therein. The nanoparticles according to the '376 patent, and used in the inventive method, are safe, efficient and cost effective non-viral vectors that are less than 100 nm in diameter and comprise plasmid genetic material (ie., pDNA) encapsulated in calcium nanoparticles (CaPi NP), magnesium nanoparticles (MgPi NP), or manganese phosphate nanoparticles (MnPi NP). The transfection efficiency of these non-viral vectors is high compared to that of the commercially available transfecting reagent Superfect™(Promega, USA). In addition, the genetic material is completely encapsulated therein and is effectively protected from degradation until it is exposed to the target site within the cell. Further, ligands may be attached to the surfaces of the nanoparticles in order target specific tissues of interest and to achieve cell-specific delivery. [0012] The genetic material used in the method is incorporated into a self-assembling plasmid. The genetic material includes genomic DNA, cDNA, RNA, siRNA, antisense DNA, antisense RNA, ribozyme, complementary RNA/DNA package with or without expression elements, a minigene, gene fragments, regulatory elements, promoters, translational start sites, and combinations thereof that may affect the expression of a protein, DNA, or RNA of interest. When a promoter is utilized, the promoter may be tissue-specific or non-tissue specific. Further, suitable promoters include promoters original to the gene of interest, artificial promoters, viral promoters, CMV promoter (cytomegalovirus), MMV promoter (mirabilis mosaic virus), SV40 promoter (simian virus 40), albumin promoter and ubiquitin promoter. The translational start may be either classical ribosome binding site or an internal ribosome binding site (IRS).

[0013] Preferably, the subject of the inventive method is a mammal, most preferably, a human. Even more preferably, the subject is a human suffering from one or more diseases or conditions that may be corrected or ameliorated using gene therapy. Preferably, the introduction of the genetic material contained in the non- viral vector results in the correction of a disease or condition. Such correction may be achieved by replacement of a mutant gene, by augmentation of the expression of a gene, or by suppression of the expression of a gene. The augmentation of a gene may achieve a therapeutic effect by increasing the expression level of an RNA/protein. For instance, a non- viral vector containing pDNA driving the expression of an RNA/protein of interest, such as the apoAl gene, in patients suffering from atherosclerosis, may lead to increased production of high density lipoprotein (HDL) and therefore have a therapeutic benefit of reducing plaque size. In contrast, suppression of a gene of interest may achieve a therapeutic effect by suppressing certain RNA/protein. For example, hyperlipidemia is associated with increased synthesis of cholesterol. Therefore a non-viral vector containing genetic material may be delivered to the liver to suppress cholesterol synthesis. For instance, ribozyme against apolipoprotein B, the structural protein of low density lipoprotein (LDL) has been shown to reduce LDL cholesterol in hypercholesterolemic rabbits (Enjoji M, Wang F, Nakamuta M, Chan L, Teng BB. Hum Gene Ther. 2000 Nov 20;11(17):2415-30. "Hammerhead ribozyme as a therapeutic agent for hyperlipidemia: production of truncated apolipoprotein B and hypolipidemic effects in a dyslipidemia murine model.").

[0014] Further, pharmacological suppression of tumor promoting proteins, such as vascular endothelial growth factor (VEGF), has been shown to be effective in treating cancer. Therefore, both augmentation and suppression of gene expression are important therapeutic tools that may supplement or replace currently used pharmacological treatments, and are made possible using the inventive method.

[0015] The correction of a disease or condition may result in one or more changes, such as, increased or decreased cytokine production, increased or decreased hormone production, as well as enhanced or suppressed enzymatic activity, production of stem cells, and immune modulators.

[0016] The correction of the disease or condition utilizing the inventive method may result in a cure of the disease or condition, or an amelioration of one or more symptoms associated with the disease or condition.. For instance, Niemann-Pick A (NPA) disease, a fatal lysosomal storage disorder, is caused by a deficiency in acid sphingomyelinase (ASM) activity resulting in cellular accumulation of sphingomyelin and cholesterol in the brain. A non- viral vector containing pDNA driving the expression of ASM may be introduced into a patient suffering from NPA thereby restoring normal ASM.

[0017] Although not exhaustive, diseases and conditions that may be treated using the inventive method include cystic fibrosis, hemophilia, SCID syndrome, Neimann-Pick A disease, atherosclerosis, myocardial infarction, hyperlipidemia, hepatitis, degenerative diseases, spinal cord injuries, autoimmune diseases, cancer, as well as diseases, conditions and disorders of the digestive, endocrine, reproductive, urinary, cardiovascular, and pulmonary systems.

[0018] In the inventive method, the non-viral vector is administered by any suitable route.

For instance, the treatment comprising the non-viral vector may be administered via intravenous injection, intraperitoneal injection, via site-specific delivery, and combinations thereof.

[0019] The non-viral vector utilized in the inventive method may further comprise a ligand to allow site-specific delivery of the non- viral vector to a target cell type. Suitable target cells include, without limitation, cells located in the heart, liver, pancreas, stomach, intestines, kidney, ovaries, testes, prostate, bladder, urinary tract, lungs, vasculature, brain, bone, bone marrow, spleen, thymus, lymph nodes, thyroid, adrenal gland, and combinations thereof.

[0020] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. EXAMPLE 1

[0021] This example demonstrates the use of calcium phosphate nanoparticles as a non- viral vector for gene therapy of experimental autoimmune hepatitis (EAH). [0022] Autoimmune hepatitis is a type of acute chronic hepatitis resulting from auto- antibodies produced against hepatocyte cell membrane lipoprotein hepatocyte nucleoprotein. Interleukin-2 (IL-2) is important for normal T-cell growth and function and is deficient in autoimmune hepatitis. Therefore, an experimental model of autoimmune hepatitis was developed to assess the effectiveness of gene therapy utilizing a non- viral vector. [0023] In this study, pUMVC3-mIL2 was used for therapeutic replacement of IL-2 in both free form, as well as after encapsulation in calcium phosphate nanoparticles. The disease status was assessed by estimation of serum anti-liver specific protein (anti-LSP), via ELISA and double immuno-diffusion technique. Antinuclear antibodies were also detected using immuno-fluorescence and estimation of circulating immune complex was determined using histopathological studies of liver tissue.

[0024] The disease model was developed using liver tissue from Bulb/c mice. The tissue was removed and washed in cold, sterile PBS at pH 7.2, homogenized and centrifuged at 5000 rpm for 30 minutes. The supernatant was ultracentrifuged fat 1,000,000 g for 1 hour and fractionated by gel chromatography (Sepharose G-50 column). The protein concentration was determined at 280 nm absorbance and further fractionated using Sephadex G-100 using saline tris buffer (pH 8.0). The first protein fraction was further purified by ultrafiltration using the LSP antigen for the development of EAH by intravenous inoculation for 8 weeks.

[0025] The animals were divided into 4 groups (n=6) designated as: B-l, B-2, B-3, and B-4. The B-l group served as an untreated control. The B-2 group represented the diseased animals. The B-3 group included diseased animals treated with pDNA loaded CaPi NP (5 μg pDNA/animal intravenously, with the second dose after 3 weeks) The B-4 group included diseased animals treated with free pDNA (5 μg pDNA/animal intravenously, with the second dose after 3 weeks). Treated animals were sacrificed 5 days after the last intravenous injection.

[0026] The ELISA index (Figure 1) and serum anti-LSP counts (Figure 2) indicated high levels of LSP in untreated, diseased animals (Group B-2) and in diseased animals treated with free pDNA (Group B-4), which is indicative of EAH. However, the levels were significantly reduced in the control animals (Group B-l) and in diseased animals treated with pDNA loaded CaPi NP (Group B-3). The histopathological results of hepatocyte degeneration and lymphocyte infiltration, shown in Table 1, were in accordance with the ELISA findings. [0027] Therefore, treatment using pDNA loaded CaPi NP is an effective means of treating EAH.

Table 1

[0028] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein. [0029] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. [0030] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

CLAIM(S):

1. A method of treating a disease or condition using gene therapy comprising: administering a non-viral vector to a subject, where in the non-viral vector comprises genetic material contained in an artificial plasmid that is encapsulated in a CaPi NP, MgPi NP, or MnPi NP of less than 100 nm in diameter.

2. The method of claim 1 wherein the genetic material is selected from the group consisting of genomic DNA, cDNA, RNA, siRNA, antisense DNA, antisense RNA, ribozyme, complimentary RNA/DNA package with or without expression elements, a mini- gene, gene fragments, regulatory elements, promoters, and combinations thereof.

3. The method of claim 2 wherein the promoter is tissue specific or non-tissue specific.

4. The method of claim 3 wherein the promoter is selected from the group consisting of an original promoter, artificial promoter, viral promoter, CMV promoter, MMV promoter, S V40 promoter, albumin promoter, and ubiquitin promoter.

5. The method of claim 1 wherein the subject is a mammal.

6. The method of claim 5 wherein the subject is a human.

7. The method of claim 2 wherein the treatment is selected from the group consisting of correction of a disease or condition.

8. The method of claim 7 wherein the correction of a disease or condition is achieved by one or more selected from the group consisting of augmentation of gene expression, suppression of gene expression, and replacement of a mutant gene.

9. The method of claim 8 wherein the disease or condition is selected from the group consisting of cystic fibrosis, hemophilia, SCID syndrome, Neimann-Pick A disease, atherosclerosis, degenerative diseases, myocardial infarction, hepatitis, spinal cord injury, hyperlipidemia, autoimmune disease, cancer, and disorders of the digestive, endocrine, reproductive, urinary, cardiovascular, and pulmonary systems.

10. The method of claim 8 wherein the non- viral vector is administered by a route selected from the group consisting of intravenous injection, intraperitoneal injection, site- specific injection, and combinations thereof.

11. The method of claim 10 wherein the non- viral vector further comprises a ligand for site-specific delivery of the non- viral vector to a target.

12. The method of claim 11 wherein the target is cells in a location selected from the group consisting of the heart, liver, pancreas, stomach, intestines, kidney, ovaries, testes, prostate, bladder, urinary tract, lungs, vasculature, brain, bone, bone marrow, spleen, thymus, lymph nodes, thyroid, adrenal gland, and combinations thereof.

13. The method of claim 8 wherein the correction of a disease or condition results in one or more of changes selected from the group consisting of increased cytokine production, decreased cytokine production, enhanced enzymatic activity, suppressed enzymatic activity, increased hormone production, decreased hormone production, stem cell production, and production of immune modulators.

14. The method of claim 1 wherein the treatment of the disease or condition results in a cure, or amelioration of one or more symptoms associated with the disease or condition.

15. A method of suppressing gene expression comprising: administering a non- viral vector to a subject, where in the non- viral vector comprises genetic material contained in an artificial plasmid that is encapsulated in a CaPi NP, MgPi NP, or MnPi NP of less than 100 nm in diameter and wherein the genetic material is selected from the group consisting of genomic DNA, cDNA, RNA, siRNA, antisense DNA, antisense RNA, ribozyme, complimentary RNA/DNA package with or without expression elements, a mini-gene, gene fragments, regulatory elements, promoters, and combinations thereof, wherein the genetic material administered to the subject has the effect of suppressing the expression of the gene of interest.

16. The method of claim 15 wherein suppression of the expression of the gene of interest results in a cure or an amelioration of one or more symptoms associated with a disease or condition.

17. A method of augmenting gene expression comprising: administering a non- viral vector to a subject, where in the non- viral vector comprises genetic material contained in an artificial plasmid that is encapsulated in a CaPi NP, MgPi NP, or MnPi NP of less than 100 nm in diameter and wherein the genetic material is selected from the group consisting of genomic DNA, cDNA, RNA, siRNA, antisense DNA, antisense RNA, ribozyme, complimentary RNA/DNA package with or without expression elements, a^'mini-gene, gene fragments, regulatory elements, promoters, and combinations thereof, wherein the genetic material administered to the subject has the effect of augmenting the expression of the gene of interest.

18. The method of claim 17 wherein augmentation of the expression of the gene of interest results in a cure or an amelioration of one or more symptoms associated with a disease or condition.

19. A method of treating a disease or condition comprising: administering a non- viral vector to a subject, where in the non- viral vector comprises genetic material contained in an artificial plasmid that is encapsulated in a CaPi NP, MgPi NP, or MnPi NP of less than 100 nm in diameter and wherein the genetic material is selected from the group consisting of genomic DNA, cDNA, RNA, siRNA, antisense DNA, antisense RNA, ribozyme, complimentary RNA/DNA package with or without expression elements, a mini-gene, gene fragments, regulatory elements, promoters, and combinations thereof, wherein the genetic material administered to the subject has the effect of modulating expression of the gene of interest such that one or more symptoms of the disease or condition is ameliorated.