siRNA Inhibition Of Human Antigen R Expression For Treatment of Cancer

Abstract

DNA dendrimers linked to both a targeting moiety and a siRNA which inhibits expression of Human antigen R (HuR), pharmaceutical compositions comprising the DNA dendrimers and methods of treatment and use employing the DNA dendrimers. Useful siRNA sequences are also disclosed.

Claims

1. A DNA dendrimer linked to a targeting moiety and to a siRNA which inhibits expression of human antigen R (HuR), wherein the siRNA is selected from the group consisting of SEQ ID NO: 1/SEQ ID NO: 3 and SEQ ID NO: 2/SEQ ID NO: 4.

2. (canceled)

3. The DNA dendrimer according to claim 1, wherein the targeting moiety is selected from the group consisting of folate, transferrin, anti-mesothelin antibody, and combinations thereof.

4. The DNA dendrimer according to claim 3, wherein the targeting moiety is folate and the siRNA is SEQ ID NO: 2/SEQ ID NO: 4.

5. A pharmaceutical composition comprising a DNA dendrimer according to claim 1 and at least one pharmaceutically acceptable excipient.

6. The pharmaceutical composition according to claim 5, wherein the targeting moiety is folate and the siRNA is SEQ ID NO: 2/SEQ ID NO: 4.

7. The pharmaceutical composition according to claim 5, which is formulated for injection or infusion.

8. A method for inhibiting proliferation of tumor cells which overexpress HuR and/or inhibiting growth of a tumor which overexpresses HuR, the method comprising administering to the tumor cells or tumor a composition comprising the DNA dendrimer of claim 1 in an amount effective to inhibit tumor cell proliferation, inhibit tumor growth and/or treat cancer.

9. (canceled)

10. The method according to claim 8, wherein the targeting moiety is selected from the group consisting of folate, transferrin, anti-mesothelin antibody, and combinations thereof.

11. The method according to claim 8, wherein the DNA dendrimer is administered in vitro.

12. The method according to claim 8, wherein the DNA dendrimer is administered in vivo.

13. The method according to claim 8, wherein the DNA dendrimer is administered to a patient with ovarian cancer.

14. The method according to claim 8, wherein the DNA dendrimer is administered by injection or infusion.

15. (canceled)

16. (canceled)

17. (canceled)

18. (canceled)

19. The method according to claim 10, wherein the targeting moiety is folate and the siRNA is SEQ ID NO: 2/SEQ ID NO: 4.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The FIGURE illustrates the effect of treatment with siHuR on ovarian cancer cell growth.

DETAILED DESCRIPTION

[0023] Before describing several exemplary embodiments of the invention, it is to be understood that the invention is not limited to the details of construction or process steps set forth in the following description. The invention is capable of other embodiments and of being practiced or being carried out in various ways.

[0024] Reference throughout this specification to “one embodiment,” “certain embodiments,” “various embodiments,” “one or more embodiments” or “an embodiment” means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Thus, the appearances of the phrases such as “in one or more embodiments,” “in certain embodiments,” “in various embodiments,” “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily referring to the same embodiment of the invention. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.

[0025] As used herein, the term “linked to a DNA dendrimer” and its equivalents refer to attachment of a non-dendrimer moiety to an arm of the DNA dendrimer by covalent or non-covalent binding. The linkage may involve formation of a chemical bond between the arm of the DNA dendrimer and the non-dendrimer moiety, or linkage may occur through hybridization of a nucleic acid attached to the non-dendrimer moiety to a complementary nucleic acid sequence on the dendrimer arm. Such hybridization bonding may be directly between the sequence on the non-dendrimer moiety and the sequence of the dendrimer arm, or it may be indirect hybridization in which a capture sequence hybridized to both the dendrimer arm and the sequence attached to the non-dendrimer moiety forms a hybridization “bridge” between the two.

[0026] As used herein, the term “inhibition of expression” and its equivalents refer to reduction in the level of mRNA transcribed from a gene. The reduction in mRNA may be either due to a lower level of transcription or to an increase in post-transcriptional destruction of the mRNA.

[0027] As used herein, the term “cancer-associated mRNA” and its equivalents refer to an mRNA that is increased in cancer cells as compared to normal cells. The increase in the mRNA may be due to an increased rate of transcription in cancer cells as compared to normal cells, amplification of a gene, or a reduction in post-transcriptional degradation of the mRNA resulting in longer mRNA half-life.

[0028] As used herein, the term “targeting moiety” and its equivalents refer to a molecule which recognizes and binds to a complementary molecule on the surface of a cell or tissue. Non-limiting examples of targeting moieties include antibodies, antibody fragments, binding proteins and peptides, receptors and ligands for receptors. A specific binding partner for the targeting moiety means that the targeting moiety does not bind substantially to other binding partners. In some cases the targeting moiety/receptor complex or the targeting moiety/specific binding partner complex may be internalized by the cell.

[0029] As used herein, the terms “inhibition of tumor cell proliferation,” “inhibition of tumor growth” and their equivalents refer to slowing of the overall rate of cell division in the tumor (which does not necessarily mean slowing of the rate of cell division in every cell in the tumor) with treatment such that the overall increase in tumor size over time is reduced compared to an increase in tumor size without treatment.

[0030] As used herein, the term “treatment of cancer” and its equivalents refer to inhibiting growth or spread of cancer, alleviating the symptoms of cancer, and/or substantially eliminating cancer from a patient.

[0031] Compositions comprising a DNA dendrimer linked to both a siRNA which inhibits expression of a cancer-associated mRNA and a targeting moiety are one aspect of the present invention. In general, the targeting moiety will specifically bind to a cell surface receptor or a cell surface protein on a cancer cell to deliver the siRNA to the targeted cancer cell. Any cell surface receptor or cell surface protein may be utilized for binding the targeting moiety of the DNA dendrimer composition. Use of targeting moieties that bind to cell surface receptors or cell surface proteins which are internalized after binding to the targeting moiety are advantageous to deliver the siRNA into the cell where it can more effectively degrade the complementary mRNA. Use of targeting moieties that bind to cell surface receptors and cell surface proteins that are over-expressed on the cancer cell are advantageous to provide improved specificity of siRNA action on degradation of the complementary mRNA in cancer cells as compared to normal cells which may also express the cell surface receptor or cell surface protein, albeit at lower amounts than on tumor cells.

[0032] The targeting moiety may be linked to an available arm of the DNA dendrimer by any of several known chemistries depending on the chemical nature of the targeting moiety. Examples include formation of a disulfide bridging bond, by N-hydroxysuccinimide ester dependent condensation, by use of a bifunctional cross-linking reaction, by direct or indirect hybridization of the targeting moiety to the arm of the DNA dendrimer, or by use of polycationic compounds to bridge the targeting moiety to the DNA dendrimer via charge-charge interactions. These linking reactions are described in WO 2010/017544 with respect to RNA, but may be applied to other appropriate targeting moieties. A nucleic acid targeting moiety may also be linked to the DNA dendrimer by blunt-end ligation using a bridging oligonucleotide as described in U.S. Pat. No. 8,685,899. Appropriate options for hybridization of the targeting moiety to the DNA dendrimer include appending a nucleic acid capture sequence to the targeting moiety which is complementary to a sequence at the end of the arm of the DNA dendrimer, as described in WO 02/33125.

[0033] The siRNA will generally be linked to a different available arm of the DNA dendrimer than the targeting moiety. The siRNA may be linked to an available arm of the DNA dendrimer by formation of a disulfide bridging bond, by N-hydroxysuccinimide ester dependent condensation, by use of a bifunctional cross-linking reaction, by direct or indirect hybridization of the siRNA to the arm of the DNA dendrimer, or by use of polycationic compounds to bridge the siRNA to the DNA dendrimer via charge-charge interactions. These linking reactions are described in WO 2010/017544. The siRNA may also be linked to the DNA dendrimer by blunt-end ligation using a bridging oligonucleotide as described in U.S. Pat. No. 8,685,899. Appropriate options for hybridization of the siRNA to the DNA dendrimer include appending a capture sequence to the RNA which is complementary to a sequence at the end of the arm of the DNA dendrimer, as described in WO 02/33125.

[0034] Any targeting moiety which binds to a cell surface receptor or cell surface protein on a cell type of interest may be linked to the DNA dendrimer. For anti-cancer therapeutic uses of the DNA dendrimer compositions, it will generally be desirable to select a targeting moiety which binds to a cell surface receptor or cell surface protein specifically expressed on cancer cells or over-expressed on cancer cells. Examples of such targeting moieties include folate, transferrin, and anti-mesothelin antibodies, among other suitable targeting moieties known to the person skilled in the art.

[0035] Any siRNA which hybridizes to and degrades a complementary cancer-associated mRNA expressed in a cancer cell of interest may be linked to the DNA dendrimer. Non-limiting examples of such mRNAs include HuR, which is over-expressed in ovarian cancer cells. Any siRNA which binds to HuR mRNA and results in its degradation is appropriate for use in the DNA dendrimer compositions. Specific non-limiting examples of useful siRNAs that may be linked to the DNA dendrimer along with the targeting moiety include SEQ ID NO:1/SEQ ID NO:3 and SEQ ID NO:2/SEQ ID NO:4. In further non-limiting examples, siRNAs SEQ ID NO:1/SEQ ID NO:3 or SEQ ID NO:2/SEQ ID NO:4 may be linked to the DNA dendrimer with folate linked to the DNA dendrimer as the targeting moiety.

[0036] In any of the foregoing DNA dendrimer compositions, the linkage of the targeting moiety and the siRNA to the DNA dendrimer may be independently selected from the group consisting of direct linkage to the arms of the DNA dendrimer via chemical conjugation and hybridization between a capture oligonucleotide ligated to the arm of the DNA dendrimer and a complementary carrier oligonucleotide linked to the targeting moiety and/or siRNA.

[0037] In specific embodiments of any of the foregoing DNA dendrimer compositions, the targeting moieties and/or the siRNAs may be linked to the DNA dendrimer via a capture oligonucleotide associated with the arm of the DNA dendrimer. The capture oligonucleotide is generally associated with the terminus of the DNA dendrimer arm. Typically it is ligated to the terminus of the arm of the DNA dendrimer, but it may also be hybridized to the terminus and optionally crosslinked thereto or associated with the arm by use of an extension oligonucleotide as described below. The capture oligonucleotide provides a specific, defined sequence present in a defined quantity for hybridization to a complementary carrier oligonucleotide linked to the targeting moiety and/or the siRNA. The capture oligonucleotide also provides a means for controlling the number of targeting moieties and siRNAs linked to the DNA dendrimer, as the carrier oligonucleotides can be hybridized to the capture oligonucleotide at a defined concentration which results in the desired number of DNA dendrimer arms being occupied by each component. Upon hybridization of the complementary carrier oligonucleotide, the targeting moiety and/or siRNA becomes linked to the arm of the DNA dendrimer through Watson-Crick base pairing.

[0038] Any of the foregoing DNA dendrimer compositions may be formulated as pharmaceutical formulations with pharmaceutically acceptable excipients. Such excipients may include one or more component selected from the group consisting of a solvent, a dispersing agent, a coating (e.g., lecithin), a surfactant (e.g., hydroxypropylcellulose), a preservative (e.g., paraben, phenol, thimerosal, sorbic acid, chlorobutanol), an emulsion, an alcohol (e.g., ethanol), a polyol (e.g., glycerol, propylene glycol), and an isotonic agent (e.g., sugars, sodium chloride).

[0039] The pharmaceutical formulations comprising the DNA dendrimer compositions may also be adapted for specific routes of administration as is known in the art. As non-limiting examples, the pharmaceutical formulations may be formulated as solutions for injection or infusion, or as tablets for oral administration. Solutions or dispersions of the DNA dendrimer compositions may be prepared, for example, in water or saline.

[0040] Pharmaceutical formulations comprising the DNA dendrimer compositions are useful for treatment of cancer in a patient, inhibiting tumor cell proliferation and/or inhibiting tumor growth. Methods for inhibiting tumor cell proliferation and/or inhibiting tumor growth may be applied either in vivo, for treatment of cancer patients, or in vitro, such as for research or diagnostic purposes. In general, the amount of the pharmaceutical formulation administered to a cancer patient in vivo will be an amount effective to inhibit tumor cell proliferation, inhibit tumor growth, and/or treat the cancer in the patient. The effective amount will be based on a combination of the dose administered, the duration of administration, the administration protocol, and the route of administration. One skilled in the art will recognize that individualization of dosage based on a patient's body composition or his/her response to treatment may be medically necessary or desirable.

[0041] Methods for treatment of cancer in a patient may include administration to the subject by parenteral, intraperitoneal, intravenous, intratumoral, or oral routes. Particularly useful routes of administration include injection, infusion, or oral administration. For treatment of cancer, a typical course of administration may include IV administration continuously or intermittently over a course of several weeks, often followed by a period of no administration and then at least one repetition of the intermittent administration schedule.

Examples

[0042] In Vitro Assays for siRNA Stability, Functionality and Biodistribution:

[0043] siHuR stability was evaluated in PBS, serum free medium and medium+10% serum. 10% TBU PAGE of siHuR showed that the sense and antisense strands were not degraded in any of the samples.

[0044] For evaluation of functionality, 2-layer DNA dendrimers linked to folate, the siHuR sequence and a fluorescent label (Cy3) in various combinations were prepared. siRNA stability was tested by transfecting cells with the following: siHuR+Lipofectamine, DNA dendrimer linked to folate, and DNA dendrimer linked to both folate and siHuR. Controls were untransfected cells and Lipofectamine only.

[0045] siHuR (SEQ ID NO:2/SEQ ID NO:4) was combined with either lipofectamine or the targeted DNA dendrimer and incubated overnight with A2780 cells (a human ovarian tumor cell line) in a medium containing 10% serum. HuR mRNA expression in each case was compared to untransfected cells (100% HuR expression). Results are shown in the following Table:

TABLE-US-00002 Targeted Targeted Untransfected Lipofectamine Lipofectamine + Dendrimer Dendrimer + Cells only siHuR only siHuR % HuR 100 96 35.4 94 26.8 Expression

[0046] Targeting of siHuR to ovarian cancer cells using folate linked to a DNA dendrimer significantly improved HuR mRNA knock-down compared to transfection of siHuR alone.

[0047] A biodistribution study in ovarian tumor bearing mice done with systemically administered DNA dendrimer linked to CY3 and to folate showed very high fluorescence in ovarian tumors with little to no fluorescence in normal tissues (brain, liver, spleen, lung, heart and normal ovary). These results suggest that specificity of cancer treatment may be achieved using folate as the targeting moiety linked to the DNA dendrimer for cancers that over-express the folate receptor.

[0048] In Vivo Testing for Inhibition of Ovarian Tumor Growth: Ovarian tumor bearing mice were injected twice per week intraperitoneally with 2 μg siHuR (SEQ ID NO:1/SEQ ID NO:3) as: DNA dendrimer linked to folate and to siHuR, DNA dendrimer linked to siHuR, and DNA dendrimer linked to folate and a negative control siRNA. Controls were underivatized DNA dendrimer and 0.9% saline (untreated). Injections were continued for four weeks.

[0049] The model system was C57BL/6 female mice injected i.p. with ID8-Fluc murine ovarian cancer cells, six mice per treatment group. Tumors developed throughout the peritoneal cavity. Approximately 4 weeks following injection, tumor-bearing mice were optically imaged to establish baseline bioluminescence, an indicator of ID8-luc-derived tumor load, then were treated twice a week for four weeks (retro-orbital injection) with one of four DNA dendrimer formulations or with 0.9% saline (n=5-6/group). The four DNA dendrimer formulations were: 1) DNA dendrimer linked to folate and to siHuR, 2) DNA dendrimer linked to siHuR; 3) DNA dendrimer linked to folate and to a negative control siRNA; and 4) underivatized DNA dendrimer. Tumor response to treatment was assessed once a week by optical imaging. Results are shown in the FIGURE. Using baseline tumor load as the comparator, tumor growth was suppressed ˜3-fold in mice treated with DNA dendrimer linked to folate and to siHuR for 4 weeks. This was the highest suppression of all treatment groups, including DNA dendrimer linked to siHuR (p=0.13), and it was substantially better than in mice treated with DNA dendrimer linked to folate and negative control siRNA (p=0.22), underivatized DNA dendrimer (p=0.10), or 0.9% saline (p=0.10). These results support the enhanced therapeutic effect that may be observed for treatment of cancer using DNA dendrimer linked to folate and to siHuR.

[0050] Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It will be apparent to those skilled in the art that various modifications and variations can be made to the method and apparatus of the present invention without departing from the spirit and scope of the invention. Thus, it is intended that the present invention include modifications and variations that are within the scope of the appended claims and their equivalents.