WO2009104051A2 - Combinational therapeutics for treatment of prostate cancer using epoxy encapsulated magnetic particles and rnai medicine - Google Patents

Combinational therapeutics for treatment of prostate cancer using epoxy encapsulated magnetic particles and rnai medicine Download PDF

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WO2009104051A2
WO2009104051A2 PCT/IB2008/003661 IB2008003661W WO2009104051A2 WO 2009104051 A2 WO2009104051 A2 WO 2009104051A2 IB 2008003661 W IB2008003661 W IB 2008003661W WO 2009104051 A2 WO2009104051 A2 WO 2009104051A2
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sirna
genes
cocktail
pro
molecule
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WO2009104051A3 (en
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Patrick Y. Lu
Ashgar Ghias
David Evans
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Lu Patrick Y
Ashgar Ghias
David Evans
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    • C12N2320/32—Special delivery means, e.g. tissue-specific

Definitions

  • the present invention provides compositions and methods for treatment of human prostate adrenocarcinoma and other types of prostate cancers, using RNA interference (RNAi) and Epoxy-Encapsulated Magnetic Particle combinational therapeutics.
  • RNAi RNA interference
  • Epoxy-Encapsulated Magnetic Particle combinational therapeutics RNA interference
  • Prostate cancer is a significant health problem in most industrialized Western countries, where it is the most commonly diagnosed cancer affecting men after middle age.
  • the worldwide 5 -year prevalence of prostate cancer has been estimated at 1,554,700 cases. It is estimated that, in Western countries, about 30% of all men will develop microscopic prostate cancer during their lifetime.
  • the risk of developing overt clinical disease is 8% (lifetime risk), and the risk of actually dying from prostate cancer is only 3%, whereas the autopsy based prevalence is 80% by the age of 80 years. Therefore, most men die with prostate cancer, rather than from it.
  • Prostate cancer is primarily a disease of men over the age of 50 years, and the trend towards an ageing worldwide FCi/ ⁇ _. u 8 / 0 0 3 6 6 1
  • prostate cancer is increasing at an average rate of 3% a year.
  • the incidence of prostate cancer varies from country to country, with the highest incidences being found in the Western world and the lowest being found in Asia. Data for the year 2000 identify, that whereas the incidence in the USA was 140 per 100,000, for Japan it was 22 per 100,000 and for China it was 1.54 per 100,000.
  • Prostate cancer has become one of the leading male cancers in some Asian countries with the incidence having risen rapidly in the last 20 years. The reasons for this high degree of variability between ethnic groups are probably multi-factorial and include the availability of improved detection methods, increasing westernisation of lifestyle and in particular genetic risk factors.
  • the stage distribution at the time of diagnosis also varies around the world.
  • Pelvic lymphadenectomy A surgical procedure to remove the lymph nodes in the pelvis.
  • Radical prostatectomy A surgical procedure to remove the prostate, surrounding tissue, and seminal vesicles. There are 2 types of radical prostatectomy:
  • Retropubic prostatectomy A surgical procedure to remove the prostate through an incision (cut) in the abdominal wall. Removal of nearby lymph nodes may be done at the same time.
  • Perineal prostatectomy A surgical procedure to remove the prostate through an incision (cut) made in the perineum (area between the scrotum and anus). Nearby lymph nodes may also be removed through a separate incision in the abdomen.
  • Transurethral resection of the prostate A surgical procedure to remove tissue from the prostate using a resectoscope (a thin, lighted tube with a cutting tool) inserted through the urethra. This procedure is sometimes done to relieve symptoms caused by a tumor before other cancer treatment is given. Transurethral resection of the prostate may also be done in men who cannot have a radical prostatectomy because of age or illness. Impotence and leakage of urine from the bladder or stool from the rectum may occur in men treated with surgery. In some cases, doctors can use a technique known as nerve-sparing surgery. This type of surgery may save the nerves that control erection. However, men with large tumors or tumors that are very close to the nerves may not be able to have this surgery.
  • Radiation therapy is a cancer treatment that uses high-energy x-rays or other types of radiation to kill cancer cells or keep them from growing. There are two types of radiation therapy. External radiation therapy uses a machine outside the body to send radiation toward the cancer. Internal radiation therapy uses a radioactive substance sealed in needles, seeds, wires, or catheters that are placed directly into or near the cancer. The way the radiation therapy is given depends on the type and stage of the cancer being treated. Impotence and urinary problems may occur in men treated with radiation therapy.
  • Hormone therapy is a cancer treatment that removes hormones or blocks their action and stops cancer cells from growing. Hormones are substances produced by glands in the body and circulated in the bloodstream. In prostate cancer, male sex hormones can cause prostate cancer to grow. Drugs, surgery, or other hormones are used to reduce the production of male hormones or block them from working. Hormone therapy used in the treatment of prostate cancer may include the following: Luteinizing hormone-releasing hormone agonists can prevent the testicles from producing testosterone. Examples are leuprolide, goserelin, and buserelin.
  • Antiandrogens can block the action of androgens (hormones that promote male sex characteristics). Two examples are flutamide and nilutamide. Drugs that can prevent the adrenal glands from making androgens include ketoconazole and aminoglutethimide.
  • Orchiectomy is a surgical procedure to remove one or both testicles, the main source of male hormones, to decrease hormone production.
  • Estrogens hormones that promote female sex characteristics
  • Estrogens can prevent the testicles from producing testosterone.
  • estrogens are seldom used today in the treatment of prostate cancer because of the risk of serious side effects.
  • Hot flashes, impaired sexual function, loss of desire for sex, and weakened bones may occur in men treated with hormone therapy.
  • Cryosurgery is a treatment that uses an instrument to freeze and destroy prostate cancer cells. This type of treatment is also called cryotherapy.
  • Chemotherapy is a cancer treatment that uses drugs to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing.
  • chemotherapy is taken by mouth or injected into a vein or muscle, the drugs enter the bloodstream and can reach cancer cells throughout the body (systemic chemotherapy).
  • systemic chemotherapy When chemotherapy is placed directly into the spinal column, an organ, or a body cavity such as the abdomen, the drugs mainly affect cancer cells in those areas (regional chemotherapy). The way the chemotherapy is given depends on the type and stage of the cancer being treated.
  • Biologic therapy is a treatment that uses the patient's immune system to fight cancer. Substances made by the body or made in a laboratory are used to boost, direct, or restore the body's natural defenses against cancer. This type of cancer treatment is also called biotherapy or immunotherapy.
  • High-intensity focused ultrasound is a treatment that uses ultrasound (high-energy sound waves) to destroy cancer cells.
  • ultrasound high-energy sound waves
  • an endorectal probe is used to make the sound waves.
  • TGF- ⁇ Transforming Growth Factor
  • TGF- ⁇ transforming growth factor- ⁇
  • PSA serum prostate-specific antigen
  • TGF- ⁇ pathway plays dual roles in cancer, inhibiting epithelial cell growth under normal physiologic conditions, but promoting invasion and metastasis once growth inhibitory responses are lost (N. Sharifi, 2007).
  • TGF-beta is a pleiotropic growth factor. It plays an important role in the regulation of growth and differentiation in many cells. In benign prostatic epithelia, its action is mediated through a paracrine mechanism. It inhibits proliferation and induces apoptosis in prostatic epithelia.
  • TGF-beta may regulate the development of prostatic smooth muscle nodules in benign prostatic hyperplasia.
  • TGF-beta action two major events occur regarding TGF-beta action. These include the loss of expression of functional TGF-beta receptors and overproduction of TGF-beta in malignant cells. The loss of expression of functional TGF-beta receptors provides a growth advantage to cancer cells over their benign counterparts. The overproduction of TGF-beta by cancer cells has a multitude of adverse consequences.
  • TGF-beta can promote extracellular matrix production, induce angiogenesis, and inhibit host immune function. The biological consequence of these activities is an enhanced tumorigenicity in prostate cancer. Results of studies with a rat prostate cancer model suggest that the immunosuppressive effect of TGF-beta seems to be the primary cause of tumor progression. This is because, if these cancer cells were engineered to reduce the production of TGF-beta, tumor growth was inhibited in syngeneic hosts but not in immune compromised hosts (Lee C, 1999).
  • Cyclooxygenase is a key enzyme in the production of prostaglandins (PGs) and other eicosanoids from arachidonic acid.
  • PGs prostaglandins
  • COX-2 was initially found as an early growth responsive gene that modulates cell adhesion, apoptosis, proliferation, and differentiation.
  • a variety of cytokines and growth factors, as well as several oncogenes, can bind to the promoter region of COX-2 to upregulate its transcription in most cells. More recently, it has been suggested that PGs and other eicosanoids produced by COX play an important role in the development and progression of human cancers.
  • COX-2 potential mechanistic roles of COX-2 in tumorigenesis and tumor progression include (1) decreased apoptosis; (2) increased angiogenesis; (3) increased tumor invasiveness; and (4) decreased immune surveillance.
  • Overexpression of COX-2 has been observed in many cancers, including prostate cancer. Consequently, suppression of COX-2 by specific inhibitors or other types of nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit the activity of COX-2, may lead to inhibition of tumor growth.
  • NSAIDs nonsteroidal anti-inflammatory drugs
  • COX-2 expression has been studied in prostate cancer tissues, and many studies showed increased expression of COX-2 in prostate cancer and prostatic intraepithelial neoplasia (PIN) compared with normal or hyperplastic prostate. In vitro and animal studies have suggested the involvement of COX-2 in prostate tumorigenesis and cancer progression.
  • COX-2 The enzyme activity and protein expression of COX-2 were found to be significantly higher in the dorsolateral prostate of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model at 8, 16, and 24 wk of age, compared with their nontransgenic littermates.
  • LNCaP cells with stably overexpressed COX-2 increased cell proliferation in vitro and tumor growth in vivo as compared with parental LNCaP cells, which was associated with increased expression of vascular endothelial growth factor (VEGF). Based on these results, it has been suggested that inhibition of COX-2 may be a useful chemopreventive/ therapeutic option for prostate cancer.
  • VEGF vascular endothelial growth factor
  • COX-2 expression was detected in androgen-sensitive LNCaP cells, as well as in androgen-insensitive PC-3 and DU 145 cells. Inhibition of COX-2 by selective inhibitors has been shown to induce growth arrest and apoptosis in both LNCaP and PC-3 cells. In animal models of prostate carcinogenesis and metastasis, COX-2 inhibitors suppressed tumor growth and metastases. These in vivo studies suggest that tumor growth suppression is achieved by a combination of tumor cell apoptosis induction and decrease in angiogenesis via downregulation of PGE2 and VEGF. However, a COX-2 independent pathway might be involved in proapoptotic and antitumor effects of NSAIDs and selective COX-2 inhibitors. Epidemiological studies and clinical trials indicated that in prostate cancer COX-2 inhibitors may have chemopreventive effects (Miyamoto et al., 2005)
  • MMP-9 Matrix Metallopeptidase 9
  • MMP-2 and MMP-9 belong to the gelatinase subfamily, are known to specifically cleave type IV collagen, the major component of basement membranes.
  • MMP activity is regulated by the transcription factor, nuclear factor-kB (NF-kB), a member of a family of dimeric transcription factors that contains a conserved ReI homology domain, allowing for dimerization, and a DNA binding domain.
  • NF-kB nuclear factor-kB
  • IkB inhibitor protein
  • IkB kinase is activated and phosph orylates IkB. NF-kB is then released, translocates to the nucleus, and binds to DNA.
  • NF-kB induces expression of target genes such as MMPs.
  • MMPs play a key role in multiple steps of tumor prog ression, including tumor promotion, angiogenesis, invasion, and metastasis.
  • a number of clinical and experimental studies have suggested that changes in MMP levels affect the invasive behavior of tumor cells and their metastatic potential.
  • MMPs can be produced not only by tumor cells but also by surrounding stromal cells and infiltrating inflammatory cells. It was demonstrated a positive correlation between increased expression/activity of MMP-2/MMP-9 and malignant potential of prostate cancer, as judged by Gleason score, pathological stage, or patient survival.
  • PDGF Platelet-derived growth factor
  • PDGF was one of the first polypeptide growth factors identified that signals through a cell surface tyrosine kinase receptor (PDGF-R) to stimulate various cellular functions including growth, proliferation, and differentiation(George_D. 2001). Since then, several related genes have been identified constituting a family of ligands (primarily PDGF A and B) and their cognate receptors (PDGF-R alpha and beta). To date, PDGF expression has been shown in a number of different solid tumors, from glioblastomas to prostate carcinomas. In these various tumor types, the biologic role of PDGF signaling can vary from autocrine stimulation of cancer cell growth to more subtle paracrine interactions involving adjacent stroma and even angiogenesis.
  • PDGF-R cell surface tyrosine kinase receptor
  • the tyrosine kinase inhibitor imatinib mesylate (Gleevec, Novartis) with blocks activity of the Bcr-Abl oncoprotein, is also a potent inhibitor of the PDGF-R kinase and is currently being evaluated for the treatment of PDGF-responsive tumors such as prostate cancer. More clinical trials that investigate both established clinical endpoints of response and benefit, as well as surrogate endpoints that may describe the biologic significance of PDGF-R inhibition in vivo are needed to expand the applications that target the PDGF axis.
  • a study characterizing PDGFR-beta expression in a wide spectrum of PCa samples has provided empirical data as part of a rational treatment strategy (Hofer MD, et al 2004). However, through a survey of five published prostate expression array studies, including 100 clinically localized PC and protein expression of
  • Fibroblast Growth Factor 2 Members of the fibroblast growth factor (FGF) family are believed to play critical roles during organogenesis and carcinogenesis via signaling between epithelial and stromal compartments. Recent studies (Abate-Shen C, et al. 2007) underscore the importance of FGF signaling in mediating epithelial-stromal interactions during prostate carcinogenesis. The experimental results show that deregulated FGF signaling in mouse models of prostate cancer leads to cancer progression and promotes an epithelial-mesenchymal transition, suggesting that FGF receptor inhibitors may have therapeutic value for prostate cancer treatment. Transforming growth factor-beta (TGF-beta) is overexpressed in most adenocarcinomas including prostate cancer.
  • TGF-beta Transforming growth factor-beta
  • TGF-beta regulates cell proliferation, phenotype and matrix synthesis. Additionally, decreased cellular fibroblast growth factor-2 (FGF-2) immunostaining was associated with attenuated TGF-beta signaling in stroma. In vitro, TGF-beta stimulated stromal FGF-2 expression and release. However, stromal cells with attenuated TGF-beta signaling were refractory to TGF-beta-stimulated FGF-2 expression and release. Re-expression of FGF-2 in these stromal cells in the mouse xenografts resulted in restored tumor mass and microvessel density.
  • FGF-2 fibroblast growth factor-2
  • Phosphoinositide 3-Kinase PI3K
  • Akt Phosphoinositide 3-Kinase
  • Akt/PKB Activated PI3K and its downstream target Akt/PKB are important signaling molecules and key survival factors involved in the control of cell proliferation, apoptosis and oncogenesis (Shukla S, et al. 2007).
  • PI3K-Akt signaling pathway was investigated the role of the PI3K-Akt signaling pathway in the invasion of prostate cancer cell lines and activation of this pathway in primary human prostate tumors.
  • Akt also significantly inhibited invasion and downregulated protein expression of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9, markers for cell invasion, indicating a central role of the PI3K-Akt pathway in this process.
  • uPA urokinase-type plasminogen activator
  • MMP matrix metalloproteinase
  • RNA interference is a sequence-specific RNA degradation process that provides a relatively easy and direct way to knockdown, or silence, theoretically any gene (13, 14).
  • RNA interference a double stranded RNA is cleaved by an RNase Ill/helicase protein, Dicer, into small interfering RNA (siRNA) molecules, a dsRNA of 19-23 nucleotides (nt) with 2-nt overhangs at the 3' ends.
  • siRNA small interfering RNA
  • RISC RNA-induced-silencing-complex
  • siRNA-directed endonuclease digests the RNA, thereby inactivating it.
  • siRNA sequences that can be used to treat disease there is a need to test these reagents in a variety of in vitro and in vivo experiments to ensure potency and efficacy. Since the therapeutics are initially aimed at humans it is obviously important that the initial screens in vitro use a cell model that is derived from human tissues and are representative of the disease etiology. siRNAs are introduced into these cells and an effect on the cell phenotype (viability, protein expression or gene expression alterations) can be monitored.
  • the degree of sequence complementarity between the siRNA and the target gene should be the same between the two species.
  • the siRNA designed needs to limit potential cross reactivity versus other genes in either species.
  • siRNA duplexes make it possible to be combined with multiple siRNA duplexes to target multiple disease causing genes in the same treatment, since all siRNA duplexes are chemically homogenous with same source of origin and same manufacturing process (13, 16). Many types of human diseases, including cancer, inflammatory conditions, autoimmune diseases and infectious diseases are able to be treated with much better clinical efficacy using such potent siRNA inhibitors with minimum toxicity and safety concerns (14, 15).
  • RNAi to silence genes involved in the angiogenesis, anti-apoptosis, cell proliferation and tumorigenesis, such as the profibrotic factor TGF- ⁇ , the inflammation promoter COX-2, Matrix Metallopeptidase 9 (MMP-9) , Platelet-derived growth factor (PDGF), Fibroblast Growth Factor 2 (FGF2), Phosphoinositide 3-Kinase (PI3K) and Akt, we can achieve enhanced anti-tumor activity.
  • TGF- ⁇ profibrotic factor
  • MMP-9 Matrix Metallopeptidase 9
  • PDGF Platelet-derived growth factor
  • FGF2 Fibroblast Growth Factor 2
  • PI3K Phosphoinositide 3-Kinase
  • siRNA cocktail with three siRNA duplexes targeting three genes, TGF- ⁇ , COX-2 and PDGFR, or COX-2, PDGFR and FGF2, or COX-2, PDGFR and PI3K will represent very power therapeutic approaches to treat prostate cancer.
  • Successful siRNA-mediated therapy not only depends on identification of the targets and sequence of active siRNA molecules, but more importantly on efficient in vivo delivery to the target tissues and into the cytoplasm (11-13).
  • the routes of delivery of siRNA cocktail formulation for treatment of skin wound healing should be local and topical with appropriate clinically validated carriers.
  • lipid-based carriers including histidine-lysine polymers (HKP) (14), pegylated PEI (15), PAMAM dendrimer (16) and DOTAP/DOPS which were developed in our lab and from our collaborators, for further enhancement of therapeutic efficacy of the treatment.
  • HTP histidine-lysine polymers
  • PEI pegylated PEI
  • PAMAM dendrimer 16
  • DOTAP/DOPS DOTAP/DOPS
  • Nanotechnology deals with the understanding and control of matter at dimensions of roughly 1 to 100 run. At the nanoparticle level materials often exhibit unique properties affecting their physical, chemical, and biologic properties. The challenge in using nanotechnology is to find application in science and remove any potential health hazards. Only after the substances are benign can we apply the nanoparticles in health care as useful products. Using submicron Fe3O4 nanoparticle aggregates encapsulated in a hydrophobic epoxy matrix that is benign and nontoxic, but possesses super-paramagnetic properties. One application of these particles can be the utilization in anti-angiogenesis therapy to pinch off the blood supply of a tumor. In addition, the magnetic nanoparticle can also be used for siRNA delivery.
  • the present invention provides compositions and methods for treatment of prostate cancer using siRNA cocktail sequences targeting multiple disease causing genes, and a combined regimen with both RNAi therapeutics and EEMP encapsulated magnetic nanoparticle therapeutics.
  • the invention includes: 1) The algorithm for designing siRNA drug API (active pharmaceutical ingredient); 2)The compositions of multiple siRNA inhibiting expression of the multiple cancer causing genes; 3) The methods of designing siRNA duplexes with different lengths, different ends and to target the same genes from both human and mouse cells; 4) The methods of formulating various siRNA cocktail which are able to inhibit tumorigenic and angiogenesis; 5) The methods of using histidine-lysine copolymer (HKP), PAMAM dendrimer and Liposomal (DOTAP) carrier to enhance siRNA delivery into the cutaneous tissue; and 6) The methods of using two disease relevant mouse xenograft models to validate the siRNA cocktail for its efficacy on controlling tumor growth.
  • siRNA drug API active pharmaceutical ingredient
  • the small interfering RNA (siRNA) duplexes can be blunt ended or with over hangs at the length of 19 to 27 nt.
  • the siRNA sequences are able to target the same gene of both human and mouse, or non-human primate.
  • three siRNA can be used as a cocktail containing at least three siRNA duplexes and targeting at least three different genes.
  • the efficacy of a siRNA cocktail must be tested and confirmed in the animal disease models.
  • five therapeutic benefit of a siRNA cocktail must be better thanthe single siRNA agent which is part of the cocktail components.
  • the metal- based nanoparticle can be used for siRNA drug and other drug delivery.
  • the Epoxy-Encapsulated Magnetic Particle is used in the treatment of prostate adrenocarcinoma with siRNA cocktail combinational therapeutics.
  • the invention provides siRNA sequences targeting 1) gene expression promote cell proliferation; 2) gene expression promote angiogenesis activity; 3) gene expression promote anti-apoptotic activity and 4) gene expression promote oncogenic effects.
  • the invention also provides method to design the proper ratio of each duplex in order to allow the siRNA cocktail to achieve the most potent synergistic effect.
  • the invention also provides the siRNA cocktail for treatment of prostate cancer with EEMP-mediated anti-angiogenesis activity.
  • the invention further provides the nanoparticle-enhanced siRNA cocktail delivery for treatment of prostate cancer.
  • FIG. 1 shows RT-PCR detection of siRNA-mediated knockdown of target gene (TGF-beta T) expression in PC-3 cells.
  • Figure 2 shows RT-PCR detection of siRNA-mediated knockdown of target gene (Cox-2) expression in PC-3 cells.
  • Figure 3 shows RT-PCR detection of siRNA-mediated knockdown of target gene (Cox- 2) expression in the C 166 Yolk-Sac Endothelial mouse cells.
  • Figure 4 shows RT-PCR detection of VEGFa, VEGFR2, PDGFa, FGF2 and MMP-2 gene expression in both human (PC-3) and mouse (CT-26) cells.
  • Figure 5 shows RT-PCR detection of siRNA-mediated knockdown of target gene (MMP-9) expression in the CT26 cells.
  • Figure 6 shows RT-PCR detection of siRNA-mediated knockdown of target gene (MMP-9) expression in the PC3 cells.
  • Figure 7 shows step-by-step procedures for preparation of Epoxy-Encapsulated Magnetic Nanoparticle.
  • FIG. 8 shows step-by-step flow chart for preparation of Epoxy-Encapsulated Magnetic
  • Figure 9 shows a schematic drawing of Epoxy-Encapsulated Magnetic Nanoparticle following acid wash.
  • Figure 10 shows a comparison of mouse skin excision wound healing rate.
  • Figure 11 shows RT-PCR analyses of total RNA from wound tissue harvested at day 7 post wounding.
  • Figure 12 shows a comparison of wound closure rate between HKP TGF ⁇ l siRNA treated and control wounds.
  • Figure 13 shows the effects of HKP Cox-2 siRNA on collagen structure in healing wounds.
  • Figure 14 shows the GUI for a sequence comparator software tool for use with the invention.
  • Figure 15 shows a GUI for a sequence comparator software tool for use with the invention DETAILED DESCRIPTION OF THE INVENTION
  • siRNA duplexes have homology to both human and mouse sequences of the same gene. That means each of the designed siRNA duplexes will be able to knockdown the same gene target in either human or mouse cells. For example, a potent siRNA specific to Cox-2 gene will be able to knockdown both human Cox-2 and mouse Cox-2 gene expression.
  • the siRNA oligos can be obtained in either blunt end or sticky end form, based upon the synthesis design and annealing. One consideration is that the sticky end siRNA oligos may be sensitive to degradation while the blunt end may activate the cellular interferon response.
  • the siRNA-mediated mRNA knockdown can be measured using Quantitive RT-PCR with primers specific against the targeted sequences. The primer pairs can also be used for the amplification of both human and mouse sequences.
  • oligonucleotides and similar terms based on this relate to short oligos composed of naturally occurring nucleotides as well as to oligos composed of synthetic or modified nucleotides, as described in the immediately preceding paragraph. Oligonucleotides may be 19 or more nucleotides in length, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or more nucleotides in length, up to 35 or more, nucleotides in length.
  • An oligonucleotide that is an siRNA may have any number of nucleotides between 19 and 35 nucleotides. In many embodiments a siRNA may have any number of nucleotides between 19 and 27 nucleotides. In many embodiments, a siRNA may have two blunt ends, or two sticky ends, or one blunt end with one sticky end. The over hang nucleotides of a sticky end can range from one to four or more.
  • Figs. 1 -13 the various results from and procedures associated with the present invention are shown.
  • a 'library' of siRNA reagents in a suitable cell model for understanding a disease process we perform an assay examining the effect of gene silencing by each reagent on the phenotype observed for the test cell, Figs 1 - 3.
  • This in vitro assay then identifies genes which may be important in the disease etiology.
  • a statistically relevant cutoff for activity produced by the siRNAs can be based on Z' factors, Z scores, robust Z scores, ranked product or even B scores. These statistical methods along with additional normalization schemes are used to select a list of siRNAs that have the required effects in the cell model.
  • a suitable control cell model is also assessed under identical conditions to the test cell. It is desirable to identify siRNAs that produce a phenotype change in the test cell and not in the control cells.
  • the control cell type is used to ensure that any phenotype changes observed upon gene silencing in the test cells are specific for the disease etiology and not a process that is present in other cell types. Obviously, the larger number of test and control cells that can be examined for the phenotype of interest, the better chance that identified siRNAs are specific for the disease vs control cells.
  • Upon identification of an siRNA with the desired effect the gene that this siRNA targets is identified and this gene is added to a list of the genes of interest (a "hitlist").
  • a pathway may consist of an enzymatic cascade where one enzyme converts a substrate to a product and removal of the enzyme (by gene silencing) results in accumulation of the substrate.
  • the pathway could consist of a series of events where modification of a substrate (e.g. by phosphorylation or de-phosphorylation) alters the sensitivity/activity of the phosphorylated protein.
  • Pathways can consist of signaling cascades or metabolic pathways (Kegg pathways). Tools used to evaluate the location of these genes in pathways include software packages that are commercially available.
  • the ideal series of siRNAs for therapeutic benefit are those that, when applied simultaneously to the test cell type produce a more pronounced phenotype change than each added alone at the same concentration. It is important to be able to identify the optimal combinations of siRNAs that can meet these requirements and this must be performed empirically. As with the initial assay the mixture of siRNAs must also have little effect on the control cells. It may be advantageous to select species of siRNAs from distinct pathways identified in step 1 in order to increase the potency/efficacy of the cocktails relative to the single siRNA species.
  • the lists of siRNAs of interest are categorized based on the pathways they are in within the cell.
  • the invention provides siRNA of 21, 23 and 25 base pairs with blunt ends.
  • polynucleotide and "oligonucleotide” are used synonymously herein.
  • the user can select the location of the files containing the human and mouse sequences by clicking buttons and respectively. Upon opening the files ihe string containing the human sequence is inserted into the text box and the mouse (or other 2 nd species) is loaded into text box. The user can select how long (# bases) the predicted siRNA sequence will be by entering the number into the text box. Furthermore, the user can select the degree of complementarity between the siRNA sequence between the 2 species by entering a number in box.
  • the user can select the check box on the form. This specifies that the lengths of the sequences to match will be set at the typical lengths for siRNAs (19mer,21mer,25mer and 27mer) and all sequences will be examined using these lengths. If the degree of complementarity set at the time of running the comparator is 100% all of the subsequent matches at each siRNA length will match these criteria.
  • sequences that meet the search criteria are saved to a file and this file can then be used as the source of additional steps and sequence searches (see Tables below).
  • Another tool that has been developed is a BLAST analysis tool. The GUI for this interface is shown in Fig. 15.
  • Tables 1 - 7 are the target mRNA coding sequences which can be used for siRNA sequence design by changing the "t" into “u”, such as the sequences listed in Table 8.
  • Table 1 Sequence design for Transforming Growth Factor- beta 1 :
  • Organism Gene homology Length No. Sense Sequences
  • Organism Gene homology Length No. Sense Sequences
  • Organism ' Gene homology Length No. , Sense Sequences
  • Organism Gene homology Length No. Sense Sequences
  • the HK polymer-siRNA nanoparticle mediated local delivery has achieved potent anti-angiogenesis activity.
  • the tumor growth curves have showed significant anti-tumor efficacy with clear down regulation of the target gene expression.
  • mice with visible tumors were separated into treatment groups. Each group had four mice with eight tumors and tumor size was assessed in two dimensions and calculated. Mice received 4 ⁇ g/tumor of siRNA with each intratumoral injection every 5 days.
  • mice with tumors were divided into these groups: untreated, b-galactosidase siRNA and Raf-1 siRNA.
  • HK polymer has been validated as an effective local siRNA delivery carrier.
  • biodegradable polyesters such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(lactic-co-glycolic acid) (PLGA), have been extensively studied for a wide variety of pharmaceutical and biomedical applications.
  • the biodegradable polyester family has been regarded as one of the few synthetic biodegradable polymers with controllable biodegradability, excellent biocompatibility, and high safety.
  • block copolymers eg, diblock, triblock, multiblock, and star-shaped block
  • PEG poly(ethylene glycol)
  • PAMAM dendrimers represent a new class of macromolecular architecture called "dense star" polymers. Unlike classical polymers, dendrimers have a high degree of molecular uniformity, narrow molecular weight distribution, specific size and shape characteristics, and a highly- functionalized terminal surface. The manufacturing process is a series of repetitive steps starting with a central initiator core. Each subsequent growth step represents a new "generation" of polymer with a larger molecular diameter, twice the number of reactive surface sites, and approximately double the molecular weight of the preceding generation.
  • Polyamidoamine (PAMAM) dendrimers are the most common class of dendrimers suitable for many materials science and biotechnology applications. PAMAM dendrimers consist of alkyl-diamine core and tertiary amine branches.
  • PLGA poly(ethylene glycol)
  • PAMAM poly(ethylene glycol)
  • DOTAP DOTAP
  • the formulations for each of these carrier-siRNA nanoparticles are defined.
  • several peptide ligands have been identififed which can be used with either polymer or liposome, achieving targeted delivery to the Jurn ⁇ r 4 site.
  • siRNA control sequence was selected targeting a non-related sequence and without homologue in both human and mouse:
  • Lu25-a sense 5 '-r(GAGGAGCCUUC AGGAUUAC AAGAUU)-3'
  • hmTF-1 sense 5'-r(GGAUCCACGAGCCCAAGGGCUACCA)-3' antisense 5 '-r(UGGUAGCCCUUGGGCUCGUGGAUCC)-3 ' hmTF-2: sense 5'-r(CCCAAGGGCUACCAUGCCAACUUCU)-3' antisense 5 '-r(AGAAGUUGGCAUGGUAGCCCUUGGG)-3 ' hmTF-3: sense 5 '-r(GAGCCCAAGGGCUACCAUGCCAACU)-3 ' antisense 5 '-r(AGUUGGCAUGGUAGCCCUUGGGCUC)-3 '
  • hmPD-1 sense 5 '-r(GAUGGUACUGAAUUUCGCCGCCACA)-3 ' antisense 5'-r(UGUGGCGGCGAAAUUCAGUACCAUC)-3' hmPD-2: sense 5 '-r(GGGAUGGUACUGAAUUUCGCCGCCA)-3 ' antisense 5 '-r(UGGCGGCGAAAUUCAGUACC AUCCC)-3 ' hmPD-3: sense 5'-r(GCCUGCUGCUCCUCGGCUGCGGAUA)-3' antisense 5'-r(UAUCCGCAGCCGAGGAGCAGCAGGC)-3' II. Transfection of siRNA duplexes into the specific cell cultures
  • the PC3 cell expressing TGF-beta, Cox-2, MMP-9, PDGFa and FGF2 were transfected by three siRNA duplexes with Lipofectamin 2000.
  • human foreskin fibroblasts (HFF) is cultured on 10-cm plates in DMEM supplemented with 10% fetal bovine serum (FBS), 100 Dg/ml streptomycin, and 100 U/ml penicillin and transfected with the siRNA duplexes using Lipofectamin 2000.
  • the cells should be washed twice with PBS and incubated in FBS-free medium for 24 h. FBS-free medium was replaced with medium containing 10% FBS to initiate the cell cycle.
  • FBS-free medium was replaced with medium containing 10% FBS to initiate the cell cycle.
  • TGF-D gene expression knockdown at both mRNA and protein levels using four selected siRNA duplexes the mouse tumor line CT26 was transfected with siRNA-Lipofectamin 2000 followed by RT-PCR analysis.
  • RNA from each of those transfected cell lines including PC3 cells , PDGF (human) expressing cells and mouse embryonic endothelial cells are isolated and purified for RT-PCR analysis.
  • PDGF up 5' -CTCCAGCTCCTGTGCCTTAT-3'
  • mPDGF down 5'-ACT GGCCATAGGCTGGTATG-3'.
  • Cox-2 amplicon human
  • an RT reaction is followed with a PCR reaction using a pair of primers: hCox-2 up: 5'-CGGGCTGGGCCATGGGGTGGA-S' hCox-2 down: 5'-CCTATCAGTATTAGCCTGCTT -3'.
  • TGF- ⁇ amplicon for detection of TGF- ⁇ amplicon (mouse), an RT reaction is followed with a PCR reaction using forward primers: mTGF- ⁇ up: 5'-CTACTGTGTGCTGAGCACCTT-S' mTGF- ⁇ down: 5'-CGCTGCTCGGCCACTCTGGCT-S'.
  • hmFGF2 up 5'-CACTTCAAGGACCCCAAGCGGCT-S'
  • hmFGF2 down 5'-CAGCCCAGTTCGTTTCAGTGCCA-S'
  • RT-PCR analysis should be done in conjunction with the transfection experiment so that a proper condition can be optimized for efficient transfection for particular cell line, in order to achieve sufficient amount of total RNA for the PCR analysis.
  • RT-PCR results showed that the potent siRNA duplexes targeting TGF-beta, Cox-2 and MMP-9 are identified ( Figure 1-6).
  • the Western blot analysis and ELISA analysis is preferably used.
  • the cell lysates or cell culture media would be used for the protein detection.
  • the human COX-2 is analyzed using COX2 ELISA kit (Zymed, San Francisco, CA) which is an enzyme-linked immunosorbent sandwich assay for quantitative detection of human COX-2 in cell culture supernatants and cell lysates. Since Cyclooxygenase (COX) is a membrane-bound enzyme, which has a molecular weight of 71 kDa, the cell lysate should be prepared for the ELISA analysis.
  • the mouse TGF- is analyzed using Human/ Mouse TGFbI (Transforming Growth Factor beta 1, TGF-betal, TGF-bl) ELISA Ready-SET-Go Kit (with Pre-Coated Plates).
  • the selection of the most potent siRNA duplex for each gene is based on three repeated experiments. It should be noted that sometimes the most potent siRNA duplex selected from the mRNA knockdown is not correlated with the one selected from the protein knockdown. When that happens, the user can rely on the data from the mRNA level knockdown, since that it is the direct reflection of RNAi mechanism of action. The discrepancy of the protein level knockdown some times may be due to the non-specific or so call "off-target" effect, which is not the result of the RNAi mechanism of action.
  • COX-2 and TGF- ⁇ are combined together as the siRNA cocktail with several ratios of the combinations which can be used such as 1:1 :1, 2:1:1 and 3:1:1, etc.
  • the user must evaluate the appropriate mouse models and siRNA nanoparticle formulations, to define the most suitable siRNA-nanoparticle formulation for potential therapeutic protocol.
  • To establish a polymer-siRNA nanoparticle we decided to first test the Histidine-Lysine branched polymer for this formulation.
  • the biopolymer core facility at the University of Maryland will synthesize polymers on a Ranin Voyager synthesizer (PTI,
  • the HKP can be dissolved in aqueous solution and then mixed with siRNA aqueous solution at a ratio of 4:1 by mass, forming nanoparticles of average size of 150-200 nm in diameter.
  • the HKP-siRNA aqueous solutions were semi- transparent without noticeable aggregation of precipitate, and can be stored at 4 ⁇ C for at least three months.
  • the invention provides the therapeutic siRNA cocktail targeting multiple disease controlling genes in the same treatment.
  • the invention provides for RNAi agents, such as siRNA oligonucleotides, that are chemically similar to the same source of supply and the same manufacturing process, and they are comprised of four types of nucleotides with different sequences.
  • the invention provides the siRNA cocktail drug for improvement of scarless wound healing by targeting genes involved in the wound healing process, including TGF- ⁇ , Cox-2, MMP-9 and others.
  • the siRNA cocktail must contain at least three siRNA duplexes targeting at least three genes (not three sequences of the same gene) at a ratio of therapeutic requirement.
  • siRNA cocktail design for each combination must follow the understanding of the role of each gene in a background of the system biology network, such as these genes are functioning either in the same pathway or in a different one.
  • the chemical property of each siRNA duplexes in the cocktail must be the same in terms of source of supply, manufacturing process, chemical modification, storage conditions and formulation procedures.
  • Each individual siRNA duplex in the cocktail can be different in their lengths, with either blunt or sticky end, as long as their potencies have been defined.
  • siRNA cocktail Since siRNA cocktail is targeting multiple genes and a single cell type usually does not express all those factors, the efficacy of a siRNA cocktail must be tested in a relevant disease model, either a multiple cell model, a tissue model or a animal model, after the confirmation of the potency of each individual siRNA duplex in the cell culture.
  • a relevant disease model either a multiple cell model, a tissue model or a animal model, after the confirmation of the potency of each individual siRNA duplex in the cell culture.
  • Each validated siRNA cocktail can be used for addressing one or more pathological conditions, for treatment of one or multiple types of diseases, such as, siRNA cocktail for suppressing inflammation, siRNA cocktail for antiangiogenesis activity and siRNA cocktail for autoimmune conditions.
  • siRNA cocktail must be administrated through the same route of delivery in the same formulation, although the regimen of dosing for each cocktail will be defined based on either the experimental design or therapeutic requirement.
  • Each siRNA cocktail can be applied either independently, or in combination with other drug modalities such as small molecule inhibitors, monoclonal antibodies, protein and peptides, and other siRNA cocktail drug(s).
  • Table A The siRNA cocktail targeting Cox-2, MMP-9 and TGF- ⁇ 2.
  • siRNA Cocktail Combinations targeting Hoxbl3, Cox-2 and VEGFA.
  • TGF- ⁇ l 5 '-ggauccacgagcccaagggcuacca-3 '
  • TGF- ⁇ 2 D 5'-cagauccugagcaagcugaagcuca-3'
  • TGF- ⁇ l 5'-ggauccacgagcccaagggcuacca-3 '
  • siRNA sequences targeting various tumorigenic genes can be arranged into different combinations for enhancing the anti-tumor activity in xenograft mouse models, or in human oncology drug testing.
  • mice were used in the experiment with a paired 5 mm diameter full-thickness excision skin wounds on both sides of the dorsal midline the depilitated dorsum of the Balb/c mouse).
  • the conventional methylcellulose was used as the topical administration carrier with or without HKP-siRNA.
  • Two wounds on each mouse were either treated with only methylcellulose or methylcellulose plus HKP-siRNA daily for the first 5 days. The observations were taken on day 0, 5, 9 and 15 th .
  • siRNA cocktail targeting TGF ⁇ -1, Hoxbl3 and Cox-2 packaged with HKP demonstrated similar knockdown activity as TGF ⁇ -1 siRNA package with HKP, while siRNA duplexes specific to either Hoxbl3 or Cox-2 packaged with HKP did show TGF ⁇ -1 down regulation.
  • nanoparticle-enhanced TGF ⁇ -1 was responsible for the therapeutic benefit for the skin wound closure, it should be determined if this benefit would also happen to other gene targets, such as Cox-2.
  • the experiment was conducted using the same Balb/c mice with the skin excision wound model.
  • the treatment groups were methylcellulose plus siRNAc ox- 2 and methylcellulose plus HKP-siRNAcox-2 in addition to the control groups with only methylcellulose and methylcellulose plus HKP-siRNA control.
  • the similar patterns of wound closure rates were observed using the Scion Imaging Program.
  • Epoxy Ferric Oxide Nano-particles are non toxic and super magnetic. Epoxy Ferric Oxide
  • Nano-particles have the size of RBC, which can be utilized for several medical application, including homestasis, anti-angiogenesis, and delivery vehicle.
  • a proposed in vitro Studies should be : 1. Simulation Studies: introduction of Epoxy grains solution with different concentrations into flexible tubes but with various diameters which to simulate the blood stream. At the bottom of the narrow area there will be a strong magnetic field applied by the use of an adhesive Bandage containing high field magnets to attract the epoxy grain embedded nanoparticles. 2. Using an angiogenesis model to test the ability of Ferric oxide- epoxy coated particle to be concentrated in a single spot. For these larger quantities of epoxy resin encapsulated nanoparticles are need. In Vivo studies include 1). Animal studies to measure toxicity; 2). pharmacokinetics, and 3). efficacy. Finally, it should be tested in clinical setting with Human subjects such as healthy volunteers.

Abstract

The present invention provides compositions and methods for treatment of prostate cancer using siRNA cocktail sequences targeting multiple disease causing genes, and a combined regimen with both RNAi therapeutics and EEMP encapsulated magnetic nanoparticle therapeutics. The invention includes: 1) The algorithm for designing siRNA drug API (active pharmaceutical ingredient); 2)The compositions of multiple siRNA inhibiting expression of the multiple cancer causing genes; 3) The methods of designing siRNA duplexes with different lengths, different ends and to target the same genes from both human and mouse cells; 4) The methods of formulating various siRNA cocktail which are able to inhibit tumorigenic and angiogenesis; 5) The methods of using histidine-lysine copolymer (HKP), PAMAM dendrimer and Liposomal (DOTAP) carrier to enhance siRNA delivery into the cutaneous tissue; and 6) The methods of using two disease relevant mouse xenograft models to validate the siRNA cocktail for its efficacy on controlling tumor growth.

Description

n^,_ u g / 0 0 3 6 6 1
Inventor 1: Asghar Ghias, Ph.D., Rockville, MD 20850, USA Inventor 2: Patrick Y. Lu, Ph.D., Rockville, MD 20855, USA Inventor 3: David Evans, Ph.D., Gaithersburg, MD 20879, USA
Attorney Docket no. EG123108
COMBINATIONAL THERAPEUTICS FOR TREATMENT OF PROSTATE CANCER USING EPOXY ENCAPSULATED MAGNETIC PARTICLES AND RNAI MEDICINE
FIELD OF THE INVENTION
The present invention provides compositions and methods for treatment of human prostate adrenocarcinoma and other types of prostate cancers, using RNA interference (RNAi) and Epoxy-Encapsulated Magnetic Particle combinational therapeutics.
BACKGROUND OF THE INVENTION
I. Prevalence of Prostate Cancer
Prostate cancer is a significant health problem in most industrialized Western countries, where it is the most commonly diagnosed cancer affecting men after middle age. The worldwide 5 -year prevalence of prostate cancer has been estimated at 1,554,700 cases. It is estimated that, in Western countries, about 30% of all men will develop microscopic prostate cancer during their lifetime. However, as most prostate cancers tend to grow slowly, the risk of developing overt clinical disease is 8% (lifetime risk), and the risk of actually dying from prostate cancer is only 3%, whereas the autopsy based prevalence is 80% by the age of 80 years. Therefore, most men die with prostate cancer, rather than from it. Based on US data, for a 50 year-old man with a life expectancy of 25 years, there is a 42% lifetime risk of having microscopic cancer, a 9.5% risk of having clinically evident cancer and a 2.9% risk of dying from prostate cancer. In recent years, the veritable epidemic of prostate cancer has probably resulted from the widespread use of PSA testing which allows the earlier diagnosis in men who have not yet developed symptoms. As an example, it is estimated that in 2005 in the USA there will be approximately 235,000 new diagnoses of prostate cancer and 29,000 deaths (approx 1 every 15 minutes). Prostate cancer is primarily a disease of men over the age of 50 years, and the trend towards an ageing worldwide FCi/^_. u 8 / 0 0 3 6 6 1
population is likely to lead to an increased incidence of cases of prostate cancer. It is estimated that the incidence of prostate cancer is increasing at an average rate of 3% a year. The incidence of prostate cancer varies from country to country, with the highest incidences being found in the Western world and the lowest being found in Asia. Data for the year 2000 identify, that whereas the incidence in the USA was 140 per 100,000, for Japan it was 22 per 100,000 and for China it was 1.54 per 100,000. Prostate cancer has become one of the leading male cancers in some Asian countries with the incidence having risen rapidly in the last 20 years. The reasons for this high degree of variability between ethnic groups are probably multi-factorial and include the availability of improved detection methods, increasing westernisation of lifestyle and in particular genetic risk factors. The stage distribution at the time of diagnosis also varies around the world. In the USA in 2001 only 13% of tumors were diagnosed as Stage 3 or 4, whereas the corresponding figures in 2000 for South Korea were 72% and for Taiwan in 1998 they were 58%. However, in most Asian countries there is evidence that there is a trend towards diagnosing cancer with more favorable prognosis. Again this is probably a reflection of improved diagnostic and screening procedures. After lung cancer, prostate cancer is the second most common cause of cancer death in men being responsible for approximately 13% of all cancer deaths.
II. Three Types of Conventional Treatment of Prostate Cancer
1. Surgery: Patients in good health are usually offered surgery as treatment for prostate cancer. The following types of surgery are used:
Pelvic lymphadenectomy: A surgical procedure to remove the lymph nodes in the pelvis.
A pathologist views the tissue under a microscope to look for cancer cells. If the lymph nodes contain cancer, the doctor will not remove the prostate and may recommend other treatment. Radical prostatectomy: A surgical procedure to remove the prostate, surrounding tissue, and seminal vesicles. There are 2 types of radical prostatectomy:
Retropubic prostatectomy: A surgical procedure to remove the prostate through an incision (cut) in the abdominal wall. Removal of nearby lymph nodes may be done at the same time. Perineal prostatectomy: A surgical procedure to remove the prostate through an incision (cut) made in the perineum (area between the scrotum and anus). Nearby lymph nodes may also be removed through a separate incision in the abdomen.
Transurethral resection of the prostate (TURP): A surgical procedure to remove tissue from the prostate using a resectoscope (a thin, lighted tube with a cutting tool) inserted through the urethra. This procedure is sometimes done to relieve symptoms caused by a tumor before other cancer treatment is given. Transurethral resection of the prostate may also be done in men who cannot have a radical prostatectomy because of age or illness. Impotence and leakage of urine from the bladder or stool from the rectum may occur in men treated with surgery. In some cases, doctors can use a technique known as nerve-sparing surgery. This type of surgery may save the nerves that control erection. However, men with large tumors or tumors that are very close to the nerves may not be able to have this surgery.
2. Radiation therapy Radiation therapy is a cancer treatment that uses high-energy x-rays or other types of radiation to kill cancer cells or keep them from growing. There are two types of radiation therapy. External radiation therapy uses a machine outside the body to send radiation toward the cancer. Internal radiation therapy uses a radioactive substance sealed in needles, seeds, wires, or catheters that are placed directly into or near the cancer. The way the radiation therapy is given depends on the type and stage of the cancer being treated. Impotence and urinary problems may occur in men treated with radiation therapy.
3. Hormone therapy
Hormone therapy is a cancer treatment that removes hormones or blocks their action and stops cancer cells from growing. Hormones are substances produced by glands in the body and circulated in the bloodstream. In prostate cancer, male sex hormones can cause prostate cancer to grow. Drugs, surgery, or other hormones are used to reduce the production of male hormones or block them from working. Hormone therapy used in the treatment of prostate cancer may include the following: Luteinizing hormone-releasing hormone agonists can prevent the testicles from producing testosterone. Examples are leuprolide, goserelin, and buserelin.
Antiandrogens can block the action of androgens (hormones that promote male sex characteristics). Two examples are flutamide and nilutamide. Drugs that can prevent the adrenal glands from making androgens include ketoconazole and aminoglutethimide.
Orchiectomy is a surgical procedure to remove one or both testicles, the main source of male hormones, to decrease hormone production.
Estrogens (hormones that promote female sex characteristics) can prevent the testicles from producing testosterone. However, estrogens are seldom used today in the treatment of prostate cancer because of the risk of serious side effects.
Hot flashes, impaired sexual function, loss of desire for sex, and weakened bones may occur in men treated with hormone therapy.
III. Several Experimental Treatment Approaches
1. Cryosurgery
Cryosurgery is a treatment that uses an instrument to freeze and destroy prostate cancer cells. This type of treatment is also called cryotherapy.
2. Chemotherapy
Chemotherapy is a cancer treatment that uses drugs to stop the growth of cancer cells, either by killing the cells or by stopping them from dividing. When chemotherapy is taken by mouth or injected into a vein or muscle, the drugs enter the bloodstream and can reach cancer cells throughout the body (systemic chemotherapy). When chemotherapy is placed directly into the spinal column, an organ, or a body cavity such as the abdomen, the drugs mainly affect cancer cells in those areas (regional chemotherapy). The way the chemotherapy is given depends on the type and stage of the cancer being treated.
3. Biologic therapy Biologic therapy is a treatment that uses the patient's immune system to fight cancer. Substances made by the body or made in a laboratory are used to boost, direct, or restore the body's natural defenses against cancer. This type of cancer treatment is also called biotherapy or immunotherapy.
4. High-intensity focused ultrasound
High-intensity focused ultrasound is a treatment that uses ultrasound (high-energy sound waves) to destroy cancer cells. To treat prostate cancer, an endorectal probe is used to make the sound waves.
III. Molecular Targets for Prostate Cancer Treatment
Transforming Growth Factor (TGF-β)
The correlation of elevated transforming growth factor-β (TGF- β) with increasing serum prostate-specific antigen (PSA) levels in metastatic stages of prostate cancer has also been well documented (Hong- Yo Kang, 2001). TGF-β pathway plays dual roles in cancer, inhibiting epithelial cell growth under normal physiologic conditions, but promoting invasion and metastasis once growth inhibitory responses are lost (N. Sharifi, 2007). TGF-beta is a pleiotropic growth factor. It plays an important role in the regulation of growth and differentiation in many cells. In benign prostatic epithelia, its action is mediated through a paracrine mechanism. It inhibits proliferation and induces apoptosis in prostatic epithelia. It provides a mechanism to maintain epithelial homeostasis in the prostate. In prostatic stroma, its continual action leads to smooth muscle differentiation. This effect of TGF-beta may regulate the development of prostatic smooth muscle nodules in benign prostatic hyperplasia. As prostatic epithelial cells undergo malignant transformation, two major events occur regarding TGF-beta action. These include the loss of expression of functional TGF-beta receptors and overproduction of TGF-beta in malignant cells. The loss of expression of functional TGF-beta receptors provides a growth advantage to cancer cells over their benign counterparts. The overproduction of TGF-beta by cancer cells has a multitude of adverse consequences. TGF-beta can promote extracellular matrix production, induce angiogenesis, and inhibit host immune function. The biological consequence of these activities is an enhanced tumorigenicity in prostate cancer. Results of studies with a rat prostate cancer model suggest that the immunosuppressive effect of TGF-beta seems to be the primary cause of tumor progression. This is because, if these cancer cells were engineered to reduce the production of TGF-beta, tumor growth was inhibited in syngeneic hosts but not in immune compromised hosts (Lee C, 1999).
Cyclooxygenase-2 (COX-2)
Cyclooxygenase (COX) is a key enzyme in the production of prostaglandins (PGs) and other eicosanoids from arachidonic acid. COX-2 was initially found as an early growth responsive gene that modulates cell adhesion, apoptosis, proliferation, and differentiation. A variety of cytokines and growth factors, as well as several oncogenes, can bind to the promoter region of COX-2 to upregulate its transcription in most cells. More recently, it has been suggested that PGs and other eicosanoids produced by COX play an important role in the development and progression of human cancers. Potential mechanistic roles of COX-2 in tumorigenesis and tumor progression include (1) decreased apoptosis; (2) increased angiogenesis; (3) increased tumor invasiveness; and (4) decreased immune surveillance. Overexpression of COX-2 has been observed in many cancers, including prostate cancer. Consequently, suppression of COX-2 by specific inhibitors or other types of nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit the activity of COX-2, may lead to inhibition of tumor growth. COX-2 expression has been studied in prostate cancer tissues, and many studies showed increased expression of COX-2 in prostate cancer and prostatic intraepithelial neoplasia (PIN) compared with normal or hyperplastic prostate. In vitro and animal studies have suggested the involvement of COX-2 in prostate tumorigenesis and cancer progression. The enzyme activity and protein expression of COX-2 were found to be significantly higher in the dorsolateral prostate of the transgenic adenocarcinoma of the mouse prostate (TRAMP) model at 8, 16, and 24 wk of age, compared with their nontransgenic littermates. LNCaP cells with stably overexpressed COX-2 increased cell proliferation in vitro and tumor growth in vivo as compared with parental LNCaP cells, which was associated with increased expression of vascular endothelial growth factor (VEGF). Based on these results, it has been suggested that inhibition of COX-2 may be a useful chemopreventive/ therapeutic option for prostate cancer. In prostate cancer cell lines, COX-2 expression was detected in androgen-sensitive LNCaP cells, as well as in androgen-insensitive PC-3 and DU 145 cells. Inhibition of COX-2 by selective inhibitors has been shown to induce growth arrest and apoptosis in both LNCaP and PC-3 cells. In animal models of prostate carcinogenesis and metastasis, COX-2 inhibitors suppressed tumor growth and metastases. These in vivo studies suggest that tumor growth suppression is achieved by a combination of tumor cell apoptosis induction and decrease in angiogenesis via downregulation of PGE2 and VEGF. However, a COX-2 independent pathway might be involved in proapoptotic and antitumor effects of NSAIDs and selective COX-2 inhibitors. Epidemiological studies and clinical trials indicated that in prostate cancer COX-2 inhibitors may have chemopreventive effects (Miyamoto et al., 2005)
Matrix Metallopeptidase 9 (MMP-9) MMPs constitute a broad family of zinc-binding endopeptidases that play a key role in the degradation of the extracellular matrix and basement membrane. Currently, there are over 20 known members of the MMP family shown to be involved in physiological processes, such as tissue remodeling and wound healing, as well as pathological states, including tumor progression. Two important members of the MMP family, MMP-2 and MMP-9, belonging to the gelatinase subfamily, are known to specifically cleave type IV collagen, the major component of basement membranes. MMP activity is regulated by the transcription factor, nuclear factor-kB (NF-kB), a member of a family of dimeric transcription factors that contains a conserved ReI homology domain, allowing for dimerization, and a DNA binding domain. Inactive NF-kB is retained in the cytoplasm by the inhibitor protein IkB. In response to certain extracellular signals, such as tumor necrosis factor, IL-I 12-Otetradecanoylphorbolacetate, and lipopolysaccharide, IkB kinase is activated and phosph orylates IkB. NF-kB is then released, translocates to the nucleus, and binds to DNA. It has been proposed that NF-kB induces expression of target genes such as MMPs. A substantial amount of evidence supports the hypothesis that MMPs play a key role in multiple steps of tumor prog ression, including tumor promotion, angiogenesis, invasion, and metastasis. Specifically, a number of clinical and experimental studies have suggested that changes in MMP levels affect the invasive behavior of tumor cells and their metastatic potential. In addition, it is noted that MMPs can be produced not only by tumor cells but also by surrounding stromal cells and infiltrating inflammatory cells. It was demonstrated a positive correlation between increased expression/activity of MMP-2/MMP-9 and malignant potential of prostate cancer, as judged by Gleason score, pathological stage, or patient survival. Several studies analyzing primary cultures of prostatic epithelial and stromal cells also support the contention that MMP-2 and MMP-9 are differentially expressed/secreted in normal prostate epithelial and prostate cancer cells. However, as opposed to primary cultures, there are little differences in expression/ secretion of MMP-2 or MMP-9 among prostate specimens originating from normal, hyperplastic, and cancer tissues. Co-cultures of prostate cancer and stromal cells also showed significantly enhanced expression of MMP-9, confirming the importance of tumor- stromal interactions in tumor progression. Several growth factors, such as transforming growth factor b and fibroblast growth factors, have been shown to upregulate expression/secretion of MMPs in prostate cancer cells (Miyamoto et al., 2005)
Platelet-derived growth factor (PDGF)
PDGF was one of the first polypeptide growth factors identified that signals through a cell surface tyrosine kinase receptor (PDGF-R) to stimulate various cellular functions including growth, proliferation, and differentiation(George_D. 2001). Since then, several related genes have been identified constituting a family of ligands (primarily PDGF A and B) and their cognate receptors (PDGF-R alpha and beta). To date, PDGF expression has been shown in a number of different solid tumors, from glioblastomas to prostate carcinomas. In these various tumor types, the biologic role of PDGF signaling can vary from autocrine stimulation of cancer cell growth to more subtle paracrine interactions involving adjacent stroma and even angiogenesis. The tyrosine kinase inhibitor imatinib mesylate (Gleevec, Novartis) with blocks activity of the Bcr-Abl oncoprotein, is also a potent inhibitor of the PDGF-R kinase and is currently being evaluated for the treatment of PDGF-responsive tumors such as prostate cancer. More clinical trials that investigate both established clinical endpoints of response and benefit, as well as surrogate endpoints that may describe the biologic significance of PDGF-R inhibition in vivo are needed to expand the applications that target the PDGF axis. A study characterizing PDGFR-beta expression in a wide spectrum of PCa samples has provided empirical data as part of a rational treatment strategy (Hofer MD, et al 2004). However, through a survey of five published prostate expression array studies, including 100 clinically localized PC and protein expression of
PDGFR-beta, it has been revealed that only a small subset of PCas may be amenable to tyrosine kinase inhibitors specific for PDGFR.
Fibroblast Growth Factor 2 (FGF2) Members of the fibroblast growth factor (FGF) family are believed to play critical roles during organogenesis and carcinogenesis via signaling between epithelial and stromal compartments. Recent studies (Abate-Shen C, et al. 2007) underscore the importance of FGF signaling in mediating epithelial-stromal interactions during prostate carcinogenesis. The experimental results show that deregulated FGF signaling in mouse models of prostate cancer leads to cancer progression and promotes an epithelial-mesenchymal transition, suggesting that FGF receptor inhibitors may have therapeutic value for prostate cancer treatment. Transforming growth factor-beta (TGF-beta) is overexpressed in most adenocarcinomas including prostate cancer. In stromal tissues, TGF-beta regulates cell proliferation, phenotype and matrix synthesis. Additionally, decreased cellular fibroblast growth factor-2 (FGF-2) immunostaining was associated with attenuated TGF-beta signaling in stroma. In vitro, TGF-beta stimulated stromal FGF-2 expression and release. However, stromal cells with attenuated TGF-beta signaling were refractory to TGF-beta-stimulated FGF-2 expression and release. Re-expression of FGF-2 in these stromal cells in the mouse xenografts resulted in restored tumor mass and microvessel density. In summary, these data show that TGF-beta signaling in reactive stroma is angiogenic and tumor promoting and that this effect is mediated in part through a TbetaRII/Smad3- dependent upregulation of FGF-2 expression and release (Yang F, et al, 2007).
Phosphoinositide 3-Kinase (PI3K) and Akt
Activated PI3K and its downstream target Akt/PKB are important signaling molecules and key survival factors involved in the control of cell proliferation, apoptosis and oncogenesis (Shukla S, et al. 2007). We investigated the role of the PI3K-Akt signaling pathway in the invasion of prostate cancer cell lines and activation of this pathway in primary human prostate tumors. Treatment of human prostate cancer cells viz. LNCaP, PC-3 and DU 145 with PI3K pharmacological inhibitor, LY294002, potentially suppressed the invasive properties in each of these cell lines. Restoration of the PTEN gene to highly invasive prostate cancer PC-3 cells or expression of a dominant negative version of the PI3K target, Akt also significantly inhibited invasion and downregulated protein expression of urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9, markers for cell invasion, indicating a central role of the PI3K-Akt pathway in this process. Immunoblot analysis of PI3K and total/activated levels of Akt showed increased protein levels of catalytic (pi lOalpha/beta) and regulatory (p85) subunits of PI3K and constitutive Akt activation in high-grade tumors compared to low-grade tumor and benign tissue. Immunohistochemical analyses further confirmed a progressive increase in p-Akt (p-Ser473) levels but not of total- Akt (Aktl/2) in cancer tissues compared to benign specimens. A successive increase in p-Akt expression was further noted in specimens serially obtained from individuals with time-course disease progression. Taken together, these results suggest that aberrant activation of PI3K-Akt pathway may contribute to increased cell invasiveness and facilitate prostate cancer progression.
IV. siRNA Therapeutics for Cancer Treatment
RNA interference (RNAi) is a sequence-specific RNA degradation process that provides a relatively easy and direct way to knockdown, or silence, theoretically any gene (13, 14). In naturally occurring RNA interference, a double stranded RNA is cleaved by an RNase Ill/helicase protein, Dicer, into small interfering RNA (siRNA) molecules, a dsRNA of 19-23 nucleotides (nt) with 2-nt overhangs at the 3' ends. These siRNAs are incorporated into a multicomponent-ribonuclease called RNA-induced-silencing-complex (RISC). One strand of siRNA remains associated with RISC, and guides the complex towards a cognate RNA that has sequence complementary to the guider ss-siRNA in RISC. This siRNA-directed endonuclease digests the RNA, thereby inactivating it. Studies have revealed that the use of chemically synthesized 21-25-nt siRNAs exhibit RNAi effects in mammalian cells, and the thermodynamic stability of siRNA hybridization (at terminals or in the middle) plays a central role in determining the molecule's function (13, 14, 15). Importantly, it is presently not possible to predict with high degree of confidence which of many possible candidate siRNA sequences potentially targeting a mRNA sequence of a disease gene will in fact exhibit effective RNAi activity. Instead, individual specific candidate siRNA polynucleotide or oligonucleotide sequences must be generated and tested in the mammalian cell culture to determine whether the intended interference with expression of a targeted gene has occurred. In the identification of siRNA sequences that can be used to treat disease there is a need to test these reagents in a variety of in vitro and in vivo experiments to ensure potency and efficacy. Since the therapeutics are initially aimed at humans it is obviously important that the initial screens in vitro use a cell model that is derived from human tissues and are representative of the disease etiology. siRNAs are introduced into these cells and an effect on the cell phenotype (viability, protein expression or gene expression alterations) can be monitored. Selectivity and specificity of the effect for diseased cells can be evaluated using human non-diseased control cells examining similar parameters. However, after in vitro discovery of the best siRNAs for therapeutic intervention the path to develop them for clinical applications requires performing toxicity tests, PK/PD testing and in vivo efficacy tests. Since it is not possible to perform such preliminary tests in human subjects and non-human primate models are expensive, the tests need to be performed in other animal species. Typically these animal models use rats, rabbits or mice. Since siRNAs are aimed at silencing genes through degradation of the mRNA (see mechanism of siRNA action), it is important that an siRNA sequence for development should be aimed at the same gene in the other animal species used. In addition, the degree of sequence complementarity between the siRNA and the target gene should be the same between the two species. Furthermore, the siRNA designed needs to limit potential cross reactivity versus other genes in either species. With the publication of the human genome (a directory of all genes and their sequences), it is feasible to predict regions of a single gene containing the number of bases required for silencing induced by an siRNA against the appropriate mRNA sequence. Other species (e.g. mouse) also have a fairly complete annotation of the genome and these sequences are also freely available from a number of sources (OMIM, Entrez gene.NCBI, EMBL databases etc). Therefore it is relatively easy to be able to select the sequences of each gene in each species and then compare regions where an siRNA can be made that has absolute complementarity with each sequence. If such sequences can be identified the next step is to determine which of the other genes in the genome may contain this sequence (if they do then these genes may also be targeted for silencing resulting in "off-target" effects). Such off target effects are not key to the therapeutic use of the siRNA and the targets being silenced may result in loss of key proteins for regular cell function or decrease genes that are repressing other key effects (e.g. cell proliferation). Targeting such genes will therefore have unwanted side effects including toxicity or other unwanted reactions. Therefore, upon identification of an siRNA sequence that matches the same gene between multiple species it is also important to ensure that this siRNA cannot alter expression of other genes in the intended species where the siRNA(s) will be utilized as a therapeutic.
V. Multi-Targeted siRNA Cocktail For Prostate Cancer Treatment
The unique advantage of siRNA makes it possible to be combined with multiple siRNA duplexes to target multiple disease causing genes in the same treatment, since all siRNA duplexes are chemically homogenous with same source of origin and same manufacturing process (13, 16). Many types of human diseases, including cancer, inflammatory conditions, autoimmune diseases and infectious diseases are able to be treated with much better clinical efficacy using such potent siRNA inhibitors with minimum toxicity and safety concerns (14, 15). Therefore, using RNAi to silence genes involved in the angiogenesis, anti-apoptosis, cell proliferation and tumorigenesis, such as the profibrotic factor TGF-β, the inflammation promoter COX-2, Matrix Metallopeptidase 9 (MMP-9) , Platelet-derived growth factor (PDGF), Fibroblast Growth Factor 2 (FGF2), Phosphoinositide 3-Kinase (PI3K) and Akt, we can achieve enhanced anti-tumor activity. We further hypothesize that siRNA cocktail with three siRNA duplexes targeting three genes, TGF-β, COX-2 and PDGFR, or COX-2, PDGFR and FGF2, or COX-2, PDGFR and PI3K, will represent very power therapeutic approaches to treat prostate cancer. Successful siRNA-mediated therapy not only depends on identification of the targets and sequence of active siRNA molecules, but more importantly on efficient in vivo delivery to the target tissues and into the cytoplasm (11-13). The routes of delivery of siRNA cocktail formulation for treatment of skin wound healing should be local and topical with appropriate clinically validated carriers. We use three polymer-based and two lipid-based carriers, including histidine-lysine polymers (HKP) (14), pegylated PEI (15), PAMAM dendrimer (16) and DOTAP/DOPS which were developed in our lab and from our collaborators, for further enhancement of therapeutic efficacy of the treatment. By adding peptide ligands onto those cationic polymer or liposome, we also achieved targeted activity when the nanoparticle packaged siRNA are delivered through IV injection.
VI. Epoxy-Encapsulated Magnetic Particles (EEMP)
Nanotechnology deals with the understanding and control of matter at dimensions of roughly 1 to 100 run. At the nanoparticle level materials often exhibit unique properties affecting their physical, chemical, and biologic properties. The challenge in using nanotechnology is to find application in science and remove any potential health hazards. Only after the substances are benign can we apply the nanoparticles in health care as useful products. Using submicron Fe3O4 nanoparticle aggregates encapsulated in a hydrophobic epoxy matrix that is benign and nontoxic, but possesses super-paramagnetic properties. One application of these particles can be the utilization in anti-angiogenesis therapy to pinch off the blood supply of a tumor. In addition, the magnetic nanoparticle can also be used for siRNA delivery.
SUMMARY OF THE INVENTION
The present invention provides compositions and methods for treatment of prostate cancer using siRNA cocktail sequences targeting multiple disease causing genes, and a combined regimen with both RNAi therapeutics and EEMP encapsulated magnetic nanoparticle therapeutics. The invention includes: 1) The algorithm for designing siRNA drug API (active pharmaceutical ingredient); 2)The compositions of multiple siRNA inhibiting expression of the multiple cancer causing genes; 3) The methods of designing siRNA duplexes with different lengths, different ends and to target the same genes from both human and mouse cells; 4) The methods of formulating various siRNA cocktail which are able to inhibit tumorigenic and angiogenesis; 5) The methods of using histidine-lysine copolymer (HKP), PAMAM dendrimer and Liposomal (DOTAP) carrier to enhance siRNA delivery into the cutaneous tissue; and 6) The methods of using two disease relevant mouse xenograft models to validate the siRNA cocktail for its efficacy on controlling tumor growth.
In embodiment one the small interfering RNA (siRNA) duplexes can be blunt ended or with over hangs at the length of 19 to 27 nt. In embodiment two, the siRNA sequences are able to target the same gene of both human and mouse, or non-human primate. In embodiment three siRNA can be used as a cocktail containing at least three siRNA duplexes and targeting at least three different genes. In embodiment four the efficacy of a siRNA cocktail must be tested and confirmed in the animal disease models. In embodiment five therapeutic benefit of a siRNA cocktail must be better thanthe single siRNA agent which is part of the cocktail components. In embodiment six the metal- based nanoparticle can be used for siRNA drug and other drug delivery. In embodiment seven, The Epoxy-Encapsulated Magnetic Particle (EEMP) is used in the treatment of prostate adrenocarcinoma with siRNA cocktail combinational therapeutics. The invention provides siRNA sequences targeting 1) gene expression promote cell proliferation; 2) gene expression promote angiogenesis activity; 3) gene expression promote anti-apoptotic activity and 4) gene expression promote oncogenic effects. The invention also provides method to design the proper ratio of each duplex in order to allow the siRNA cocktail to achieve the most potent synergistic effect. The invention also provides the siRNA cocktail for treatment of prostate cancer with EEMP-mediated anti-angiogenesis activity. The invention further provides the nanoparticle-enhanced siRNA cocktail delivery for treatment of prostate cancer.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows RT-PCR detection of siRNA-mediated knockdown of target gene (TGF-beta T) expression in PC-3 cells.
Figure 2 shows RT-PCR detection of siRNA-mediated knockdown of target gene (Cox-2) expression in PC-3 cells.
Figure 3 shows RT-PCR detection of siRNA-mediated knockdown of target gene (Cox- 2) expression in the C 166 Yolk-Sac Endothelial mouse cells.
Figure 4 shows RT-PCR detection of VEGFa, VEGFR2, PDGFa, FGF2 and MMP-2 gene expression in both human (PC-3) and mouse (CT-26) cells. Figure 5 shows RT-PCR detection of siRNA-mediated knockdown of target gene (MMP-9) expression in the CT26 cells.
Figure 6 shows RT-PCR detection of siRNA-mediated knockdown of target gene (MMP-9) expression in the PC3 cells.
Figure 7 shows step-by-step procedures for preparation of Epoxy-Encapsulated Magnetic Nanoparticle.
Figure 8 shows step-by-step flow chart for preparation of Epoxy-Encapsulated Magnetic
Nanoparticle for medical use.
Figure 9 shows a schematic drawing of Epoxy-Encapsulated Magnetic Nanoparticle following acid wash. Figure 10 shows a comparison of mouse skin excision wound healing rate.
Figure 11 shows RT-PCR analyses of total RNA from wound tissue harvested at day 7 post wounding.
Figure 12 shows a comparison of wound closure rate between HKP TGFβl siRNA treated and control wounds. Figure 13 shows the effects of HKP Cox-2 siRNA on collagen structure in healing wounds.
Figure 14 shows the GUI for a sequence comparator software tool for use with the invention.
Figure 15 shows a GUI for a sequence comparator software tool for use with the invention DETAILED DESCRIPTION OF THE INVENTION
I. Design of siRNA Sequences Targeting Tumorieenic Genes The present invention provides a novel approach to the design of siRNA targeting sequences. There are four important aspects of the invention which differ from known approaches:
(1) The sequences designed to be targeted by siRNA duplexes have homology to both human and mouse sequences of the same gene. That means each of the designed siRNA duplexes will be able to knockdown the same gene target in either human or mouse cells. For example, a potent siRNA specific to Cox-2 gene will be able to knockdown both human Cox-2 and mouse Cox-2 gene expression.
(2) The sequences are designed in three different lengths: 21-mer, 23-mer and 25-mer. One consideration is that 23-mer and 25-mer are usually more potent than 21-mer siRNA but 25-mer may have more chance to induce unwanted interferon response. Therefore, siRNA duplexes at various lengths will provide the best chance to achieve potent inhibitor with less interferon response.
(3) The siRNA oligos can be obtained in either blunt end or sticky end form, based upon the synthesis design and annealing. One consideration is that the sticky end siRNA oligos may be sensitive to degradation while the blunt end may activate the cellular interferon response. (4) The siRNA-mediated mRNA knockdown can be measured using Quantitive RT-PCR with primers specific against the targeted sequences. The primer pairs can also be used for the amplification of both human and mouse sequences.
As used herein, "oligonucleotides" and similar terms based on this relate to short oligos composed of naturally occurring nucleotides as well as to oligos composed of synthetic or modified nucleotides, as described in the immediately preceding paragraph. Oligonucleotides may be 19 or more nucleotides in length, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or more nucleotides in length, up to 35 or more, nucleotides in length.
An oligonucleotide that is an siRNA may have any number of nucleotides between 19 and 35 nucleotides. In many embodiments a siRNA may have any number of nucleotides between 19 and 27 nucleotides. In many embodiments, a siRNA may have two blunt ends, or two sticky ends, or one blunt end with one sticky end. The over hang nucleotides of a sticky end can range from one to four or more.
II. High-Throughput siRNA Design Algorithm
Referring now to Figs. 1 -13, the various results from and procedures associated with the present invention are shown. Using a 'library' of siRNA reagents in a suitable cell model for understanding a disease process, we perform an assay examining the effect of gene silencing by each reagent on the phenotype observed for the test cell, Figs 1 - 3. This in vitro assay then identifies genes which may be important in the disease etiology. A statistically relevant cutoff for activity produced by the siRNAs can be based on Z' factors, Z scores, robust Z scores, ranked product or even B scores. These statistical methods along with additional normalization schemes are used to select a list of siRNAs that have the required effects in the cell model. A suitable control cell model is also assessed under identical conditions to the test cell. It is desirable to identify siRNAs that produce a phenotype change in the test cell and not in the control cells. The control cell type is used to ensure that any phenotype changes observed upon gene silencing in the test cells are specific for the disease etiology and not a process that is present in other cell types. Obviously, the larger number of test and control cells that can be examined for the phenotype of interest, the better chance that identified siRNAs are specific for the disease vs control cells. Upon identification of an siRNA with the desired effect the gene that this siRNA targets is identified and this gene is added to a list of the genes of interest (a "hitlist").
Each of the genes in this gene 'hit list' is then evaluated for its position within distinct pathways within the cell. A pathway may consist of an enzymatic cascade where one enzyme converts a substrate to a product and removal of the enzyme (by gene silencing) results in accumulation of the substrate. Alternatively, the pathway could consist of a series of events where modification of a substrate (e.g. by phosphorylation or de-phosphorylation) alters the sensitivity/activity of the phosphorylated protein. Pathways can consist of signaling cascades or metabolic pathways (Kegg pathways). Tools used to evaluate the location of these genes in pathways include software packages that are commercially available. The ideal series of siRNAs for therapeutic benefit are those that, when applied simultaneously to the test cell type produce a more pronounced phenotype change than each added alone at the same concentration. It is important to be able to identify the optimal combinations of siRNAs that can meet these requirements and this must be performed empirically. As with the initial assay the mixture of siRNAs must also have little effect on the control cells. It may be advantageous to select species of siRNAs from distinct pathways identified in step 1 in order to increase the potency/efficacy of the cocktails relative to the single siRNA species. The lists of siRNAs of interest are categorized based on the pathways they are in within the cell. Prior to testing the siRNAs in the hitlist, it must be established that the siRNA sequences that will have efficacy in silencing the gene of interest in both human cells and mouse cells can be identified. This can be done using readily available software tools to obtain the gene sequence of the target gene of interest for each of the siRNAs in the hitlist. Tools such as Entrez gene from NCBI (National Institute for Health) ("http://www.ncbi.nlm.nih.gov/sites/entrez?db=:gene) may be used. The gene sequence is obtained for both human and mouse (or an appropriate second species defined by the development path for the therapeutic). Preferably a gene reformatter software tool is used to allow the sequences from these genes to be quickly formatted for searching. The reformatter tool removes any unwanted numbering schemes and gaps in the sequence string and saves the output as a distinct file structure. The files are then used by importing them into the appropriate fields of the sequence ID tool The GUI for the sequence comparator tools is shown in Fig. 14.
In a preferred embodiment, the invention provides siRNA of 21, 23 and 25 base pairs with blunt ends. The terms "polynucleotide" and "oligonucleotide" are used synonymously herein. In this form the user can select the location of the files containing the human and mouse sequences by clicking buttons and respectively. Upon opening the files ihe string containing the human sequence is inserted into the text box and the mouse (or other 2nd species) is loaded into text box. The user can select how long (# bases) the predicted siRNA sequence will be by entering the number into the text box. Furthermore, the user can select the degree of complementarity between the siRNA sequence between the 2 species by entering a number in box. This allows the user to specify which bases must be forced to be identical within the 5' to 3' region (text boxes below 1Od). In the example shown the default values are bases 10 and U from the 5' end but these values can be changed. By selecting another check box ("3' to 5' also?") allows the user to also find base identity in the reverse sequence (3' to 5') at bases in the same location numbered for the reverse end. It is believed that the nucleotides at locations 10 and 11 from the 5' end are the cleavage sites for dicer and hence these sites need to be a perfect match in both species to ensure that processing by dicer (cleavage of the mRNA) is identical in both species. To provide a quick visual validation that the gene sequences were loaded properly the 2 text boxes and hold the values for the length of each string in boxes respectively. Additional criteria for matching sequences between the 2 species are provided as check boxes on the form [13]. Selection of these check boxes allows refinement of the output expected. Options include:
1) Forcing the first 2 bases in the gene to be a "G" or a "C"
2) Excluding sequences as a match if the last 2 bases are not "A" or "T" 3) Excluding specific gene sequences that have been shown to have immune modulating features in their own right. Such sequences include ""UGUGU"" and "GUCCUUCAA". 4) An appropriate siRNA may be one with a specific GC content between 35 and 65%. This option can be selected with another check box. Finally, the user can select to prevent trinucleotide repeat sequences in the selected gene sequences with homology. After selecting the criteria, the user presses the button "Run Comparator" and the software examines the 2 genes for regions with the expected complementarity along the length of sequence specified. A progress bar shows the current estimate for the amount of the sequence that has been searched. The text box below the progress indicator shows the number of matches and updates as the number increases. Any matches are printed to a file specified by the user based on a dialog box.
Finally, to speed up selection of siRNAs with various lengths, the user can select the check box on the form. This specifies that the lengths of the sequences to match will be set at the typical lengths for siRNAs (19mer,21mer,25mer and 27mer) and all sequences will be examined using these lengths. If the degree of complementarity set at the time of running the comparator is 100% all of the subsequent matches at each siRNA length will match these criteria.
The sequences that meet the search criteria are saved to a file and this file can then be used as the source of additional steps and sequence searches (see Tables below). Another tool that has been developed is a BLAST analysis tool. The GUI for this interface is shown in Fig. 15.
The sequences listed in Tables 1 - 7 are the target mRNA coding sequences which can be used for siRNA sequence design by changing the "t" into "u", such as the sequences listed in Table 8. Table 1. Sequence design for Transforming Growth Factor- beta 1 :
Organism , Gene ■ homology Length No. Sense Sequences
Human : TGF-b1 Human 21-mer 1 ggtcacccgcgtgctaatggt
Mouse Mouse 2 , cacccgcgtgctaatggtgga
3 i ccaactattgcttcagctcca t
4 gcggcagctgtacattgactt
5 ccacgagcccaagggctacca
I 6 gcccaagggctaccatgccaa
I 7 ; ccaagggctaccatgccaact
8 cgcaagcccaaggtggagcag
I 9 cgctcctgcaagtgcagctga
10 I caagggctaccatgccaactt
23-mer 1 gaggtcacccgcgtgctaatggt
I 2 ' gtcacccσcgtgctaatggtgga
I
3. gtgcggcagctgtacattgactt
I 4 cgagcccaagggctaccatgcca
5 : gcccaagggctaccatgccaact
6 caagggctaccatgccaacttct
I 7 ■ gtgcgctcctgcaagtgcagctg
8 I cccaagggctaccatgccaactt
9 accaactattgcttcagctccac
10 ccgcccggcccgctgcccgaggc
I
I 25-mer 1 ! ggatccacgagcccaagggctacca
2 ' gatccacgagcccaagggctaccat
3 ' cacgagcccaagggctaccatgcca
4 gagcccaagggctaccatgccaact ; 5 cccaagggctaccatgccaacttct
6 gaggtcacccgcgtgctaatggtgg
I I 7 gtacaacagcacccgcgaccgggtg
I 8 ' ggcgcccjcctcccccatgccgccct
9.
10
Table 2. Sequence design for Transforming Growth Factor- beta 2:
Figure imgf000022_0001
I I 10, ccagtggtgatcagaaaactataaa |
Table 3. Sequence design for Cox-2:
Organism Gene homology Length No. Sense Sequences
Human Cox-2 Human 21-mer 1 caaaagctgggaagccttctc
Mouse Mouse 2 gatgtttgcattctttgccca
3 cattctttgcccagcacttca
4 catcagtttttcaagacagat
5 cagtttttcaagacagatcat
6 gtttttcaagacagatcataa i 7 ctgcgccttttcaaggatgga
8 gtctttggtctggtgcctggt
9 ctttggtctggtgcctggtct
10 ggagcaccattctccttgaaa
I
23-mer 1 gatgtttgcattctttgcccagc
I 2 catcagtttttcaagacagatcε
I 3 cagtttttcaagacagatcataa
4 ctgcgccttttcaaggatggaaa
I 5 gtctttggtctggtgcctggtct
6 ctttggtctggtgcctggtctga
7 ggtctgqtgcctggtctgatgat
8 ctggtgcctggtctgatgatgta
9 gcctggtctgatgatgtatgcca
10 gagcaccattctccttgaaagga
!
25-mer 1 gatgtttgcattctttgcccagcac
2 catcagtttttcaagacagatcata
3 gtttttcaagacagatcataagcga
4 gtctttggtctggtgcctggtctga
5 ggtctggtgcctggtctgatgatgt
6 gtgcctggtctgatgatgtatgcca
I 7 gagcaccattctccttgaaaggact
8 caecattctccttgaaaggacttat
I 9 cctcaattcagtctctcatctgcaa
10 caattcagtctctcatctgcaataa
Table 4. Sequence design for MMP-2
Organism , Gene homology Length No. Sense Sequences
Human xMMP-2 Human 21-mer 1 cccttgtttccgctgcatcca
Mouse Mouse 2 catcatcaagttccccggcga 3 gacaaagagttggcagtgcaa
4 gcaacccagatgtggccaact
5 caagcccaagtgggacaagaa
6 gcccaagtgggacaagaacca
I 7 caactttgagaaggatggcaa
1
8 gatgqcatcgctcagatccgt
I cctggatgccgtcgtggacct
I gccagggatctcttcaatgct
I
23-mer 1 cccttgtttccgctgcatccaga
I 2 ccatcatcaagttccccggcgat
3 gacaaagagttggcagtgcaata
4 ggcaacccagatgtggccaacta
5 cgcaagcccaagtgggacaagaa
6 gcccaagtgggacaagaaccaga
7 ggacaagaaccagatcacataca
I . 8 caactttgagaaggatggcaagt
9 ggcatcgctcagatccgtggtga
I 10 ctggatgccgtcgtggacctgca
I
I 25-mer 1 cccttgtttccgctgcatccagact
I 2 ccatcatcaagttccccggcgatgt
3 gagttggcagtgcaatacctgaaca
4 gcaacccagatgtggccaactacaa
5 gcaagcccaagtgggacaagaacca
6 cccaagtgggacaagaaccagatca
7 gacaagaaccagatcacatacagga
I 8 ggcatcgctcagatccgtggtgaga
I 9 gagcgtgaagtttggaagcatcaaa
10 gagatcttcttcttcaaggaccggt
Table 5. Sequence design for MMP-9
Organism ! Gene homology Length No. Sense Sequences
Human I MMP-9 Human 21-mer 1 catccagtttggtgtcgcgga
Mouse ' Mouse 2 ccagtttggtgtcgcggagca
3 gcggagcacggagacgggtat j 4 cggagacgggtatcccttcga
5 gagctgtgcgtcttccccttc
I 23-mer 1 gtcatccagtttggtgtcgcgga I
I 2 gcgcggagcacggagacgggtat
I 3 > ggagcacggagacgggtatccct
I 25-mer 1 ccagtttggtgtcgcggagcacgga
2 ' cgcgcgcggagcacggagacgggta
I 3 cggaσ.:acggagacgggtatccctt
Table 6. Sequence design for PDGF a:
Organism ' Gene homology Length No. , Sense Sequences
Human ! PDGF a Human 21-mer 1 caccctcctccgggccgcgct
Mouse , Mouse 2 ■ ctcctccgggccgcgctccct
3 gtactgaatttcgccgccaca
I 4 , ctgaatttcgccgccacagga
5 ggagcgcccgccccgcggcct i 6 ctgctgctcctcggctgcgga
7 gctgctcctcggctgcggata
8 gatccacagcatccgggacct
9 ccacagcatccgggacctcca
I
10 i catccgggacctccagcgact
i ' 23-mer ' 1 : gccaccctcctccgggccgcgct
! 2 I ccctcctccgggccgcgctccct
I 3 • gatggtactgaatttcgccgcca
I 4 ctggagcgcccgccccgcggcct
I 5 gcgcccgccccgcggcctcgcct
! '
6 gcctcgggacgcgatgaggacct
! I 7 ggcttgcctgctgctcctcggct
8 • gcctgctgctcctcggctgcgga i 9 cagatccacagcatccgggacct
I i 10 gaccaggacggtcatttacgaga
I
! 25-mer ' 1 I gcgccaccctcctccgggccgcgct
I 2 i caccctcctccgggccgcgctccct
3 I gggatggtactgaatttcgccgcca
I 4 1 gatggtactgaatttcgccgccaca i 5 ggtaci-gaatttcgccgccacagga
! 6 ggctggagcgcccgccccgcggcct
I
7 gagcgcccgccccgcggcctcgcct
! 8 ccagcgcctcgggacgcgatgagga
I 9 gcgcctcgggacgcgatgaggacct I 10 gcctgctgctcctcggctgcggata j
Table 7. Sequence design for FGF-2:
Organism Gene homology Length No. Sense Sequences
Human FGF-b Human 21-mer 1 cttcaaggaccccaagcggct
Mouse i Mouse 2 ggccacttcaaggaccccaag
, 3 ggcttcttcctgcgcatccat
4 caagcagaagagagaggagtt
! I 5 cagaagagagaggagttgtgt
6 gagaggagttgtgtctatcaa
7 gaagagagaggagttgtgtct
8 gaatctaataactacaatact
9 cagttggtatgtggcactgaa
10 cactgaaacgaactgggcagt
23-mer 1 cacttcaaggaccccaagcggct
2 caagcagaagagagaggagttgt
3 gcagaegagagaggagttgtgtt „
4 cagaagagagaggagttgtgtct
5 gaagagagaggagttgtgtctat
6 gagagaggagttgtgtctatcaa
7 ggaatctaataactacaatactt
8 ggtatgtggcactgaaacgaact g gttggtatgtggcactgaaacga
I 10 gtggcactgaaacgaactgggca
25-mer 1 gccacttcaaggaccccaagcggct
' 2 caagcagaagagagaggagttgtgt
3 gaagagagaggagttgtgtctatea
, 4 cagaagagagaggagttgtgtctat
5 gaagagagaggagttgtgtctatca
6 ggaatctaataactacaatacttac
7 ctaataactacaatacttaccggtc
8 cagttggtatgtggcactgaaacga
9 gtggcactgaaacgaactgggcagt
10 tcttccaatgtctgctaagagctga
III. Polymer Enhanced siRNA Cocktail Delivery
As shown in the study using siRNA cocktail to inhibit ocular neovascularization induced by HSV viral sequence, the HK polymer-siRNA nanoparticle mediated local delivery has achieved potent anti-angiogenesis activity. In a separate study using HK polymer to enhance siRNA delivery intratumorally, the tumor growth curves have showed significant anti-tumor efficacy with clear down regulation of the target gene expression. At 10 days after the injection of MDA- MB-435 cells into the mammary fat pad, mice with visible tumors were separated into treatment groups. Each group had four mice with eight tumors and tumor size was assessed in two dimensions and calculated. Mice received 4 μg/tumor of siRNA with each intratumoral injection every 5 days. To confirm the antitumor efficacy of siRNA Raf-1 with the optimal polymer in greater detail, mice with tumors were divided into these groups: untreated, b-galactosidase siRNA and Raf-1 siRNA. Clearly, HK polymer has been validated as an effective local siRNA delivery carrier. We plan to use HK polymer to facilitate the local siRNA delivery onto the skin wounds with the appropriate formulations.
Over the past few decades, biodegradable polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(lactic-co-glycolic acid) (PLGA), have been extensively studied for a wide variety of pharmaceutical and biomedical applications. The biodegradable polyester family has been regarded as one of the few synthetic biodegradable polymers with controllable biodegradability, excellent biocompatibility, and high safety. The need for a variety of drug formulations for different drugs and delivery pathways resulted in development of various types of block copolymers (eg, diblock, triblock, multiblock, and star-shaped block) consisting of the biodegradable polyesters and poly(ethylene glycol) (PEG).
PAMAM dendrimers represent a new class of macromolecular architecture called "dense star" polymers. Unlike classical polymers, dendrimers have a high degree of molecular uniformity, narrow molecular weight distribution, specific size and shape characteristics, and a highly- functionalized terminal surface. The manufacturing process is a series of repetitive steps starting with a central initiator core. Each subsequent growth step represents a new "generation" of polymer with a larger molecular diameter, twice the number of reactive surface sites, and approximately double the molecular weight of the preceding generation. Polyamidoamine (PAMAM) dendrimers are the most common class of dendrimers suitable for many materials science and biotechnology applications. PAMAM dendrimers consist of alkyl-diamine core and tertiary amine branches.
Since the introduction of DOTAP, there has been a steadily increasing number of scientific articles referring to this lipid. To date, at least 400 scientific papers have been published regarding different aspects of DOTAP. There are abundant literatures regarding in vitro and in vivo applications of TOTAP for nucleic acid delivery including siRNA delivery. We compare the in vivo delivery efficiencies between (S)-DOTAP Chloride and ®-D0TAP- Chloride after they are packaged with active siRNA oligos with xenograft tumor models with PC-3 cells. The lipoplex-siRNA nanoparticles are analyses for the particle size, Zeta potential and other characteristics related to the stability and deliverability.
To test HK polymer, PLGA, PAMAM and DOTAP for their role in enhancement of siRNA delivery in vivo, the formulations for each of these carrier-siRNA nanoparticles are defined. In accordance with one aspect of the invention, several peptide ligands have been identififed which can be used with either polymer or liposome, achieving targeted delivery to the Jurnρr4site.
1). RGDfH-ACRGDMFGCA-OH;
21-RyO-^VH-YTIWMP4ENPRPGTPCDIFTNSRGKRA1SNG-OH;
3>SapC fusogenic? H-SDVYCEVCEFLVKE^VTKCΪDNNKTEKEILDAFDKMGSK-OH.
IV. Identify The Most Potent siRNAs For Silencing TGF- β, COX-2 And MMP-9 In Vitro
As described in the Table 1, having already designed in silico 10 siRNA sequences for each of the targeted gene, COX-2, TGF- β and MMP-9, the next step is to take three siRNX,sequences from
Figure imgf000028_0001
target gene knbckdown in the respected cell culture. A. The siRNA control sequence was selected targeting a non-related sequence and without homologue in both human and mouse:
Lu25-a: sense 5 '-r(GAGGAGCCUUC AGGAUUAC AAGAUU)-3'
Antisense 5'-r(AAUCUUGUAAUCCUGAAGGCUCCUC)-3' B. Sequences Targeting TGF-beta: hmTF-1: sense 5'-r(GGAUCCACGAGCCCAAGGGCUACCA)-3' antisense 5 '-r(UGGUAGCCCUUGGGCUCGUGGAUCC)-3 ' hmTF-2: sense 5'-r(CCCAAGGGCUACCAUGCCAACUUCU)-3' antisense 5 '-r(AGAAGUUGGCAUGGUAGCCCUUGGG)-3 ' hmTF-3: sense 5 '-r(GAGCCCAAGGGCUACCAUGCCAACU)-3 ' antisense 5 '-r(AGUUGGCAUGGUAGCCCUUGGGCUC)-3 '
C. Sequences Targeting Cox-2: hmCX-1: sense 5 '-r(GGUCUGGUGCCUGGUCUGAUGAUGU)-3 ' antisense 5'-r(ACAUCAUCAGACCAGGCACCAGACC)-3' hmCX-2: sense 5'-r(GAGCACCAUUCUCCUUGAAAGGACU)-3 ' antisense 5 '-r(AGUCCUUUC AAGGAGAAUGGUGCUC)-3 ' hmCX-3: sense 5'-r(CCUCAAUUCAGUCUCUCAUCUGCAA)-3 ' antisense 5'-r(UUGCAGAUGAGAGACUGAAUUGAGG)-3'
D. Sequences Targeting MMP-9: hmM9-l: sense 5'-r(CCAGUUUGGUGUCGCGGAGCACGGA)-3' antisense 5 '-r(UCCGUGCUCCGCGACACCAAACUGG)-3 ' hmM9-2: sense 5 '-r(CGCGCGCGGAGC ACGGAGACGGGUA)-3 ' antisense 5'-r(UACCCGUCUCCGUGCUCCGCGCGCG)-3' hmM9-3: sense 5'-r(CGGAGCACGGAGACGGGUAUCCCUU)-3' antisense 5 '-r(AAGGGAUACCCGUCUCCGUGCUCCG)-3 '
E. Sequence Targeting PDGFa: hmPD-1 : sense 5 '-r(GAUGGUACUGAAUUUCGCCGCCACA)-3 ' antisense 5'-r(UGUGGCGGCGAAAUUCAGUACCAUC)-3' hmPD-2: sense 5 '-r(GGGAUGGUACUGAAUUUCGCCGCCA)-3 ' antisense 5 '-r(UGGCGGCGAAAUUCAGUACC AUCCC)-3 ' hmPD-3: sense 5'-r(GCCUGCUGCUCCUCGGCUGCGGAUA)-3' antisense 5'-r(UAUCCGCAGCCGAGGAGCAGCAGGC)-3' II. Transfection of siRNA duplexes into the specific cell cultures
For measuring gene expression knockdown at both mRNA and protein levels using three selected siRNA duplexes, the PC3 cell expressing TGF-beta, Cox-2, MMP-9, PDGFa and FGF2 were transfected by three siRNA duplexes with Lipofectamin 2000. Similarly, for measurement of Cox-2 gene expression knockdown at both mRNA and protein levels using four selected siRNA duplexes, human foreskin fibroblasts (HFF) is cultured on 10-cm plates in DMEM supplemented with 10% fetal bovine serum (FBS), 100 Dg/ml streptomycin, and 100 U/ml penicillin and transfected with the siRNA duplexes using Lipofectamin 2000. The cells should be washed twice with PBS and incubated in FBS-free medium for 24 h. FBS-free medium was replaced with medium containing 10% FBS to initiate the cell cycle. For measuring TGF-D gene expression knockdown at both mRNA and protein levels using four selected siRNA duplexes, the mouse tumor line CT26 was transfected with siRNA-Lipofectamin 2000 followed by RT-PCR analysis.
III. Measurement of mRNA levels using RT-PCR
Total RNA from each of those transfected cell lines including PC3 cells , PDGF (human) expressing cells and mouse embryonic endothelial cells are isolated and purified for RT-PCR analysis.
A. For detection of PDGF amplicon (mouse), an RT reaction is followed with a PCR reaction using a pair of primers: mPDGF up: 5' -CTCCAGCTCCTGTGCCTTAT-3' mPDGF down: 5'-ACT GGCCATAGGCTGGTATG-3'.
B. For detection of Cox-2 amplicon (human), an RT reaction is followed with a PCR reaction using a pair of primers: hCox-2 up: 5'-CGGGCTGGGCCATGGGGTGGA-S' hCox-2 down: 5'-CCTATCAGTATTAGCCTGCTT -3'.
C. For detection of TGF-β amplicon (mouse), an RT reaction is followed with a PCR reaction using forward primers: mTGF-β up: 5'-CTACTGTGTGCTGAGCACCTT-S' mTGF-β down: 5'-CGCTGCTCGGCCACTCTGGCT-S'.
D. Human and mouse homologues for PCR detection of Cox-2 : hmCox-2 up: 5'- GATGTTTGCATTCTTTGCCCAGCA -3 ' hmCox-2 down: 5'- CATCAGACCAGGCACCAGACCA-3' E. Human and mouse homologues for PCR diction of TGF-b 1 : hmTGF-βl up: 5'-GTGCGGCAGCTGTACATTGACTT-S' hmTGF-βl down: 5'-CAGCTGCACTTGCAGGAGCGCA-S'
F. Human and mouse homologues for PCR detection of MMP-9: hmMMP-9 up 5'-GGAGCACGGAGACGGGTATCCCTT-S' hmMMP-9 down: 5'-GAAGGGGAAGACGCACAGCT-S'
G. Human and mouse homologues for PCR detection of MMP-2: hmMMP-2 up: 5'-GATGGCATCGCTCAGATCCGTGGT-S' hmMMP-2 down: 5'-CCTGCAGGTCCACGACGGCATCCA-S'
H. Human and mouse homologues for PCR detection of FGF-2: hmFGF2 up: 5'-CACTTCAAGGACCCCAAGCGGCT-S' hmFGF2 down: 5'-CAGCCCAGTTCGTTTCAGTGCCA-S'
I. Human and mouse homologues for PCR detection of PDGFA-2: hmPDGFA up: 5'-GCAAGACCAGGACGGTCATTT-S' hmPDGFA down: 5 '-CAC AGTTTTTCACGGAGGAGA-3 ' The PCR products should be loaded on a 1% agarose gel and stained with ethidium bromide. The PCR product should exhibit the levels of the knockdown of each particular mRNA using the particular siRNA duplexes. As a result of this procedure, the potency of each siRNA duplex is determined if a particular siRNA duplex should be the most potent one. The RT-PCR analysis should be done in conjunction with the transfection experiment so that a proper condition can be optimized for efficient transfection for particular cell line, in order to achieve sufficient amount of total RNA for the PCR analysis. RT-PCR results showed that the potent siRNA duplexes targeting TGF-beta, Cox-2 and MMP-9 are identified (Figure 1-6).
III. Measurement of protein levels using ELISA To measure protein levels of the cells transfected with corresponding siRNA duplexes, the Western blot analysis and ELISA analysis is preferably used. The cell lysates or cell culture media would be used for the protein detection. The human COX-2 is analyzed using COX2 ELISA kit (Zymed, San Francisco, CA) which is an enzyme-linked immunosorbent sandwich assay for quantitative detection of human COX-2 in cell culture supernatants and cell lysates. Since Cyclooxygenase (COX) is a membrane-bound enzyme, which has a molecular weight of 71 kDa, the cell lysate should be prepared for the ELISA analysis. The mouse TGF- is analyzed using Human/ Mouse TGFbI (Transforming Growth Factor beta 1, TGF-betal, TGF-bl) ELISA Ready-SET-Go Kit (with Pre-Coated Plates). The selection of the most potent siRNA duplex for each gene is based on three repeated experiments. It should be noted that sometimes the most potent siRNA duplex selected from the mRNA knockdown is not correlated with the one selected from the protein knockdown. When that happens, the user can rely on the data from the mRNA level knockdown, since that it is the direct reflection of RNAi mechanism of action. The discrepancy of the protein level knockdown some times may be due to the non-specific or so call "off-target" effect, which is not the result of the RNAi mechanism of action.
IV. Select The Most Efficacious siRNA Cocktail In Vivo
After selection of the most potent siRNA duplex for each of the following three genes, MMP-9,
COX-2 and TGF-β based on the cell culture studies, they are combined together as the siRNA cocktail with several ratios of the combinations which can be used such as 1:1 :1, 2:1:1 and 3:1:1, etc. In addition, the user must evaluate the appropriate mouse models and siRNA nanoparticle formulations, to define the most suitable siRNA-nanoparticle formulation for potential therapeutic protocol. To establish a polymer-siRNA nanoparticle, we decided to first test the Histidine-Lysine branched polymer for this formulation. The biopolymer core facility at the University of Maryland will synthesize polymers on a Ranin Voyager synthesizer (PTI,
Tucson, AZ). The branched HK polymer, effective for in vivo siRNA transfer, was complexed with siRNA duplexes for local administration. The polymer will be purified by HPLC (Beckman, Fullerton, CA). The second branched H (histidine) and K (Lysine) polymers used in this study should be R-KR-KR-KR, where R =[HHHKHHHKHHHKHHH]2KH4NH4]. H3K4b is a branched polymer with the same core and structure described above except the R branches differ: R = KHHHKHHHKHHHKHHHK. The HKP can be dissolved in aqueous solution and then mixed with siRNA aqueous solution at a ratio of 4:1 by mass, forming nanoparticles of average size of 150-200 nm in diameter. The HKP-siRNA aqueous solutions were semi- transparent without noticeable aggregation of precipitate, and can be stored at 4~ C for at least three months. In addition to HK polymers, we may also test two different types of polymer carriers, pegylated PEI and PAMAM dendrimer, with our siRNA cocktail for efficient delivery into the surrounding areas of the skin wounds. All these siRNA polymer formulations will be dissolved in the RNAse free D5W solution.
V. Method for siRNA cocktail Design
Human disease is a complicate pathological process showing various severities of disease symptoms. Many human diseases are caused by abnormal over expressions of disease causing or disease control genes from human body itself, or from foreign infectious organisms, or both. The disease progression and development of drug resistance can also circumvent the effect of single drug treatment. One strategy to overcome those hurdles is using combination of multiple drugs. The invention provides the therapeutic siRNA cocktail targeting multiple disease controlling genes in the same treatment. The invention provides for RNAi agents, such as siRNA oligonucleotides, that are chemically similar to the same source of supply and the same manufacturing process, and they are comprised of four types of nucleotides with different sequences. The invention provides the siRNA cocktail drug for improvement of scarless wound healing by targeting genes involved in the wound healing process, including TGF-β, Cox-2, MMP-9 and others.
A key aspect of the invention is the identification of the following characteristics of the siRNA cocktail and its applications in the experimental and therapeutic settings:
(1) The siRNA cocktail must contain at least three siRNA duplexes targeting at least three genes (not three sequences of the same gene) at a ratio of therapeutic requirement.
(2) The siRNA cocktail design for each combination must follow the understanding of the role of each gene in a background of the system biology network, such as these genes are functioning either in the same pathway or in a different one. (3) The chemical property of each siRNA duplexes in the cocktail must be the same in terms of source of supply, manufacturing process, chemical modification, storage conditions and formulation procedures.
(4) Each individual siRNA duplex in the cocktail can be different in their lengths, with either blunt or sticky end, as long as their potencies have been defined.
(5) Since siRNA cocktail is targeting multiple genes and a single cell type usually does not express all those factors, the efficacy of a siRNA cocktail must be tested in a relevant disease model, either a multiple cell model, a tissue model or a animal model, after the confirmation of the potency of each individual siRNA duplex in the cell culture. (6) Each validated siRNA cocktail can be used for addressing one or more pathological conditions, for treatment of one or multiple types of diseases, such as, siRNA cocktail for suppressing inflammation, siRNA cocktail for antiangiogenesis activity and siRNA cocktail for autoimmune conditions.
(7) The siRNA cocktail must be administrated through the same route of delivery in the same formulation, although the regimen of dosing for each cocktail will be defined based on either the experimental design or therapeutic requirement.
(8) Each siRNA cocktail can be applied either independently, or in combination with other drug modalities such as small molecule inhibitors, monoclonal antibodies, protein and peptides, and other siRNA cocktail drug(s).
Table A. The siRNA cocktail targeting Cox-2, MMP-9 and TGF-β 2.
Figure imgf000034_0001
Cocktail 3 MMP-9, 5'-cgcgcgcggagcacggagacgggua-3'
Cox-2, 5 '-gagcaccauucuccuugaaaggacu-3 '
TGF-β2, 5 '-cgcccacuuucuacagacccuacuu-3 '
Cocktail 4 MMP-9, 5 '-ccaguuuggugucgcggagcacgga-3 '
Cox-2, 5'-ggucuggugccuggucugaugaugu-3 '
TGF-β2, 5 '-cgcccacuuucuacagacccuacuu-3 '
Table B. The second siRNA cocktail targeting Hoxbl3, Cox-2 and VEGFA. siRNA Cocktail Combinations (targeted sequences)
MMP-9
Cox-2 Humar i and Mouse homologues
PDGFA
Cocktail 1 MMP-9, 5'-cgcgcgcggagcacggagacgggua-3'
Cox-2, 5 '-gucuuuggucuggugccuggucuga-3 '
PDGFA, 5'- gaugguacugaauuucgccgccaca-31
Cocktail 2 MMP-9, 5 '-caaggauaucgaaggcuugcuggga-3 '
Cox-2, 5'-ggucuggugccuggucugaugaugu-3'
PDGFA, 5'- gggaugguacugaauuucgccgcca-3'
Cocktail 3 FGF2, 5'-gaagagagaggagttgtgtctatca-3'
Cox-2, 5 '-gagcaccauucuccuugaaaggacu-3 '
PDGFA, 5 '-ccaugccaaguggucccaggcugca-3 '
Table C. An Alternative siRNA cocktail targeting PDGFa, Cox-2, TGF-βl &TGF-β2:
Figure imgf000035_0001
Cocktail 2 FGF-2, 5 '-gaagagagaggagttgtgtctatca-3 '
Cox-2, 5 '-gagcaccauucuccuugaaaggacu-3 '
TGF-βl, 5 '-ggauccacgagcccaagggcuacca-3 '
TGF-β2, D 5'-cagauccugagcaagcugaagcuca-3'
Cocktail 3 MMP-9, 5'-cgcgcgcggagcacggagacgggua-3'
Cox-2, 5'-ggucuggugccuggucugaugaugu-3 '
TGF-βl, 5'-ggauccacgagcccaagggcuacca-3 '
TGF-β2, 5'-cagauccugagcaagcugaagcuca-3 '
Besides the above examples, the siRNA sequences targeting various tumorigenic genes can be arranged into different combinations for enhancing the anti-tumor activity in xenograft mouse models, or in human oncology drug testing.
VI. Demonstration of in vivo siRNA delivery and target gene knockdown in mouse model
An experiment was done with the skin excision wound model to analyze the therapeutic benefit of TGFβ-siRNA with HKP-mediated topical administration. Ten mice were used in the experiment with a paired 5 mm diameter full-thickness excision skin wounds on both sides of the dorsal midline the depilitated dorsum of the Balb/c mouse). The conventional methylcellulose was used as the topical administration carrier with or without HKP-siRNA. Two wounds on each mouse were either treated with only methylcellulose or methylcellulose plus HKP-siRNA daily for the first 5 days. The observations were taken on day 0, 5, 9 and 15th. When the images were put together (Figure 10), we found an evident therapeutic benefit on the closure of the skin wounds treated with the nanoparticle-enhanced siRNA delivery. The speed of wound closure at day 5th was much faster in treated group than those in the control group. On day 9th , almost all treated wound were pretty much closed while the control group still had many opened wounds. On day 15th, the superficial observation showed no significant difference between the two groups.
VII. Evidence of target gene knockdown in samples from the skin excision wound After observing that HKP-siRNAτGFβ-i illustrated the therapeutic benefit for the skin wound closure, it must be determined if the improvement of wound healing is really the result of the target gene silencing in the surround tissue of the wounds. Tc answer such a question, the total RNA samples were isolated from the representative samples of each group followed by RT-PCR analysis. As showed in Figure 11, the specific knockdown of TGFβ-1 expression in the wound tissue treated with the HKP-siRNAτσFβ-i is very compelling compared to the control groups. The siRNA cocktail targeting TGFβ-1, Hoxbl3 and Cox-2 packaged with HKP demonstrated similar knockdown activity as TGFβ-1 siRNA package with HKP, while siRNA duplexes specific to either Hoxbl3 or Cox-2 packaged with HKP did show TGFβ-1 down regulation.
VIII. Quantification of wound closures after treatments
The next experiment was to quantify the wound closure at each time point. During the experiment, it was also determined if the therapeutic benefit is the result of HK polymer or siRNA itself. Four groups under different treatments with J 0 samples each were conducted in the study: 1) Methylcellulose only, 2) Methylcellulose plus HKP-siRNA TGFβ-i, 3) Methylcellulose plus naked siRNA TGFP-I, and 4) Methylcellulose plus HKP- siRNAcoπtroi- The daily treatment was applied for the first 5 days. The wound images were collected at day 5 and day 9, and further quantified using Scion Imaging Program for Windows (Scion Corp., Frederic, MD) and presented with a percentage of the initial wound area. By averaging the measurements of the wound samples of each group on day 5th and 9th (Figure 12), significant differences (P< 0.05) between group 2 and the other three groups were found, although some effects were seen with group 3. The therapeutic benefit for faster skin wound closure is the results of HKP- siRNA
TGFβ-1.
IX. HKP-Cox-2-siRNA delivery in vivo
Once it was determined that nanoparticle-enhanced TGFβ-1 was responsible for the therapeutic benefit for the skin wound closure, it should be determined if this benefit would also happen to other gene targets, such as Cox-2. The experiment was conducted using the same Balb/c mice with the skin excision wound model. The treatment groups were methylcellulose plus siRNAcox-2 and methylcellulose plus HKP-siRNAcox-2 in addition to the control groups with only methylcellulose and methylcellulose plus HKP-siRNA control. The similar patterns of wound closure rates were observed using the Scion Imaging Program. To further evaluate the characteristics of the wound healing process and the underlining property of the wounds treated with various reagents, we collected the skin tissue samples from the wounded areas and than conducted studies on the histopathologic changes of those samples (Figure 13). The images are showing that HKP Cox-2 siRNA was the potent contributor resulting similar skin structure as the normal tissue. On the other hand, the control groups and the naked Cox-2 siRNA treatment group did not show any significant benefit to the recovery from the skin wounds which were demonstrated through three different magnifications, x40, xl 60 and x400.
X. Use EEMP for Cancer Treatment
Epoxy Ferric Oxide Nano-particles are non toxic and super magnetic. Epoxy Ferric Oxide
Nano-particles have the size of RBC, which can be utilized for several medical application, including homestasis, anti-angiogenesis, and delivery vehicle. A proposed in vitro Studies should be : 1. Simulation Studies: introduction of Epoxy grains solution with different concentrations into flexible tubes but with various diameters which to simulate the blood stream. At the bottom of the narrow area there will be a strong magnetic field applied by the use of an adhesive Bandage containing high field magnets to attract the epoxy grain embedded nanoparticles. 2. Using an angiogenesis model to test the ability of Ferric oxide- epoxy coated particle to be concentrated in a single spot. For these larger quantities of epoxy resin encapsulated nanoparticles are need. In Vivo studies include 1). Animal studies to measure toxicity; 2). pharmacokinetics, and 3). efficacy. Finally, it should be tested in clinical setting with Human subjects such as healthy volunteers.

Claims

What is claimed is: 1. A composition comprising at least one small interfering RNA (siRNA) double stranded oligonucleotide (duplex) and a pharmaceutical carrier, wherein said siRNA binds to a single stranded RNA molecule, said single stranded RNA molecule can be a mRNA encodes at least part of a peptide or protein whose activity promotes proliferation, angiogenesis, tumorigenesis, or said single stranded RNA molecule can be a micro RNA (miRNA) functioning as a regulatory molecule whose activity promotes proliferation, angiogenesis, tumorigenesis in human.
2. The composition according to claim 1 where the pharmaceutical carrier is selected from the group consisting of saline, sugars, polymer, lipid, cream, gel, micelle materials and metal nanoparticle, such as Epoxy Encapsulated Fe3O4 magnetic nanoparticles.
3. The composition according to claim 1 or claim 2 wherein said tumorigenesis is selected from the group consisting of various types of tumors including prostate adrenocarcinoma.
4. A composition according to any of claims 1-3 wherein said mRNA molecule encodes a gene selected from the group of pro-tumorigenic pathway genes, pro-angiogenesis pathway genes, pro-cell proliferation pathway genes, and viral infectious agent genome RNA, and viral infectious agent genes.
5. A composition according to Claim 1 comprising at least three siRNA duplexes, siRNA cocktail, where the siRNA duplexes bind at least three mRNA molecule that encode genes selected from the group of pro-tumorigenic pathway genes, pro-angiogenesis pathway genes, pro-cell proliferation pathway genes, and viral infectious agent genome RNA, and viral infectious agent genes.
6. A composition according to Claim 5 comprising an siRNA cocktail with at least three siRNA duplexes wherein each siRNA molecule binds to at least an mRNA molecule that encodes at least one protein.
7. A composition according to Claim 5 comprising an siRNA cocktail with at least three siRNA molecules wherein each siRNA molecule binds to at least a mRNA molecule of human gene that encodes at least one protein.
8 A composition according to Claim 5 comprising an siRNA cocktail with at least three siRNA molecules wherein each siRNA molecule binds to both human mRNA molecule and mouse mRNA molecule with homologues encode a same or similar protein.
9. A composition according to Claim 6 comprising an siRNA cocktail with at least three siRNA molecules (Table A) wherein an siRNA (sense: 5'-ccaguuuggugucgcggagcacgga-3", antisense: 5'-uccgugcuccucgcacaccaaacugg-3') binds to an mRNA molecule that encodes human and mouse both MMP-9 protein, at least one siRNA molecule (sense: 5'- gucuuuggucuggugccuggucuga -3', antisense: 5'-ucagaccaggcaccagaccaaagac-3') binds to an mRNA molecule that encodes both human and mouse Cox-2 protein, and at least one siRNA molecule (sense: 5'- ccccggaggugauuuccaucuacaa -3', antisense: 5'- uuguagauggaaaucaccuccgggg-3') binds to an mRNA molecule that encodes both human and mouse TGFβl.
10. A composition according to Claim 5 wherein said mRNA molecules encode one or more MMP pathway genes, Cox-2 pathway genes, TGFβ 1 pathway genes or a combination thereof.
12. A composition according to Claim 5 wherein said mRNA molecules encode one or more pro-angiogenesis genes, pro-inflammatory genes, or a combination thereof.
13. A composition according to Claim 5 wherein said mRNA molecules encode one or more pro-inflammation genes, or a combination thereof.
14. A composition according to Claim 5 comprising at least three siRNA molecules that bind to at least two or more different mRNA molecules. 15. A composition according to Claim 2 where said carrier is selected from the group consisting of saline solution, glucose solution, polycationic binding agent, cationic lipid, cationic micelle, cationic polypeptide, hydrophilic polymer grafted polymer, non-natural cationic polymer, cationic polyacetal, hydrophilic polymer grafted polyacetal, ligand functionalized cationic polymer, and ligand functionalized-hydrophilic polymer grafted polymer.
16. A composition according to Claim 15 where said polymers are biodegradable Histitine-Lysine polymers, biodegradable polyesters, such as pcly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(lactic-co-glycolic acid) (PLGA), and Polyamidoamine (PAMAM) dendrimers. 17. A composition according to Claim 2 where said carrier is identified as a histidine- lysine copolymer which forms nanoparticle with siRNA molecule in β size of 100 -400 nm in diameter.
18. The composition according to any preceding claim wherein said siRNA molecule is a dsRNA oligonucleotide, at length of 19 base pairs, or 20, or 21, or 22, or 23, or 24, or 25, or 26 or 27 base pairs.
19. The composition according to any preceding claim wherein said siRNA molecule is a dsRNA oligonucleotide, with blunt ends at both ends, or sticky ends at both ends, or one of each.
20. The composition according to any preceding claim wherein said siRNA molecule is a dsRNA oligonucleotide, with chemical modification or without chemical modification at individual nucleotide level or oligo backbone level.
21. A method for treating ocular disease in a subject, wherein said disease is characterized at least in part by inflammation and neovascularization, comprising administering to said subject a composition comprising a siRNA cocktail and a pharmaceutically acceptable carrier, wherein said siRNA cocktail inhibits expression of multiple genes that promotes disease pathology in ocular tissues of said subject.
22. The method according to claims 20, wherein said siRNA cocktail must be tested in animal disease models.
23. The method according to claims 20, wherein said siRNA cocktail must be first tested in animal disease models where said ocular disease is in at least the anterior of the eye.
24. A method according to claim 20 where said composition is administered at a site distal to the eye selected from the group of subconjunctival, intravenous, subcutaneous and intravenous routes.
25. A method according to claim 20 wherein said composition is administered topically to the eye.
26. A method according to Claim 20 where said pharmaceutical carrier is selected from the group of a saline, glucose, polymer, lipid, or micelle solutions. 27. A method according to Claim 20 where the ocular disease is selected from the group of herpetic stromal keratits. uveitis, rubeosis, conjunctivitis, keratitis, blepharitis, sty, chalazion, iritis, age-related macular degeneration, proliferate diabetic retinopathy and retinopathy of premature.
28. A method according to Claim 20 wherein said the siRNA cocktail inhibits expression of at least one gene selected from the group of pro-inflammatory pathway genes, pro- angiogenesis pathway genes, pro-cell proliferation pathway genes, and viral infectious agent genome RNA, and viral infectious agent genes.
29. A method according to Claim 20 wherein said siRNA cocktail inhibits expressions of multiple genes. 30. A method according to Claim 27 wherein said siRNA cocktail, with sequences in
Table 3, 4 and 5, inhibits expressions of MMP-9, TGF-betal or 2 and Cox-2 in both human and mouse cells.
31. A method according to Claim 27 wherein said siRNA cocktail, with sequences in Table 1, 2 and 16, inhibits expressions of PDGFa, FGF-2 and Lamin Bl proteins in both human and mouse cells.
32. A method according to Claim 27 wherein said siRNA cocktail inhibits expression of pro-angiogenesis genes, pro-inflammatory genes, or a combination.
33. A method according to Claim 27 wherein said siRNA cocktail inhibits expression of pro-angiogenesis genes, promote scar formation genes, or a combination thereof. 34. The method according to Claim 28 wherein said siRNA cocktail inhibits expression of pro-angiogenesis genes, proinflammation genes, or a combination thereof. 35. The method according to Claim 41 wherein said siRNA cocktail compose of at least three siRNA duplexes at proper ratio, wherein said siRNA cocktail compose of at least three siRNA duplexes at a 1:1:1 ratio, or 1:1.5:0.5, or 0.5:0.5:2 or other ratios according to the potency of each siRNA duplex and therapeutic requirement during the application.
36. A method according to Claim 2 wherein said carrier is selected from the group of polycationic binding agent, cationic lipid, cationic micelle, cationic polypeptide, hydrophilic polymer grafted polymer, non-natural cationic polymer, cationic polyacetal, hydrophilic polymer grafted polyacetal, ligand functicmalized cationic polymer, and ligand functionalized-hydrophilic polymer grafted polymer.
37. Epoxy Encapsulated Magnetic Nanoparticle can be use directly or in combination with siRNA therapeutics for treatment of various tumors.
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