METHODS FOR INTRACELLULAR DELIVERY AND ENHANCED GENE TARGETING
1. A method of enhancing gene targeting comprising:
- administering a targeting molecule to a cell, wherein the targeting molecule binds a target molecule in the cell; and
administering a stressor to the cell, wherein the stressor induces a cellular stress response;
wherein co-administration of the stressor with the targeting molecule enhances the function of the targeting molecule.
Disclosed herein are methods and compositions for enhancing gene targeting. The method entails co-administrating to a cell a targeting molecule and a means of enhancing the function of the targeting molecule upon delivery to the cell. The means of enhancing the function of the targeting molecule including one or more of a stressor that induces cellular stress, a proton sponge molecule, and an endosome or lysosome inhibitor. Compositions disclosed include a targeting molecule and one or more of a stressor that induces cellular stress, a proton sponge molecule, and an endosome or lysosome inhibitor.
- 1. A method of enhancing gene targeting comprising:
administering a targeting molecule to a cell, wherein the targeting molecule binds a target molecule in the cell; and administering a stressor to the cell, wherein the stressor induces a cellular stress response; wherein co-administration of the stressor with the targeting molecule enhances the function of the targeting molecule.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
- 17. A method of enhancing gene targeting comprising:
administering a proton sponge molecule or an endosome or lysosome inhibitor to the cell; wherein co-administration of the proton sponge molecule or the endosome or lysosome inhibitor with the targeting molecule enhances the function of the targeting molecule.
- View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)
- 33. A composition comprising:
a targeting molecule, wherein the targeting molecule binds a target molecule in the cell; and a stressor, wherein the stressor induces a cellular stress response; wherein the composition enhances the function of the targeting molecule when administered to a cell.
- View Dependent Claims (34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45)
- 46. A composition comprising:
a targeting molecule, wherein the targeting molecule binds a target molecule in the cell; and a proton sponge molecule or an endosome or lysosome inhibitor; wherein the composition enhances the function of the targeting molecule when administered to a cell.
- View Dependent Claims (47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58)
- 59. A method of controlling the cellular localization of an siRNA or an endogenous miRNA, comprising administering a stressor, a proton sponge molecule, or both to a cell, such that the siRNA or the endogenous miRNA translocates from the cytoplasm to the nucleus, thereby increasing the function of the siRNA or the miRNA against its nuclear RNA target, wherein the stressor induces a cellular stress response.
This application claims priority to U.S. Provisional Application No. 62/422,057, entitled “Methods for Intracellular Delivery of Oligonucleotides,” filed Nov. 15, 2016, which is incorporated herein by reference in its entirety, as if fully set forth herein.
It is well established that oligonucleotides (ONs) are highly potent in the cell nucleus. MiRNAs, siRNAs and chemically modified oligonucleotides have been employed for decades for research and therapeutic purposes (1). MiRNAs, which may control the expression of more than half of all human genes, are active predominantly in the cytoplasm, but they also form complexes in the cell nuclei with components of the RNAi machinery (2). Various regulatory nuclear functions have been attributed to miRNAs and other non-coding RNA (ncRNAs), including the still-debated transcriptional gene silencing (3). Similarly, oligonucleotides delivered by gymnosis are active in the cytoplasm, but can also be transported to and are effective in the nucleus (4). As used herein, the term “gymnosis” refers to oligonucleotide delivery to cells that produces function in the absence of any carriers or conjugations. It is unclear why and how the oligonucleotides translocate to the cell nucleus since they can effectively function in the cytoplasm (4). Though several candidate proteins have been reported to bind oligonucleotides (4-6), the mechanism that determines whether an miRNA or an oligonucleotide exerts its function in the cytoplasm, or shuttles to the nucleus and acts at an earlier step in the gene regulation pathway, is unknown.
The recent FDA approval for the marketing of eteplirsen, a phosphoromorpholidate antisense oligonucleotide (ASO) (50,51), for the treatment of Duchenne'"'"'s muscular dystrophy, has propelled the clinical development of splice-switching oligonucleotides (SSOs) (52). At the same time, drisapersen, a phosphorothioate (PS) SSO, which like eteplirsen was designed to produce exon skipping in the dystrophin mRNA, did not fare as well, missing its primary endpoint (the 6 minute walking test) (53). Though the use of SSOs as therapeutic molecules is promising and has shown to be well tolerated, including after multiple intrathecal administrations as in the case of nusinersen (54), their potential, as for any therapeutic antisense oligonucleotide, is hampered by substandard delivery to their targeted cells. Attempts to improve efficacy by escalating oligonucleotide doses frequently lead to unacceptable toxicity. Chemical modifications, such as locked nucleic acid (LNA) have proven to increase oligonucleotide efficacy in vivo, but the required concentrations for some therapeutic applications, depending on the oligonucleotide sequence, may also produce toxicity (55,56).
Intracellular delivery of therapeutic agents such as antisense oligonucleotides, siRNA, shRNA, miRNA, splice-switching oligonucleotides, or other small molecules is not well understood. Thus, methods and compositions for improving the efficacy of gene targeting would be desired.
In certain embodiments, methods of enhancing gene targeting is provided. In one aspect such methods may include steps of administering a targeting molecule to a cell, wherein the targeting molecule binds a target molecule in the cell; and administering a stressor to the cell, wherein the stressor induces a cellular stress response; wherein co-administration of the stressor with the targeting molecule enhances the function of the targeting molecule. The method may also include a step of administering a proton sponge molecule or an endosome or lysosome inhibitor to the target cell, wherein co-administration of the proton sponge molecule or the endosome or lysosome inhibitor with the targeting molecule and the stressor further enhances the function of the targeting molecule.
In another aspect, the method may include steps of administering a targeting molecule to a cell, wherein the targeting molecule binds a target molecule in the cell; and administering a proton sponge molecule or an endosome or lysosome inhibitor to the cell, wherein co-administration of the proton sponge or the endosome or lysosome inhibitor with the targeting molecule enhances the function of the targeting molecule. The method may also include a step of administering a stressor to the target cell, wherein the stressor induces a cellular stress response, and wherein co-administration of the stressor with the targeting molecule and the proton sponge molecule or the endosome or lysosome inhibitor further enhances the function of the targeting molecule.
In some embodiments, disclosed herein are compositions that include (i) a targeting molecule, and (ii) a stressor, a proton sponge molecule or an endosome or lysosome inhibitor, both of a stressor and a proton sponge molecule, or both of a stressor and an endosome or lysosome inhibitor.
In the embodiments described herein, the targeting molecule includes an oligonucleotide, for example, an antisense oligonucleotide (ASO), an siRNA, an shRNA, or an miRNA. In certain embodiments, the antisense oligonucleotide includes a splice-switching oligonucleotide (SSO). In certain embodiments, the oligonucleotide includes a phosphorothioate oligonucleotide. In certain embodiments, the oligonucleotide includes a locked nucleic acid (LNA).
In the embodiments described herein, the stressor is capable of inducing a cellular stress, including for example, arsenic trioxide (As III or ATO), H2O2, glutathione, lipofectamine, or heat shock.
In the embodiments described herein, the proton sponge molecule includes an ammonium compound, such as ammonium chloride (NH4Cl), ammonium hydroxide (NH4OH), ammonium sulfate (NH4SO4), ammonium nitrate (NH4NO3), ammonium acetate (NH4CH4CO2), or ammonium bicarbonate (NH4HCO3).
In the embodiments described herein, an endosome or lysosome inhibitor includes Ambroxol (Amb), Cyclohexylamine (CHA) or oleic acid (OA).
The embodiments described herein can be used to treat or research an indication that requires the targeting molecule to be delivered to the nucleus, the nucleolus, or the cytoplasm of a cell. As such, the methods described herein may include in vitro, in vivo, or ex vivo administration of (i) a targeting molecule, and (ii) a stressor, a proton sponge molecule or an endosome or lysosome inhibitor, both of a stressor and a proton sponge molecule, or both of a stressor and an endosome or lysosome inhibitor.
Methods and compositions for intracellular delivery and enhanced gene targeting are provided herein. The methods and compositions relate to delivery of two or more molecules that, when co-administered to a cell, enhance cellular delivery and/or enhance the efficacy of one of the molecules.
In certain embodiments, a composition is provided. The composition may include two or more molecules that, when co-administered to a cell, enhance cellular delivery and/or enhance the efficacy of one of the molecules.
In one embodiment, the composition includes a targeting molecule. The targeting molecule binds a target molecule in the cell. In certain aspects, the targeting molecule suppresses the transcription or translation of the target molecule. For example, the targeting molecule may bind an mRNA molecule to suppress expression of a protein in the cytoplasm of the cell. Targeting molecules that can be used in accordance with the embodiments described herein include, but are not limited to, an antisense oligonucleotide (ASO) molecule, a splicing switch oligonucleotide (SSO) molecule, an siRNA molecule, an miRNA molecule, an shRNA molecule, or any other charged or modified small molecules. Those targeting molecules can target any molecule in the cell-in the nucleus or the cytoplasm-including, but not limited to, an mRNA molecule, an ncRNA molecule, a piRNA molecule, an miRNA molecule, a viral RNA molecule, or a promoter sequence.
The composition may also include a stressor. The stressor may include any suitable molecule that induces a cellular stress response including, but not limited to, a metabolic stressor (e.g., arsenite), a redox stressor of cells (e.g., hydrogen peroxide (H2O2) or glutathione), or other stressor (e.g., 6BIO, lipofectamine). In one embodiment, the stressor molecule may be arsenic (also referred to herein as arsenite or arsenic trioxide or As III or “ATO”). In some embodiments, the stressor is a heat shock. As shown in the working examples below, the combination of a targeting molecule and a stressor such as arsenic enhances delivery of the targeting molecule to the nucleus and also enhances its nuclear function.
The composition may also include a proton sponge molecule or an endosome or lysosome inhibitor. The proton sponge molecule may include any suitable proton sponge molecule including, but not limited to, ammonium (NH4+). Any suitable ammonium compound can be used, such as ammonium chloride (NH4Cl), ammonium hydroxide (NH4OH), ammonium sulfate (NH4SO4), ammonium nitrate (NH4NO3), ammonium acetate (NH4CH4CO2), or ammonium bicarbonate (NH4HCO3). In some embodiments, the endosome or lysosome inhibitor includes Ambroxol (Amb), Cyclohexylamine (CHA) or oleic acid (OA).
In certain embodiments, the two or more molecules that, when co-administered to a cell, enhance cellular delivery and/or enhance the efficacy of one of the molecules, are co-administered individually instead as part of a composition. In some embodiments, a targeting molecule can be co-administered in combination with a stressor. In some embodiments, a targeting molecule can be co-administered in combination with a proton sponge molecule or an endosome or lysosome inhibitor. In some embodiments, a targeting molecule can be co-administered in combination with a stressor and a proton sponge molecule. In some embodiments, a targeting molecule can be co-administered in combination with a stressor and an endosome or lysosome inhibitor. Co-administration of these combinations can occur simultaneously, or may be spared out at a predetermined time interval.
In some embodiments, the compositions and combinations described herein may be used in research studies to treat cells in vitro. In that case, the compositions or combinations may be administered using a pipet or any other suitable method for treating cultured cells. The cells may be any suitable cultured cell, including primary cultured cells, cell lines, immortal cell lines, stem cells.
In other embodiments, the compositions and combinations described herein may be used an in vivo or ex vivo method for use in clinical research studies, animal research studies, or for treatment of a therapeutic indication. In that case, the compositions or combinations may be administered to a cell that is part of population of cells that make up an organ or tissue. For in vivo methods, the compositions and combinations may be administered to a subject (e.g., a human or an animal) orally, via injection, absorption, inhalation, or any other suitable administration method. For ex vivo methods, the cell may be found in a subject'"'"'s circulation.
Also provided herein are methods for using the compositions and combinations described herein. In certain embodiments, a method of enhancing gene targeting is provided. In one aspect the method may include steps of administering a targeting molecule to a cell, wherein the targeting molecule binds a target molecule in the cell; and administering a stressor to the cell, wherein the stressor molecule induces a cellular stress response; wherein co-administration of the stressor molecule with the targeting molecule enhances the function of the targeting molecule. The method may also include a step of administering a proton sponge molecule or an endosome or lysosome inhibitor to the target cell, wherein co-administration of the proton sponge molecule or the endosome or lysosome inhibitor with the targeting molecule and the stressor further enhances the function of the targeting molecule.
In another aspect, the method may include steps of administering a targeting molecule to a cell, wherein the targeting molecule binds a target molecule in the cell; and administering a proton sponge molecule or an endosome or lysosome inhibitor to the cell, wherein co-administration of the proton sponge molecule or the endosome or lysosome inhibitor with the targeting molecule enhances the function of the targeting molecule. The method may also include a step of administering a stressor to the target cell, wherein the stressor induces a cellular stress response, and wherein co-administration of the stressor with the targeting molecule and the proton sponge molecule or the endosome or lysosome inhibitor further enhances the function of the targeting molecule.
In certain aspects, this disclosure relates to a mechanism of directing the oligonucleotides such as siRNAs and miRNAs into the nucleus. The cellular localization of the oligonucleotides, siRNAs (
This disclosure relates to compositions and methods for nuclear transport of small molecules such as oligonucleotides, siRNAs and miRNAs by induced cellular stress and the presence of proton sponges such as ammonium (NH4+). Also disclosed is a stress-induced response complex (SIRC) including one or more of following proteins, e.g., Ago-1, Ago-2, and transcription and splicing regulators such as YB1, CTCF, FUS, Smad1, Smad3, and Smad4. The SIRC is capable of transporting the small molecules (e.g., oligonucleotides, siRNAs, and miRNAs) to the nucleus. The induced cellular stress can significantly increase oligonucleotide- or siRNA-directed splicing switch events and the miRNA targeting of nuclear RNAs.
Because of their charge and their ensuing ability to bind heparin-binding cellular proteins, phosphorothioate oligonucleotides (e.g., phosphorothioate, locked nucleic acid oligonucleotides) can enter cells and hijack endogenous miRNA pathways (4). MiRNAs also shuttle to and function in the nuclear compartment (3, 44). A small miRNA subset has been proven to participate in the cellular stress response (45).
As demonstrated in the working examples, the translocation of these small nucleic acids into the nucleus results from a general response to cell stress, which triggers the formation of a stress-induced response complex, the SIRC. This complex contains both shuttling and gene expression modulator proteins. An interaction between Ago-2 and YB-1 increases as a response to cell stress and leads to the translocation of the SIRC into the nucleus. The SIRC can include miRNAs and oligonucleotides; a surge in nuclear shuttling corresponds to a proportional rise in the nuclear function of oligonucleotides, siRNAs and miRNAs. The cytoplasmic function of oligonucleotides and siRNAs decline concomitantly. The loss of cytoplasmic miRNA potency is consistent with the previously observed nuclear re-localization of Ago-2 and decreased cytoplasmic RNAi linked to cell stress (46). The results demonstrated in the working examples also support the initial formation of the SIRC, including the binding to oligonucleotides and possibly to miRNAs, to be occurring in cytoplasmic stress granules (SG). The data disclosed herein helps explaining why the intracellular localization of oligonucleotides appears to be different based on the mode of delivery. Gymnosis (which by itself at lower oligonucleotide concentrations and shorter treatment times is not a significant stressor) results in predominantly perinuclear localization of the oligonucleotides. This is in contrast to lipofectamine, which is a potent cell stressor, and directs the oligonucleotides to the nucleus (4).
As illustrated in
Cell stress leads to phosphorylation of the R-Smads (Smad 1/5/8) and their binding to Smad-4 (28) and the SIRC, followed by the nuclear re-localization of this complex (
Smad-1 and Smad-4 are important transcription regulators that can induce or repress a number of transcripts. These proteins together with CTCF, a master regulator of transcription, would allow the cell to have a wide-ranging stress response, which may include chromatin remodeling. The TNRC6 family contains homologies to domains of the S. pombe Tas3 and Chpl proteins, which are part of the RNA-Induced Transcriptional Silencing (RITS) complex (48).
MiRNAs play crucial roles in modulating gene expression. Their deregulation has been shown to be hallmark of cancer and other diseases. As III at low concentrations is an FDA-approved anti-leukemia drug (31, 49). As disclosed herein, small, clinically relevant concentrations of As III can induce SIRC formation and can shuttle siRNAs, miRNAs and other oligonucleotides delivered by gymnosis to the cell nucleus. The potency of nuclear targeting is significantly increased by the As III treatment in consequence. Thus, this disclosure relates to a combinatorial strategy employing small nucleic acids and As III in therapeutic applications.
It was previously demonstrated that oligonucleotides delivered by gymnosis are bound by Argonaute proteins 1-4 (4), and may hijack multiple endogenous mechanisms employed by cellular miRNAs. Ago-2 binding augments oligonucleotide function, which may due to Ago-2 facilitation of oligonucleotide transport (4). Thus, Ago-2 could be part of a transport complex that differed from RISC.
To test the hypothesis that oligonucleotides were bound to an Ago-2 transport complex and to identify additional proteins belonging to this complex, immuno-precipitations (IPs) of Ago-1 or Ago-2 were performed using cell lysates harvested from HEK 293 cells that were 1) untreated; 2) treated with a control oligonucleotide delivered by gymnosis; or 3) transfected with a control siRNA. All the oligonucleotides used in the studies were phosphorothioate, locked nucleic acid oligonucleotides (PS-LNA-ONs) (7, 8), which increase stability and cellular uptake by gymnosis.
A mass spectrometric analysis of the precipitates was performed and only those proteins that were common in both lysates of cells treated with the control siRNA and lysates treated with the oligonucleotides, but were absent in all other samples and controls were analyzed. Under the experimental conditions, apart from ribosomal proteins, tubulin, and immuno-precipitated Argonautes, nudeolin, the SRSF1 and 7 splicing factors, YB-1, DbpA, PABP1, HSP-70, KIF11 and elongation factor 1 a were found. Nucleolin is a shuttling protein (9) that has been previously reported to bind oligonucleotides (5). SRSF1 and SRSF7 are members of the SR protein family, which has been shown to act on nuclear export factor 1 (NXF1) (10). The heat shock protein HSP-70 is a chaperon, stress-response protein which, among other functions, increases the stability of nucleolin during oxidative stress (11) and it is known to associate with YB-1 during stress (12). YB-1 is involved in a myriad of cellular functions; it is also a chaperon and a key player in the cellular stress response, which leads to its translocation into the nucleus (13). YB-1 is also involved in stress granule (SG) formation where it localizes (12). DbpA (YBX3) is also a Y-box binding protein (14, 15) while PABP1 binds to the mRNA poly(A) tail and is important in mRNA translation and non-sense mediated decay. PABP1 also concentrates in stress granules (16). KIF11 is a kinesin-related protein that plays a role in cell division and enhances the efficiency of mRNA translation (17). Elongation factor 1□ also plays a role in translation and, in addition, a central role in the nuclear export of proteins (18). Therefore, nearly all of the proteins identified are involved in transport and in the cellular stress response.
As demonstrated in the working examples, nucleolin and YB1 are of importance. Nucleolin is known to bind oligonucleotides (5), but has not been previously shown to bind siRNAs. However, it has been reported to interact with the microprocessor complex and to affect the processing of specific miRNAs (19).
As shown in
Down-regulation of Ago-2, but not Ago-1 or Ago-3 resulted in oligonucleotide localization that favors the cytoplasmic compartment, as shown in
Some reports have indicated an active role for nucleolin in the binding of oligonucleotides (21), but others have not been able to find a functional role for it in oligonucleotide activity (6). It is possible that nucleolin either has a redundant function, or that its contribution to ON function is measurable only in certain cell systems and under specific experimental conditions (22). Subsequent to down-regulating nucleolin expression by an siRNA approach, and consistent with these observations, only a marginal effect (up to 30%) on oligonucleotide function was detected and then only if the oligonucleotides were delivered by gymnosis rather than lipofection. Therefore the working examples focused on the role of YB1, which was also found in the Ago-ON immuno-precipitated complexes. As demonstrated below, delivery of oligonucleotides by gymnosis results in increased Ago-2/YB-1 complex formation. Moreover, Ago-2/YB-1 directly interact as a consequence of cellular stress and co-localize in the same cellular compartments.
YB1, Ago-2 and the miRNA machinery are involved in the cellular stress response (25, 26). Furthermore, YB1 has been shown to be important in the regulation of the Smad-signaling pathway (27). Smad transcription factors are a critical piece of one of the most multifaceted cytokine signaling pathways, the transforming growth factor-β pathway (28). Once activated by phosphorylation, these proteins translocate to the nucleus where they regulate gene expression (28). Therefore, they also can be potential partners of the stress-induced YB1/Ago-2 complex. As demonstrated in the working examples, Ago-2 also directly or indirectly interacts with the Smad complex.
Further, as disclosed herein, NH4+ potentiates oligonucleotide activity via a different mechanism than As II. Because two separate pathways of oligonucleotide activation seem to exist, combining both compounds (As III and NH4+), resulted in a potent synergistic increase in oligonucleotide function.
Oligonucleotide concentrations employed for therapeutic applications vary widely, but in general are high enough to raise significant concerns for off target effects and cellular toxicity. However, lowering oligonucleotide concentrations reduces the chances of a therapeutic response, since typically only relatively small amounts of oligonucleotides are taken up by targeted cells. It is therefore imperative to identify new strategies to improve the concentration dependence of oligonucleotide function.
As disclosed herein, ammonium ion (NH4+) can be used as a non-toxic potent enhancer of oligonucleotide activity in the nucleus and cytoplasm following delivery by gymnosis. Enhancement of function can be found in attached and suspension cells, including difficult-to-transfect Jurkat and CEM T cells. The working examples also demonstrate that NH4+ can synergistically interact with arsenic trioxide to further promote oligonucleotide function without yielding any apparent increased cellular toxicity. These small, inexpensive, widely distributed molecules can be used not only in laboratory experiments but potentially in therapeutic oligonucleotide-based combinatorial strategy for clinical applications.
The locked nucleic acid (LNA) modification was first synthesized by the Wengel laboratory approximately 20 years ago (57,58). Phosphorothioated (PS) oligonucleotides containing LNA moieties are not only highly resistant to nucleases, but each LNA can increase the Tm of an RNA/PS LNA oligonucleotide duplex by up to 2-6° C. per residue (58,59). PS LNA oligonucleotides are active splice-switching oligonucleotides, having been shown to induce exon skipping in vivo as well as in vitro, especially in the colon, small intestine and liver (60). However, for therapeutic applications it is critically important to develop strategies that take advantage of these characteristics at low oligonucleotide doses so that the potential for off target effects are reduced.
The PS LNA SSO disclosed herein (SSO-654) is a 16mer. The LNA moieties are interspersed in the oligonucleotide chain. This substitution promotes nuclease stability and increases the stability of the oligonucleotide hybrid with the nuclear pre-mRNA. At the same time, LNA substitution blocks the induction of RNAse H activity, which would cleave the pre-mRNA and terminate exon skipping. To monitor oligonucleotide efficacy, a splice-switching model was used, in which HeLa cells have been engineered to express the enhanced green fluorescent protein (EGFP; HeLa EGFP-654) (61,62). In this model, a mutated β-globin intron has been inserted into the EGFP coding sequence to create an internal additional exon, which prevents canonical splicing and EGFP translation. Treatment of these cells with an SSO targeted to one of the internal splice sites causes exon skipping and the reconstitution of the EGFP correct reading frame (62).
The general principle that PS LNA oligonucleotides and other highly stabilized oligonucleotides such as 2′F-arabinose nucleic acids (16) can enter cells in the absence of any transfection vehicles and can also silence gene expression was previously reported (63-65). This process is called gymnosis from the Greek word for naked, and is different from the process of “free uptake” which only refers to the absence of transfection reagents (66), and has never been associated with silencing of gene expression. The process of gymnosis in tissue culture more resembles in vivo oligonucleotide uptake in saline formulations than does the process of transfection (63), and is often used for both ASO and SSO experiments.
The concentrations of oligonucleotide in the media for an optimal gymnosis experiment are often in the 250 nM-5 μM range. However, while gymnotic delivery of oligonucleotides generally results in excellent oligonucleotide function, it still can be associated with inherent potential toxicity and with sub-optimal in vivo delivery. Thus, disclosed herein are methods of improving the activity of oligonucleotides such as PS LNA oligonucleotides after gymnosis in order to improve the concentration dependence of oligonucleotide function.
It is desirable to identify small molecules that are capable of enhancing oligonucleotide functions at a low cost and that are non-toxic at the concentrations employed. However, there are very few such small molecules currently available. For example, a small molecule known as Retro-1, which reduces the toxicity of plant and bacterial compounds (67) emerged from a high throughput screen. Retro-1 enhanced both SSO and ASO efficacy when the oligonucleotides were delivered by gymnosis. However, the optimal concentration of Retro-1 was approximately 50-100 μM, and the compound is also poorly water-soluble. In a subsequent high throughput screen of >100,000 compounds, a series of 3-deazapteridine analogues were discovered (68) that at a concentration of 10 μM substantially increased SSO activity. Dantrolene (25-50 μM), a drug used clinically in the treatment of malignant hyperthermia, and other small molecules that target the ryanodine receptor have been demonstrated to promote SSO modulated exon skipping in myotubes in vitro and in mdx mice (69).
Ammonium, also approved for clinical use, considerably increases the oligonucleotides function, likely by acting as a proton sponge and aiding their endosomal release into the cytoplasm. This greatly enhances cytoplasmic gene targeting and function of the delivered molecules. Moreover, by increasing the cytoplasmic concentration of these molecules, the amount that translocates into the nucleus increases. Therefore ammonium can also increase nuclear function, although to a lesser extent when compared to As III.
As disclosed herein and demonstrated in the working examples, the ammonium ion (NH4+) can facilitate SSO activity in the HeLa EGFP-654 model in vitro, in the absence of toxicity. NH4+ also improves in vitro ASO activity both in attached suspension cells, including in Jurkat and CEM T cells, in which gene silencing has historically been difficult. Moreover, NH4+ can interact synergistically with arsenic trioxide (As III or ATO, arsenite in solution) to significantly promote oligonucleotide function in cells.
As described herein, the effects of NH4+ on the activity of oligonucleotides are studied because of the previous results (79), which highlighted the importance of endosomal maturation for oligonucleotide activity and because it has previously been shown that NH4+ affects the maturation and outcomes of late endosomes (73,74,80). After cell surface adsorption, or through fluid phase endocytosis, oligonucleotides become localized inside the cell in early endosomes. The maturation of early to late endosomes (LEs)/multivesicular bodies (MVBs) is at least in part under the control of PKC-α. Blocking PKC-α expression by a variety methods leads to a marked diminution of ASO gene silencing, suggesting that oligonucleotides, at least in part, exit the endosomal pathway at the level of the LE/MVBs. In this context, it is of interest that Ago-2, one of the proteins that interacts with oligonucleotides (81) and shuttles the oligonucleotides to the cell nucleus, can physically interact with the LE (82).
LEs/MVBs can also fuse with lysosomes; and SSO accumulation in lysosomes, a process deemed to be non-productive with respect to oligonucleotide activity, was reported (81,83). The ability of NH4+ to block or slow the fusion of LEs/MLVs with lysosomes (73,74,80) might allow the endosomal cargo to be retained for longer times in the LE/MVB, increasing its ability to exit the endosomal pathway. The exit of oligonucleotides from the endosome might also be aided by the ability of NH4+ and of other lysosomotropic weak bases, to also cause endosomal swelling (84, 85). The way that endosomal swelling is produced by NH4+ is complex, but cannot be due to proton sponge effects, as the pKa of NH4′=9.26 (78), two orders of magnitude higher than the intracellular pH. The endosomal swelling effects of NH4+ may mimic those of the so-called cell penetrating peptide-oligonucleotides, in which the peptide moieties are usually short (9-30 amino acids) polypeptides that are often replete with arginines and lysines (86), both of which are organic amines.
However, the mechanism of action of NH4+, regardless of how it enhances oligonucleotides (e.g., ASO and SSO) function, appears to be different than that of As III (a mechanistic model is depicted in
As III enhances oligonucleotide function in the nucleus but not in the cytoplasm, while NH4+ enhances function in both cellular compartments possibly by increasing the total amount of oligonucleotides released into the cytoplasm. This is in accord with the observations that As III, through induction of cellular stress, facilitates shuttling of oligonucleotides from the cytoplasm to the nucleus. In contrast to As III, under the conditions of the experiments described below, NH4+ at 5 mM, does not appear to induce a cellular stress response and thus the formation of an oligonucleotide-binding stress-induced response complex (SIRC), which consists of Ago-2, nucleolin, and Yb-1, in addition to other proteins. Furthermore, the working examples demonstrating that the higher concentrations of NH4+/As III are synergistic, as defined by the combination index (CI) method of Chou and Talalay, also suggest that these small molecules augment oligonucleotide activity by different mechanisms. The possibility that the positively charged NH4+ augments binding of an oligonucleotide to its mRNA target by charge masking may be discounted, as experiments examining the Tm of DNA/RNA duplexes in the presence or absence of 5 mM NH4+ demonstrated no difference.
The combination of As III, which has previously been shown to be a potential anti-leukemia drug (87-90), and/or NH4+ (or other organic amines such as Amb., CHA and OA that act on the endosomal maturation pathway) is an easy, inexpensive, non-toxic and effective way to improve oligonucleotides, including SSO and ASO, activities after their delivery by gymnosis, even in non-attached difficult to transfect cells. Most likely, a synergistic increase in function is produced because these agents appear to be active by two different mechanisms: NH4+ seems to be acting at the level of the endosomes, while As III induces a cellular stress reaction that promotes cytoplasmic-nuclear oligonucleotide shuttling. These compounds and the mechanisms disclosed herein can be used to enhance oligonucleotide activity for therapeutic uses.
The following examples are intended to illustrate various embodiments of the invention. As such, the specific embodiments discussed are not to be construed as limitations on the scope of the invention. For example. the methods described in the working examples below may be used to enhance the efficacy of any nuclear targeting strategy, including si-RNA directed promoter methylation or activation. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of invention, and it is understood that such equivalent embodiments are to be included herein. Further, all references cited in the disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.
As discussed in the examples below, this work may be broadly applicable for clinical applications for the intracellular delivery of therapeutic molecules, and may also have application for the enhanced efficacy of any cellular targeting strategy, including siRNA directed promoter methylation or activation.
From the foregoing, it will be appreciated that specific embodiments of the invention have been described herein for purposes of illustration, but that various modifications may be made without deviating from the scope of the invention. Accordingly, the invention is not limited except as by the appended claims.
Materials and Methods
HeLa EGFP-654, HCT116 and SW480 cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. LNCaP cells and the Jurkat and CEM T lymphocyte cell lines were maintained in RPMI 1640 medium supplemented with 10% FBS and 2 mM L-glutamine. Cultures of all cell lines were maintained at 37° C. in a humidified 5% CO2 incubator.
Reagents and Antibodies:
Ammonium chloride (NH4Cl), arsenic trioxide (As2O3) and oleic acid (OA) were purchased from Sigma-Aldrich (Milwaukee, Wis.); Ambroxol hydrochloride and cyclohexylamine were from Santa Cruz Biotechnology (Santa Cruz, Calif.), as were the AR (N-20) and GFP (sc-9996) antibodies. The β-catenin antibody (4270) was purchased from Cell Signaling Technology (Danvers, Mass.) and the anti-α-tubulin antibody from Sigma-Aldrich; the anti-BCL2 antibody (clone 124) was from Dako (Santa Clara, Calif.). An arsenite solution was prepared by dissolving As2O3 in minimal volumes of 1 N sodium hydroxide (NaOH). The arsenite solution was then diluted with phosphate buffered saline to a concentration of 10 mM as a stock solution. Other compound solutions were prepared as per the manufacturer'"'"'s recommendation.
The sequences of oligonucleotides used herein are listed in Table 1. All are phosphorothioates, with DNA given in lower case letters and LNA modifications in upper case letters. “m”=5-methylcytosine.
Cells were harvested with trypsin digestion and washed once with PBS. Cell pellets were lysed in cold RIPA buffer containing protease inhibitors. Cellular RIPA lysates were sonicated for 2 seconds and then rested on ice for 5 min. Cell debris was removed by centrifugation at 12,000×g for 10 min at 4° C. Protein concentrations were determined using the Pierces BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, Mass.). Aliquots of cell extracts containing 30-40 μg of protein were resolved by SDS-PAGE gel electrophoresis, and then transferred to PVDF membranes. After treatment with the appropriate primary and secondary antibodies, enhanced chemiluminescence was performed. Protein signals on the blot were quantified with the Image J program and protein expression was normalized to control=100%.
RNA was extracted from cells using RNA-STAT 60 (AMS Biotechnology, Abingdon, UK) as recommended by the manufacturer. First-strand cDNA was synthesized with the Super-Script® III First-Strand Synthesis System Kit (Invitrogen, Carlsbad, Calif.). PCR was performed with Power SYBR GREEN PCR Master Mix (Thermo Fisher Scientific, Waltham, Mass.).
Cells were harvested by trypsin digestion and re-suspended in culture media or PBS buffer prior to flow cytometry. Flow cytometry data were collected by a CyAn Flow Cytometer (Beckman Coulter, Brea, Calif.), and were analyzed by the FlowJo program (Tree Star, Inc., Ashland, Oreg.) to determine fluorescence intensity vs. cell number. For cell viability assays, harvested cells were re-suspended in PBS containing 1 μg/ml DAPI (Molecular Probes, Eugene, Oreg.).
Cell Growth Assays:
Cell growth and proliferation were assayed by staining with sulforhodamine B (SRB). Briefly, cells were fixed by adding an equal volume of 10% cold trichloroacetic acid to each well. After one hour incubation at 4° C., cells were stained with 0.06% SRB for 30 min at room temperature in the dark. Cell-bound SRB was then solubilized in 10 mM Tris buffer (pH 10) and its absorbance determined at 510 nm by a microplate reader.
Quantifying the interaction between the oligonucleotide and ammonium and As III treatments was performed by a combination index (CI) plot using the Chou-Talalay method (70). All data are expressed as mean±standard deviation (s.d.); data from three or more independent experiments were analyzed with a two-tailed, unpaired Students t-test. p<0.05 was considered statistically significant.
In this example it was determined that arsenite (a metabolic stressor), heat shock, hydrogen peroxide (a redox stressor of cells), or glutathione (a redox stressor), causes translocation of a splice switching synthetic oligonucleotide (e.g., mixer oligonucleotide, siRNA, or antisense oligonucleotide) from the cytoplasm to the nucleus. This, in the case of the splice-switching oligo, results in the increased frequency of splice switching in human tumor cell lines.
As shown in
A Stress Induced Shuttling Complex (SISC) was identified by the studies described in this Example. The SISC can transport oligonucleotides, siRNAs, microRNAs, and other small molecules into the nucleus.
First, an LNA-PS-oligo or an siRNA was gymnotically delivered or transfected to stable cell lines expressing Ago1-Flag or Ago2-Flag and performed immuno-precipitation with the anti-Flag antibodies (
Next, it was determined that silencing of Nucleolin results in decreased oligo function. The Hela 654 eGFP used in these experiments is a stable cell line with an integrated eGFP gene whose coding sequence is interrupted by an internal exon. Therefore the eGFP protein is not expressed unless the splicing pattern is altered to exclude the internal exon and reconstitute the eGFP reading frame. The splicing switch oligo (SSO) may be used for this purpose, the amount of eGFP produced will be directly proportional to the SSO function. The eGFP produced by the SSO when cells were previously transfected with an anti-Nucleolin siRNA (si-Ncl) was reduced when compared to cells transfected with a control siRNA (si-CNTR) (
Because both Nd and YBX-1 are shuttling proteins it was investigated whether this argonaute-associated complex was involved in transporting the oligo into the nucleus. First, a control oligo was delivered to HEK-293 cells via gymnosis as shown in
Next, inducible HCT-116 cell lines expressing anti Ago1-shRNA, anti Ago2-shRNA or anti Ago3-shRNA were used to assess if other argonautes had a similar phenotype as Ago2 (
Next, it was determined why the oligo induces formation of the SISC. Additional immuno-precipitation experiments that complemented the original Ago-IPs were performed by using HEK 293 cell lysates and a YB1 antibody to confirm 1) the interaction of YB1 with Ago-1 and/or Ago-2 (
Specifically, it was determined whether the complex forms as a result of a stress signal (
To determine whether a direct interaction between YB1 and Ago-2 occurred, a proximity ligation assay (PLA) (23, 24) was conducted using YB1 and Ago-1 or Ago-2 specific primer-conjugated antibodies (
A more intense fluorescent signal was detected for the Ago2/YB1 interaction in the siRNA control sample (siCntr Ago2/YB1), which underwent transfection. Following this stress signal that was triggered by lipofection, an increased interaction between these two proteins was observed, consistent with the IP results (
To determine if Ago-2 also interacts with the Smad complex, a PLA assay was performed with Smad-1- and Ago-2-specific mixed probes. The fluorescent signal detected (
Confocal sections of the cells analyzed via PLA with YB-1 and Ago2 antibodies demonstrate co-localization of these two proteins in the cytoplasm and in the nuclear compartments (
To further validate the specificity of the PLA assay and the reliability of these results, Ago-2 and/or YBX-1 (the gene producing YB1) expression was silenced with specific siRNAs (siAgo-2 and siYBX-1;
Silencing of Ago2 or YBX-1 results in a loss of the fluorescent signal generated via PLA using specific Ago2 and YB-1 antibodies.
To examine if the interaction between Ago2 and YB1 was a general response to stress or specific only to the delivery of siRNA and oligonucleotides, YB1 IPs were performed after lipofecting an empty plasmid backbone into HEK-293 cells (
More specifically, to test if the intracellular delivery of the oligonucleotide is specifically required to induce the increased association of Ago2 to the YB-1 complex or if it is the result of a general stress signal (triggered by the oligo), a control vector (Cntr-Tx) was transfected and a YB-1 immuno-precipitation was performed. Similar to what was observed in presence of the oligonucleotide, ASO, the Ago2 association to YB-1 increased in the transfected (stressed) cells (See
Then, PKR activation was examined under the experimental conditions. The delivery of oligonucleotides by gymnosis triggered an increase in PKR expression as a function of time and oligonucleotide concentration (
To establish if the increased interaction between Ago-2 and YB1 translated to augmented oligonucleotide function, a splice switching oligonucleotide (SSO-654) was delivered by gymnosis to the HeLa-EGFP-654 cell line (29), which was either untreated or previously transfected with a stressor (in this case, a non-targeting siRNA). SSO-654 was designed to induce skipping of an exon which disrupts the eGFP coding sequence expressed in these cells; therefore the potency of this splice switching ON is directly proportional to the signal of the eGFP that is produced (29). The stress induced by the siRNA-transfection resulted in more effective splicing switch activity and higher eGFP expression at three separate SSO-654 concentrations (
The increase in oligonucleotide function could be recapitulated with arsenic trioxide (As III or ATO), a standard cellular stressor. Although As III can cause oxidative damage (30), at the appropriate concentrations it also has therapeutic properties (31). It was found that SSO-654 potency improved, as determined by eGFP production in HeLa-EGFP-654 cells, when low concentrations (0.5-2 μM) of As III were combined with SSO-654 treatment (
The increased production of eGFP as a function of the As III concentration (1 and 2 μM) was confirmed by flow-cytometry measurements (
Finally, to exclude the possibility that the enhanced SSO-654 function was due to changes in oligonucleotide uptake or cell viability, the following was evaluated: (1) the efficiency of oligonucleotide delivery by gymnosis in two separate cell lines (HeLa-EGFP-654,
These data corroborated that the increased oligonucleotide potency is related to a general cellular stress response. Indeed, additional cell stressors, such as hydrogen peroxide (H2O2) or heat shock also increased SSO-654 function (
As shown in
To verify that the various treatments that were used to increase oligo function were working by triggering cellular stress, which in turn resulted in the formation of a nuclear translocating complex, PKR activation was monitored following the treatments. As shown in
The increased potency of SSO-654 was a reflection of its increased concentration in the nuclear compartment, and coincided with the nuclear translocation of YB1. Nuclear/cytoplasmic fractionations of the HeLa-654 cells treated with oligonucleotides with or without As III were performed. As III induces accumulation of YB1 in the nucleus (
It has been shown that YB1 can bind to and regulate the biogenesis of specific miRNAs (32). As disclosed herein, the interaction of YB1 with Ago-2 increases during cellular stress. It was previously shown that oligonucleotides behave similar to siRNAs, and may hijack endogenous si/miRNA cellular pathways (4). Therefore, whether the nuclear translocation and improvement in function observed using oligonucleotides in stress condition could be recapitulated with si- and miRNAs was investigated. RNA was extracted from the original input lysates (
Then nuclear/cytoplasmic fractionations of untreated cells or cells treated with the oligonucleotide, or with a combination of the oligonucleotide and As III were performed. Shuttling of the miRNAs to the nucleus increased proportionally to the extent of the stress signal (
To verify that this nuclear shuttling of miRNAs translated into an increased activity in the nucleus, a splicing switching siRNA (SSsi-654) with the same sequence as SSO-654 was designed. This is a canonical siRNA and thus functions primarily in the cytoplasm; however, splice switching activity in the nucleus was detected (
The augmentation of nuclear targeting by the nuclear-translocated siRNAs or oligonucleotides is expected to occur with a concomitant decrease of their cytoplasmic function. Cytoplasmic gene silencing was monitored using a 5′ end-capped, 3′-polyadenylated eGFP mRNA (eGFP mRNA) (4). The transfected mRNA is rapidly bound by the ribosome and remains localized in the cytoplasm where it is transcribed to rapidly generate eGFP (4). Low concentrations (10 nM) of an anti-eGFP siRNA (siRNA-GFP) or a non-targeting control (siRNA-Cntr) were delivered to HEK 293-T or HT-1080 cells using lipofectamine 3K, which minimizes cellular stress. The next day, cells were re-plated and As III was added to half of the samples. The eGFP mRNA was then delivered to the cells and fluorescence images were acquired shortly thereafter (
The same phenomenon could be reproduced when delivering an eGFP-targeted oligonucleotide (ON-eGFP) prior to the eGFP mRNA in HeLa cells (
Finally, to confirm that the increased nuclear function extended to endogenous miRNAs, two separate systems that rely on miR-led target suppression in the nucleus of the CDR-1-AS and the non-coding Malat-1 RNAs were selected. CDR-1-AS (or ciRS-7) is the circular, naturally occurring antisense RNA product of the CDR-1 gene and acts as a sponge of cellular miR-7. This leads to increased expression of the miR-7 targeted transcripts (33-35). CDR-1-AS is, in turn, targeted in the nucleus by miR-671, whose binding supports Ago-2 cleavage and the subsequent destruction of the sponge (33). Reduction of CDR-1-AS results in destabilization of the CDR-1 sense strand, an mRNA localized to the cytoplasm (33). Treating cells with As III could result in increased migration of miR-671 to the nucleus, followed by the targeting of CDR-1-AS and the subsequent reduction of the CDR-1 mRNA. The second system investigated is based on miR-9 regulation of Malat1 gene expression, which has been shown to also occur in the nucleus (36). Similar to the CDR-1 mRNA, the Malat1 RNA could be suppressed by treatment of cells with As III and the resultant shuttling of miR-9 to the nucleus. As shown in
The data support the occurrence of a YB1/Ago-2 interaction that shuttles siRNAs and miRNAs into the nucleus, likely as a mechanism of gene regulation in response to cellular stress. Oligonucleotides delivered by gymnosis hijack this pathway to reach the nucleus. To examine where within the cell the interaction between the oligonucleotides and this endogenous cellular pathway occurs, we delivered 5′-Cy5-oligonucleotides to cells with and without As III treatment, and an immuno-fluorescence assay was performed using anti-Ago-2 specific antibodies (
The SG in HT1080 cells was visualized using a stress granule marker, fluorescent G3BP protein, and the 5-′Cy5-Iabeled oligonucleotide was delivered via gymnosis. The co-localization of the 5′-Cy5-oligonucleotide to the SG, and its shuttling to the nucleus, was enhanced by doubling the oligonucleotide concentration (from 1 μM to 2 μM;
SISC includes splicing regulators, translation regulators and chromatin remodeling factors such as CTCF, FUS, Smad1 and Smad3, YB-1. These proteins can bind RNA and DNA and have some preferential affinity for specific short nucleotide sequences. These short sequences can be included in the site selection process to increase the targeting of siRNAs-, shRNAs- miRNAs- and oligonucleotides-bound SISC. The technology disclosed herein can be used to affect gene splicing or to target promoter sequences and trigger gene activation or permanent silencing.
The YB1/Ago-2 complex, in addition to nudeolin and Ago-1, can include additional proteins, some of which may be involved in gene regulation as a response to cellular stress. Based on the known interactions of proteins with oligonucleotides and/or YB1 and Ago-2, Ago-1 and Ago-2 immunoprecipitations from the lysates of untreated or oligonucleotide- and As Ill-treated cells were analyzed for the presence of additional proteins present in the SIRC (
Cells may respond to stress by modulating gene expression at the transcription and/or splicing steps. The following molecules were analyzed: 1) YB-1, which is known to localize in cytoplasmic nuclear speckles (mostly stress granules) and is involved in transcription, replication and RNA processing. Its patterns of localization resembles siRNAs and oligonucleotides. 2) FUS which binds ssDNA, dsDNA, ssRNA and Spl. The latter in turn binds to CG promoter sequences. It'"'"'s involved in pre-mRNA splicing and export. It may also be implicated in mRNA/MiRNA processing, regulation of gene expression and genome integrity. 3) SMAD1, which is a very important factor for the regulation of transcription activation and repression. SMAD also interacts with CRM-1 which in turn interacts with siRNA. We have previously shown that CRM-1 shuttles the siRNAs between the nucleus and the cytoplasm. 4) CTCF, an important transcriptional repressor, which plays an important role in the epigenetic regulation.
The interaction of Ago-2 with the Smad complex was confirmed by immuno-blotting the immunoprecipitations with antibodies specific for Smad-1, 3 and 4. The binding of Smad1 and Smad 4 to Ago-2 significantly increased upon stress (
Under normal growth conditions, there is only a minimal association of FUS with the Argonaute complex (
The HeLa EGFP-654 cell line (61,62), stably expresses an EGFP pre-mRNA whose coding sequence is interrupted by the insertion of an additional exon. The binding of SSO-654 to the first 3′ splice site causes the skipping of this internal exon and restores the correct EGFP reading frame and EGFP protein expression (
HeLa EGFP-654 cells stably express an EGFP construct whose coding sequence has been interrupted by the insertion of an aberrant intron from the human β-globin gene. A mutation at position 654 in this intron creates aberrant splice sites, preventing EGFP expression. Binding of SSO-654 to the aberrant splice site restores correct splicing and EGFP expression. In
Exposure to 5 mM NH4+ for 2 days increased SSO-654 function and EGFP expression in HeLa EGFP-654 cells (
Consistent with these results, other ammonium-containing compounds such as ammonium acetate and ammonium bicarbonate produced comparable effects on SSO-654-mediated EGFP expression (
Taken together, these data demonstrate that NH4+, at a range of clinically relevant concentrations, significantly up-regulated SSO-654 activity in a dose-dependent manner in HeLa EGFP-654 cells.
NH4+ can be inhibitory to the growth of and toxic for mammalian cell cultures (71,72). Therefore, whether the exposure of NH4+ combined with an SSO oligonucleotide could affect the growth, proliferation, and viability of HeLa EGFP-654 cells was examined. In
As shown in
Whether NH4+ could facilitate the activity of PS LNA gapmer ASOs (ASOs) when delivered to a variety of cells by gymnosis was investigated. LNCaP prostate cancer cells were treated with an ASO targeting either the androgen receptor (AR-ASO), or BCL-2 (BCL2-ASO), or β-catenin (f3-Cat-ASO) mRNAs, with or without added NH4+. As shown in
LNCaP cells (
A dose response for the AR-ASO in LNCaP cells was obtained and it was demonstrated that 0.25 μM AR-ASO when combined with NH4+ induced greater AR reduction than 1 μM AR-ASO alone (
Whether NH4+ could promote gymnosis in non-adherent leukemia cells was examined next. These cells are often resistant to liposome-based transfection, a delivery method widely employed for the manipulation of gene expression. The experiment of Jurkat cells, a T lymphocyte cell line, confirmed that these cells were indeed resistant to gene silencing by Lipofectamine® 3000-mediated-transfection of the BCL2-ASO (
The effect of NH4+ on BCL2-ASO-mediated gene silencing activity in CEM T-lymphoblastoid cells was also confirmed. These cells, like Jurkat T cells, are well-studied and transfection-resistant. Nevertheless, NH4+ was still capable of augmenting the ASO silencing of BCL-2 gene expression (
Whether NH4+ could simultaneously facilitate the silencing ability of two oligonucleotides in cells in culture was investigated. As shown in
The concentration of the SSO-654 (evaluated by Western blotting,
To understand how NH4+ facilitates ASO and SSO activity, whether it increased oligonucleotide uptake in cells was examined. In
As III and NH4+ work through different mechanisms, so the two were combined. Their function is strikingly synergistic in improving nuclear and cytoplasmic gene targeting. This was determined by the following studies. NH4+ can aid nuclear SSO function since it will increase the available concentration of functional oligonucleotide (that can shuttle into the nucleus) by increasing its endosomal release. Therefore, a further increase in oligonucleotide nuclear function can be induced by combining the As III and NH4+ treatments.
As III facilitates SSO activity as shown in
As shown in
HeLa EGFP-654 cells were treated with 1 μM SSO-654 in the presence of NH4, As III, or both at fixed molar ratios for two days, prior to flow cytometry. Flow data from three independent experiments were analyzed with the FlowJo program (Tree Star, Inc., Ashland, Oreg.) to obtain EGFP MFI values. The MFI from the cells treated with 1 μM SSO-654 alone were used as the background control to calculate the MFI increase after drug treatment. A combination index (CI) plot was generated and CI values were obtained according to the method of Chou and Talalay (70).
Quantitative analysis was performed by flow cytometry, which allowed the production of a combination index (CI) plot using the Chou-Talalay method (70), in which the line parallel to the x-axis (CI=1,
In addition to mRNA gene targeting, the single or combined use of As III and NH4+ or other stressors can be employed to improve targeting of other sequences and molecules including, but not limited to, ncRNA, piRNA, miRNA, viral RNA and DNA promoter sequences.
Further, in presence of ASO (which causes a stress signal) there is a higher concentration of miRNAs and oligonucleotide in the YBX-1 complex. The YB-1 was immuno-precipitated using cellular lysates harvested from untreated cells or cells treated with a gymnotic delivered oligonucleotide. The association of miRNAs with YB-1 appears to increase when the oligonucleotide is present (
All publications and patent documents cited herein are incorporated by reference.
- 1. R. Kole, A. R. Krainer, S. Altman, RNA therapeutics: beyond RNA interference and antisense oligonucleotides. Nature reviews. Drug discovery 11, 125 (Jan. 20, 2012).
- 2. K. T. Gagnon, L. Li, Y. Chu, B. A. Janowski, D. R. Corey, RNAi factors are present and active in human cell nuclei. Cell reports 6, 211 (Jan. 16, 2014).
- 3. C. Catalanotto, C. Cogoni, G. Zardo, MicroRNA in Control of Gene Expression: An Overview of Nuclear Functions. International journal of molecular sciences 17, (Oct. 13, 2016).
- 4. D. Castanotto et al., A cytoplasmic pathway for gapmer antisense oligonucleotide-mediated gene silencing in mammalian cells. Nucleic acids research 43, 9350 (Oct. 30, 2015).
- 5. D. A. Weidner, B. C. Valdez, D. Henning, S. Greenberg, H. Busch, Phosphorothioate Oligonucleotides Bind in a Non Sequence-Specific Manner to the Nucleolar Protein C23/Nucleolin. Febs Lett 366, 146 (Jun. 12, 1995).
- 6. X. H. Liang, W. Shen, H. Sun, T. P. Prakash, S. T. Crooke, TCP1 complex proteins interact with phosphorothioate oligonucleotides and can co-localize in oligonucleotide-induced nuclear bodies in mammalian cells. Nucleic acids research 42, 7819 (Aug. 1, 2014).
- 7. N. K. Christensen et al., A novel class of oligonucleotide analogues containing 2′-O,3′-C-linked [3.2.0]bicycloarabinonucleoside monomers: Synthesis, thermal affinity studies, and molecular modeling. J Am Chem Soc 120, 5458 (Jun. 10, 1998).
- 8. C. Wahlestedt et al., Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. P Natl Acad Sci USA 97, 5633 (May 9, 2000).
- 9. M. Gama-Carvalho, M. Carmo-Fonseca, The rules and roles of nucleocytoplasmic shuttling proteins. Febs Lett 498, 157 (Jun. 8, 2001).
- 10. M. Muller-McNicoll et al., SR proteins are NXF1 adaptors that link alternative RNA processing to mRNA export. Genes & development 30, 553 (Mar. 1, 2016).
- 11. B. Jiang et al., Nucleolin/C23 mediates the antiapoptotic effect of heat shock protein 70 during oxidative stress. The FEBS journal 277, 642 (February, 2010).
- 12. T. Tanaka, S. Ohashi, S. Kobayashi, Roles of YB-1 under arsenite-induced stress: translational activation of HSP70 mRNA and control of the number of stress granules. Biochimica et biophysica acta 1840, 985 (March, 2014).
- 13. A. V. Sorokin et al., Proteasome-mediated cleavage of the Y-box-binding protein 1 is linked to DNA-damage stress response. The EMBO journal 24, 3602 (Oct. 19, 2005).
- 14. K. Matsumoto, A. P. Wolffe, Gene regulation by Y-box proteins: coupling control of transcription and translation. Trends in cell biology 8, 318 (August, 1998).
- 15. P. L. Graumann, M. A. Marahiel, A superfamily of proteins that contain the cold-shock domain. Trends in biochemical sciences 23, 286 (August, 1998).
- 16. J. R. Buchan, R. Parker, Eukaryotic stress granules: the ins and outs of translation. Molecular cell 36, 932 (Dec. 25, 2009).
- 17. K. M. Bartoli, J. Jakovljevic, J. L. Woolford, Jr., W. S. Saunders, Kinesin molecular motor Eg5 functions during polypeptide synthesis. Molecular biology of the cell 22, 3420 (September, 2011).
- 18. A. N. Sasikumar, W. B. Perez, T. G. Kinzy, The many roles of the eukaryotic elongation factor 1 complex. Wiley interdisciplinary reviews. RNA 3, 543 (July-August, 2012).
- 19. B. F. Pickering, D. H. Yu, M. W. Van Dyke, Nucleolin Protein Interacts with Microprocessor Complex to Affect Biogenesis of MicroRNAs 15a and 16. J Biol Chem 286, 44095 (Dec. 23, 2011).
- 20. D. Schmitter et al., Effects of Dicer and Argonaute down-regulation on mRNA levels in human HEK293 cells. Nucleic acids research 34, 4801 (2006).
- 21. P. J. Bates, J. B. Kahlon, S. D. Thomas, J. O. Trent, D. M. Miller, Antiproliferative activity of G-rich oligonucleotides correlates with protein binding. J Biol Chem 274, 26369 (Sep. 10, 1999).
- 22. E. M. Reyes-Reyes, Y. Teng, P. J. Bates, A new paradigm for aptamer therapeutic AS1411 action: uptake by macropinocytosis and its stimulation by a nucleolin-dependent mechanism. Cancer research 70, 8617 (Nov. 1, 2010).
- 23. S. Fredriksson et al., Protein detection using proximity-dependent DNA ligation assays. Nature biotechnology 20, 473 (May, 2002).
- 24. K. J. Leuchowius, I. Weibrecht, O. Soderberg, In situ proximity ligation assay for microscopy and flow cytometry. Current protocols in cytometry Chapter 9, Unit 9 36 (April, 2011).
- 25. I. A. Eliseeva, E. R. Kim, S. G. Guryanov, L. P. Ovchinnikov, D. N. Lyabin, Y-box-binding protein 1 (YB-1) and its functions. Biochemistry. Biokhimiia 76, 1402 (December, 2011).
- 26. A. Emde, E. Homstein, miRNAs at the interface of cellular stress and disease. The EMBO journal 33, 1428 (Jul. 1, 2014).
- 27. K. Higashi et al., Interferon-gamma interferes with transforming growth factor-beta signaling through direct interaction of YB-1 with Smad3. J Biol Chem 278, 43470 (Oct. 31, 2003).
- 28. B. Schmierer, C. S. Hill, TGFbeta-SMAD signal transduction: molecular specificity and functional flexibility. Nature reviews. Molecular cell biology 8, 970 (December, 2007).
- 29. P. Sazani et al., Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs. Nucleic acids research 29, 3965 (Oct. 1, 2001).
- 30. R. C. Lantz, A. M. Hays, Role of oxidative stress in arsenic-induced toxicity. Drug Metab Rev 38, 791 (2006).
- 31. X. Thomas, J. Troncy, Arsenic: a beneficial therapeutic poison—a historical overview. Adler Museum bulletin 35, 3 (June, 2009).
- 32. C. Blenkiron, D. G. Hurley, S. Fitzgerald, C. G. Print, A. Lasham, Links between the oncoprotein YB-1 and small non-coding RNAs in breast cancer. PloS one 8, e80171 (2013).
- 33. T. B. Hansen et al., miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA. Embo Journal 30, 4414 (Nov. 2, 2011).
- 34. T. B. Hansen et al., Natural RNA circles function as efficient microRNA sponges. Nature 495, 384 (Mar. 21, 2013).
- 35. S. Memczak et al., Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495, 333 (Mar. 21, 2013).
- 36. E. Leucci et al., microRNA-9 targets the long non-coding RNA MALAT1 for degradation in the nucleus. Scientific reports 3, 2535 (2013).
- 37. N. Kedersha et al., Stress granules and processing bodies are dynamically linked sites of mRNP remodeling. The Journal of cell biology 169, 871 (Jun. 20, 2005).
- 38. A. Wilczynska, C. Aigueperse, M. Kress, F. Dautry, D. Weil, The translational regulator CPEB1 provides a link between dcpl bodies and stress granules. Journal of cell science 118, 981 (Mar. 1, 2005).
- 39. D. Dormann et al., ALS-associated fused in sarcoma (FUS) mutations disrupt Transportin-mediated nuclear import. The EMBO journal 29, 2841 (Aug. 18, 2010).
- 40. M. Morlando et al., FUS stimulates microRNA biogenesis by facilitating co-transcriptional Drosha recruitment. The EMBO journal 31, 4502 (Dec. 12, 2012).
- 41. I. V. Chemukhin et al., Physical and functional interaction between two pluripotent proteins, the Y-box DNA/RNA-binding factor, YB-1, and the multivalent zinc finger factor, CTCF. J Biol Chem 278, 29915 (Sep. 22, 2000).
- 42. R. Bergstrom et al., Transforming Growth Factor beta Promotes Complexes between Smad Proteins and the CCCTC-binding Factor on the H19 Imprinting Control Region Chromatin. J Biol Chem 288, 19727 (Jun. 25, 2010).
- 43. K. Nishi, A. Nishi, T. Nagasawa, K. Ui-Tei, Human TNRC6A is an Argonaute-navigator protein for microRNA-mediated gene silencing in the nucleus. Rna 19, 17 (January, 2013).
- 44. D. Castanotto, R. Lingeman, A. D. Riggs, J. J. Rossi, CRM1 mediates nuclear-cytoplasmic shuttling of mature microRNAs. Proc Natl Acad Sci USA 106, 21655 (Dec. 22, 2009).
- 45. A. K. Leung, P. A. Sharp, MicroRNA functions in stress responses. Molecular cell 40, 205 (Oct. 22, 2010).
- 46. A. Detzer, C. Engel, W. Wunsche, G. Sczakiel, Cell stress is related to re-localization of Argonaute 2 and to decreased RNA interference in human cells. Nucleic acids research 39, 2727 (April, 2011).
- 47. Z. Xiao, N. Watson, C. Rodriguez, H. F. Lodish, Nucleocytoplasmic shuttling of Smad1 conferred by its nuclear localization and nuclear export signals. J Biol Chem 276, 39404 (Oct. 19, 2001).
- 48. U. Upadhyay et al., Ablation of RNA interference and retrotransposons accompany acquisition and evolution of transposases to heterochromatin protein CENPB. Molecular biology of the cell 28, 1132 (Apr. 15, 2017).
- 49. S. J. Chen et al., From an old remedy to a magic bullet: molecular mechanisms underlying the therapeutic effects of arsenic in fighting leukemia. Blood 117, 6425 (Jun. 16, 2011).
- 50. Stein, C. A. and Castanotto, D. (2017) FDA-Approved Oligonucleotide Therapies in 2017. Molecular therapy: the journal of the American Society of Gene Therapy.
- 51. Mendell, J. R., Goemans, N., Lowes, L. P., Alfano, L. N., Berry, K., Shao, J., Kaye, E. M., Mercuri, E., Eteplirsen Study, G. and Telethon Foundation, D. M. D. I. N. (2016) Longitudinal effect of eteplirsen versus historical control on ambulation in Duchenne muscular dystrophy. Annals of neurology, 79, 257-271.
- 52. Disterer, P., Kryczka, A., Liu, Y., Badi, Y. E., Wong, J. J., Owen, J. S. and Khoo, B. (2014) Development of therapeutic splice-switching oligonucleotides. Human gene therapy, 26, 587-598.
- 53. http:/us.gsk.com/en-us/media/press-releases/2014/prosensa-regains-rights-to-drisapersen-from-qsk-and-retains-rihts-to-all-other-programmes-for-the-treatment-of-duchenne-muscular-dystrophy-dmd/.
- 54. Hache, M., Swoboda, K. J., Sethna, N., Farrow-Gillespie, A., Khandji, A., Xia, S. and Bishop, K. M. (2016) Intrathecal Injections in Children With Spinal Muscular Atrophy: Nusinersen Clinical Trial Experience. Journal of child neurology, 31, 899-906.
- 55. Burdick, A. D., Sciabola, S., Mantena, S. R., Hollingshead, B. D., Stanton, R., Wameke, J. A., Zeng, M., Martsen, E., Medvedev, A., Makarov, S. S. et al. (2014) Sequence motifs associated with hepatotoxicity of locked nucleic acid-modified antisense oligonucleotides. Nucleic acids research, 42, 4882-4891.
- 56. Straarup, E. M., Fisker, N., Hedtjam, M., Lindholm, M. W., Rosenbohm, C., Aarup, V., Hansen, H. F., Orum, H., Hansen, J. B. and Koch, T. (2010) Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates. Nucleic acids research, 38, 7100-7111.
- 57. Christensen, N. K., Petersen, M., Nielsen, P., Jacobsen, J. P., Olsen, C. E. and Wengel, J. (1998) A novel class of oligonucleotide analogues containing 2′-O,3′-C-linked [3.2.0]bicydoarabinonucleoside monomers: Synthesis, thermal affinity studies, and molecular modeling. J Am Chem Soc, 120, 5458-5463.
- 58. Wahlestedt, C., Salmi, P., Good, L., Kela, J., Johnsson, T., Hokfelt, T., Broberger, C., Porreca, F., Lai, J., Ren, K. et al. (2000) Potent and nontoxic antisense oligonucleotides containing locked nucleic acids. Proceedings of the National Academy of Sciences of the United States of America, 97, 5633-5638.
- 59. Kurreck, J. (2003) Antisense technologies. Improvement through novel chemical modifications. European journal of biochemistry, 270, 1628-1644.
- 60. Roberts, J., Palma, E., Sazani, P., Orum, H., Cho, M. and Kole, R. (2006) Efficient and persistent splice switching by systemically delivered LNA oligonucleotides in mice. Molecular therapy: the journal of the American Society of Gene Therapy, 14, 471-475.
- 61. Sazani, P., Gemignani, F., Kang, S. H., Maier, M. A., Manoharan, M., Persmark, M., Bortner, D. and Kole, R. (2002) Systemically delivered antisense oligomers upregulate gene expression in mouse tissues. Nature biotechnology, 20, 1228-1233.
- 62. Sazani, P., Kang, S. H., Maier, M. A., Wei, C., Dillman, J., Summerton, J., Manoharan, M. and Kole, R. (2001) Nuclear antisense effects of neutral, anionic and cationic oligonucleotide analogs. Nucleic acids research, 29, 3965-3974.
- 63. Stein, C. A., Hansen, J. B., Lai, J., Wu, S., Voskresenskiy, A., Hog, A., Worm, J., Hedtjam, M., Souleimanian, N., Miller, P. et al. (2010) Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents. Nucleic acids research, 38, e3.
- 64. Soifer, H. S., Koch, T., Lai, J., Hansen, B., Hoeg, A., Oerum, H. and Stein, C. A. (2012) Silencing of gene expression by gymnotic delivery of antisense oligonucleotides. Methods in molecular biology, 815, 333-346.
- 65. Souleimanian, N., Deleavey, G. F., Soifer, H., Wang, S., Tiemann, K., Damha, M. J. and Stein, C. A. (2012) Antisense 2′-Deoxy, 2′-Fluoroarabino Nucleic Acid (2′F-ANA) Oligonucleotides: In Vitro Gymnotic Silencers of Gene Expression Whose Potency Is Enhanced by Fatty Acids. Molecular therapy. Nucleic acids, 1, e43.
- 66. Juliano, R. L. and Carver, K. (2015) Cellular uptake and intracellular trafficking of oligonucleotides. Advanced drug delivery reviews, 87, 35-45.
- 67. Ming, X., Carver, K., Fisher, M., Noel, R., Cintrat, J. C., Gillet, D., Barbier, J., Cao, C., Bauman, J. and Juliano, R. L. (2013) The small molecule Retro-1 enhances the pharmacological actions of antisense and splice switching oligonucleotides. Nucleic acids research, 41, 3673-3687.
- 68. Yang, B., Ming, X., Cao, C., Laing, B., Yuan, A., Porter, M. A., Hull-Ryde, E. A., Maddry, J., Suto, M., Janzen, W. P. et al. (2015) High-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides. Nucleic acids research, 43, 1987-1996.
- 69. Kendall, G. C., Mokhonova, E. I., Moran, M., Sejbuk, N. E., Wang, D. W., Silva, O., Wang, R. T., Martinez, L., Lu, Q. L., Damoiseaux, R. et al. (2012) Dantrolene enhances antisense-mediated exon skipping in human and mouse models of Duchenne muscular dystrophy. Science translational medicine, 4, 164ra160.
- 70. Chou, T. C. and Talalay, P. (1984) Quantitative analysis of dose-effect relationships: the combined effects of multiple drugs or enzyme inhibitors. Advances in enzyme regulation, 22, 27-55.
- 71. Schneider, M., Marison, I. W. and von Stockar, U. (1996) The importance of ammonia in mammalian cell culture. Journal of biotechnology, 46, 161-185.
- 72. Hansen, H. A. and Emborg, C. (1994) Influence of ammonium on growth, metabolism, and productivity of a continuous suspension Chinese hamster ovary cell culture. Biotechnology progress, 10, 121-124.
- 73. Huotari, J. and Helenius, A. (2011) Endosome maturation. The EMBO journal, 30, 3481-3500.
- 74. van Weert, A. W., Dunn, K. W., Geuze, H. J., Maxfield, F. R. and Stoorvogel, W. (1995) Transport from late endosomes to lysosomes, but not sorting of integral membrane proteins in endosomes, depends on the vacuolar proton pump. The Journal of cell biology, 130, 821-834.
- 75. Weisz, O. A. (2003) Acidification and protein traffic. International review of cytology, 226, 259-319.
- 76. Yakubov, L. A., Deeva, E. A., Zarytova, V. F., Ivanova, E. M., Ryte, A. S., Yurchenko, L. V. and Vlassov, V. V. (1989) Mechanism of oligonucleotide uptake by cells: involvement of specific receptors? Proceedings of the National Academy of Sciences of the United States of America, 86, 6454-6458.
- 77. https://www.drugbank.ca/drugs/DB06742.
- 78. www.csun.edu/˜hcchm003/321/Ka.pdf.
- 79. Castanotto, D., Lin, M., Kowolik, C., Koch, T., Hansen, B. R., Oerum, H. and Stein, C. A. (2016) Protein Kinase C-alpha is a Critical Protein for Antisense Oligonucleotide-mediated Silencing in Mammalian Cells. Molecular therapy: the journal of the American Society of Gene Therapy, 24, 1117-1125.
- 80. Dean, R. T., Jessup, W. and Roberts, C. R. (1984) Effects of exogenous amines on mammalian cells, with particular reference to membrane flow. The Biochemical journal, 217, 27-40.
- 81. Castanotto, D., Lin, M., Kowolik, C., Wang, L., Ren, X. Q., Soifer, H. S., Koch, T., Hansen, B. R., Oerum, H., Armstrong, B. et al. (2015) A cytoplasmic pathway for gapmer antisense oligonucieotide-mediated gene silencing in mammalian cells. Nucleic acids research, 43, 9350-9361.
- 82. Siomi, H. and Siomi, M. C. (2009) RISC hitches onto endosome trafficking. Nature cell biology, 11, 1049-1051.
- 83. Koller, E., Vincent, T. M., Chappell, A., De, S., Manoharan, M. and Bennett, C. F. (2011) Mechanisms of single-stranded phosphorothioate modified antisense oligonucleotide accumulation in hepatocytes. Nucleic acids research, 39, 4795-4807.
- 84. Schindler, J. F. and Devries, U. (1990) Effects of Ammonia, Chloroquine, and Monensin on the Vacuolar Apparatus of an Absorptive Epithelium. Cell Tissue Res, 269, 283-292.
- 85. Misinzo, G., Delputte, P. L. and Nauwynck, H. J. (2008) Inhibition of endosome-lysosome system acidification enhances porcine circovirus 2 infection of porcine epithelial cells. Journal of virology, 82, 1128-1135.
- 86. Juliano, R., Alam, M. R., Dixit, V. and Kang, H. (2008) Mechanisms and strategies for effective delivery of antisense and siRNA oligonucleotides. Nucleic acids research, 36, 4158-4171.
- 87. Zhang, X. W., Yan, X. J., Zhou, Z. R., Yang, F. F., Wu, Z. Y., Sun, H. B., Liang, W. X., Song, A. X., Lallemand-Breitenbach, V., Jeanne, M. et al. (2010) Arsenic trioxide controls the fate of the PML-RARalpha oncoprotein by directly binding PML. Science, 328, 240-243.
- 88. Jeanne, M., Lallemand-Breitenbach, V., Ferhi, O., Koken, M., Le Bras, M., Duffort, S., Peres, L., Berthier, C., Soilihi, H., Raught, B. et al. (2010) PML/RARA oxidation and arsenic binding initiate the antileukemia response of As203. Cancer cell, 18, 88-98.
- 89. Chen, S. J., Zhou, G. B., Zhang, X. W., Mao, J. H., de The, H. and Chen, Z. (2011) From an old remedy to a magic bullet: molecular mechanisms underlying the therapeutic effects of arsenic in fighting leukemia. Blood, 117, 6425-6437.
- 90. Hu, J., Liu, Y. F., Wu, C. F., Xu, F., Shen, Z. X., Zhu, Y. M., Li, J. M., Tang, W., Zhao, W. L., Wu, W. et al. (2009) Long-term efficacy and safety of all-trans retinoic acid/arsenic trioxide-based therapy in newly diagnosed acute promyelocytic leukemia. Proceedings of the National Academy of Sciences of the United States of America, 106, 3342-3347.