Patent Grant
9809632
siRNA is of considerable current interest because it can elicit potent, target-specific knockdown of virtually any mRNA, creating new opportunities for personalized medicine and for addressing a broad range of traditionally undruggable disease targets using small molecules. Similar to other antisense approaches, however, cell-specific delivery of siRNA technology in vivo still represents a major technical hurdle.
In a first aspect, the present invention provides a chimeric protein, comprising:
(a) one or more double stranded nucleic acid binding domains (dsNABD); and
(b) one or more polyHis domains, where each polyHis domain comprises at least3 consecutive histidine residues. In various embodiments, the one or more polyHis domains in total comprise at least 6, 12, 18, or more histidine residues. In another embodiment, only one polyHis domain is present. In a further embodiment, the one or more dsNABDs comprise one or more double stranded RNA binding domains (dsRBD). In exemplary embodiments, the one or more dsRBDs comprise an amino acid sequence selected from the group consisting of SEQ ID NOs:1-36 and 38-82.
In various other aspects, the invention provides recombinant nucleic acids encoding the chimeric protein of any embodiment or combination of embodiments of the invention, recombinant expression vectors comprising the recombinant nucleic acid of the invention, and recombinant host cells comprising the recombinant expression vectors of the invention.
In a further aspect, the invention provides compositions comprising (a) the chimeric protein of any embodiment or combination of embodiments of the invention and (b) a therapeutic comprising (i) a therapeutic double stranded nucleic acid; and (ii) a targeting ligand bound to the therapeutic double stranded nucleic acid, wherein the dsNABD of the chimeric protein is bound to the therapeutic double stranded nucleic acid. In one embodiment, the therapeutic double stranded nucleic acid comprises a therapeutic double stranded RNA, including but not limited to siRNA, small hairpin RNA (shRNA), or miRNA. In one embodiment, the therapeutic double stranded RNA comprises an siRNA. In a further embodiment, the targeting ligand is a single stranded aptamer, including but not limited an aptamer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS:85-87.
In another aspect, the invention provides a use of the composition of any embodiment or combination of embodiments of the invention for treating a subject in need of treatment with the therapeutic double stranded nucleic acid. In a further aspect, the invention provides methods for reducing translation from a mRNA of interest, comprising contacting a cell or tissue comprising the mRNA with the composition of any embodiment or combination of embodiments of the invention for a time and under conditions to promote delivery of the siRNA into the cell or tissue to interfere with translation from the mRNA target of the siRNA.
All references cited are herein incorporated by reference in their entirety. As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise. “And” as used herein is interchangeably used with “or” unless expressly stated otherwise.
All embodiments of any aspect of the invention can be used in combination, unless the context clearly dictates otherwise.
As used herein, “about” means plus or minus 5% of the recited measurement.
Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above.” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.
The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While the specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize.
In a first aspect, the present invention provides chimeric proteins, comprising or consisting of:
(a) one or more double stranded nucleic acid binding domains (dsNABD); and
(b) one or more polyHis domains, where each polyHis domain comprises at least 3 consecutive histidine residues.
The chimeric protein is one which is engineered to possess the one or more dsNABDs and the one or more polyHis domains (i.e.: does not encompass any naturally occurring protein). The inventors have surprisingly discovered that the chimeric proteins of the present invention provide a universal delivery vehicle for therapeutic double stranded nucleic acids that is capable of endosomal escape, and thus significantly improved efficacy of the therapeutic nucleic acid.
As used herein, a “polyHis” domain is a sequence of consecutive/contiguous His residues totaling at least 3. The one or more polyHis domains can be any suitable number of such domains that can be used to promote endosomal escape but no interfere with protein folding. In various embodiments, the chimeric protein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polyHis domains. In various further embodiments, the polyHis domains may comprise 3, 6, 9, 12, 15, 18, 20, 23, or more His residues in total.
In various embodiments, the one or more polyHis domains may be present at the N-terminus, the C-terminus, or both the N-terminus and the C-terminus of the chimeric protein. In other embodiments, one or more polyHis domains may be present between two (or more) dsNABD, particularly in embodiments in which the chimeric protein includes linker amino acid sequences between multiple dsNABDs.
As will be understood by those of skill in the art, the chimeric proteins may comprise amino acid residues/domains in addition to the polyHis domain and the dsNABD; such additional domains may include purification tags, detectable tags, linker domains, etc. In one embodiment, the chimeric protein may comprise a linker domain of any suitable length between an dsNABD and a polyHis domain; in this embodiment, the number of polyHis domains may be increased, as the linker can limit the effect of the polyHis domains on folding of the dsNABDs. In another embodiment, the chimeric protein may comprise a linker domain of any suitable length between two dsNABDs.
As used herein, a dsNABD is a protein domain that binds to double stranded nucleic acid, including but not limited to double stranded DNA, RNA, or modified nucleic acids. In one embodiment, the dsNABD binds to double stranded RNA (dsRBD); in another embodiment, the dsNABD binds to double stranded DNA (dsDBD), including but not limited to zinc finger, leucine zipper, and helix-turn-helix domains. The double stranded nucleic acid may be any such ds nucleic acid that is to be delivered intracellularly. Non-limiting examples of such double stranded RNAs are small interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), and micro-RNAs (miRNAs).
As will be understood by those of skill in the art, any suitable dsNABD can be used that binds appropriately to a given double stranded nucleic acid to be delivered. A wide variety of proteins containing dsRBDs are known, including but not limited to human protein kinase R (hPKR), DICER, Staufen, adenosine demainase acting on RNA (ADAR), spermatid perinuclear RNA binding protein, and a variety of other proteins as shown below in Tables 1and 2 below. These proteins typically share an evolutionarily-conserved dsRNA binding domain of about 65-68 amino acids. See, for example, Masliah et al., Cell. Mol. Life Sci. (2013) 70:1875-1895; Lupold et al, [Cancer Research 62, 4029-4033, Jul. 15, 2002]; and Nanduri et al., The EMBO Journal Vol. 17 No. 18 pp. 5458-5465, 1998. In one embodiment, the dsRBD comprises or consists of the amino acid sequence of SEQ ID NO:29 (consensus sequence of the evolutionarily-conserved dsRNA binding domain). In another embodiment, the dsRBD comprises or consists of the amino acid sequence of any one of SEQ ID NOS:4-28 and 30-36, and 38-82.
Drosophila
A. gambiae (mosquito)
C. elegans
Drosophila
A. gambiae (partial)
Drosophila
A. gambiae (partial)
Drosophila
Drosophila
Drosophila
Drosophila
A. gambiae
C. elegans
O. sativa
O. sativa
A. thaliana
A. thaliana
D. disco
O. sativa
A. thaliana
A. thaliana
A. thaliana
A. thaliana
O. sativa
A. thaliana
Vaccinia
virus E3L
Haemophilus
influenzae
Paramecium
bursaria
clorella
Coltivirus Vp8
Coltivirus Vp12
Drosophila
C virus (DCV)
Acyrthosiphon
pisum virus P1
As will be understood by those of skill in the art, the chimeric protein may contain more than one dsNABD; thus, in various embodiments, the dsNABD includes 1, 2, or more dsNABDS. When the construct includes more than one dsNABD, each dsNABD may be the same, or they may be different dsNABDs.
In one embodiment, the one or more dsNABDs comprise a dsRBD from human protein kinase R (hPKR). In one embodiment, the hPKR binding domain comprises or consists of the amino acid of SEQ ID NO:1 (hPKR RNA binding domain 1), SEQ ID NO:2 (hPKR RNA binding domain 2), SEQ ID NO:3 (consensus sequence of hPKR RNA binding domains 1 and 2). SEQ ID NO: 82 (hPKR RNA binding domains 1-2 plus linking sequences) and SEQ ID NO:81 (full length hPKR).
As used throughout the present application, the term “protein” is used in its broadest sense to refer to a sequence of subunit amino acids, whether naturally occurring or of synthetic origin. The proteins of the invention may comprise L-amino acids, D-amino acids (which are resistant to L-amino acid-specific protecases in vivo), or a combination of D- and L-amino acids. The proteins described herein may be chemically synthesized or recombinantly expressed. The proteins may be linked to other compounds to promote an increased half-life in vivo, such as by PEGylation, HESylation, PASylation, or glycosylation. Such linkage can be covalent or non-covalent as is understood by those of skill in the art. The proteins may be linked to any other suitable linkers, including but not limited to any linkers that can be used for purification or detection (such as FLAG or His tags).
In another aspect, the present invention provides isolated nucleic acids encoding the protein of any aspect or embodiment of the invention. The isolated nucleic acid sequence may comprise RNA or DNA. As used herein, “isolated nucleic acids” are those that have been removed from their normal surrounding nucleic acid sequences in the genome or in cDNA sequences. Such isolated nucleic acid sequences may comprise additional sequences useful for promoting expression and/or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the proteins of the invention.
In a further aspect, the present invention provides nucleic acid expression vectors comprising the isolated nucleic acid of any embodiment of the invention operatively linked to a suitable control sequence. “Recombinant expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the gene product. “Control sequences” operably linked to the nucleic acid sequences of the invention are nucleic acid sequences capable of effecting the expression of the nucleic acid molecules. The control sequences need not be contiguous with the nucleic acid sequences, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the nucleic acid sequences and the promoter sequence can still be considered “operably linked” to the coding sequence. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, and ribosome binding sites. Such expression vectors can be of any type known in the art, including but not limited plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed nucleic acid sequences in a mammalian system may be constitutive (driven by any of a variety of promoters, including but not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven by any of a number of inducible promoters including, but not limited to, tetracycline, ecdysone, steroid-responsive). The construction of expression vectors for use in transfecting prokaryotic cells is also well known in the art, and thus can be accomplished via standard techniques. (See, for example, Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin, Tex.). The expression vector must be replicable in the host organisms either as an episome or by integration into host chromosomal DNA. In a preferred embodiment, the expression vector comprises a plasmid. However, the invention is intended to include other expression vectors that serve equivalent functions, such as viral vectors.
In another aspect, the present invention provides recombinant host cells comprising the nucleic acid expression vectors of the invention. The host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably transfected or transduced. Such transfection and transduction of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique known in the art, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection. (See, for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R.I. Freshney. 1987. Liss, Inc. New York, N.Y.). A method of producing a polypeptide according to the invention is an additional part of the invention. The method comprises the steps of (a) culturing a host according to this aspect of the invention under conditions conducive to the expression of the polypeptide, and (b) optionally, recovering the expressed polypeptide. The expressed polypeptide can be recovered from the cell free extract, cell pellet, or recovered from the culture medium. Methods to purify recombinantly expressed polypeptides are well known to the man skilled in the art.
In a further aspect, the present invention provides compositions comprising the chimeric protein of any embodiment or combination of embodiments of the present invention; and a therapeutic comprising (i) a therapeutic double stranded nucleic acid; and (ii) a targeting ligand bound to the therapeutic double stranded nucleic acid, wherein the dsNABD of the chimeric protein is bound to the therapeutic double stranded nucleic acid.
The inventors demonstrate in the examples that follow that compositions of the invention can be used for more efficient delivery of double stranded therapeutic nucleic acids to an intended target.
The protein chimera of the composition can be any embodiment or combination of embodiments as discussed herein. For example, the chimeric protein may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more polyHis domains. In various further embodiments, the polyHis domains may comprise 3, 6, 9, 12, 15, 18, 20, 23, or more His residues in total. The chimeric proteins may comprise amino acid residues/domains in addition to the polyHis domain and the dsNABD; such additional domains may include purification tags, detectable tags, linker domains, etc. In one embodiment, the chimeric protein may comprise a linker domain of any suitable length between the dsNABD and the polyHis domain; in this embodiment, the number of polyHis domains may be increased, as the linker can limit the effect of the polyHis domains on folding of the dsNABDs. In one embodiment, the dsNABD binds to double stranded RNA (dsRBD); in another embodiment, the dsNABD binds to double stranded DNA (dsDBD). In various further embodiments, the one or more dsNABD comprises one or more domains selected from the group consisting of SEQ ID NOS: 1-36 and 38-82. In another embodiment, the dsNABD comprises a domain comprising the amino acid sequence of SEQ ID NO: 29. The chimeric protein may contain more than one dsNABD; thus, in various embodiments, the dsNABD includes 1, 2, or more dsNABDS. When the construct includes more than one dsNABD, each dsNABD may be the same, or they may be different dsNABDs. In one embodiment, the one or more dsNABDs comprise a dsRBD from human protein kinase R (hPKR). In various embodiments, the dsRBD from hPKR comprises or consists of any one or more of SEQ ID NOS:1-3 and 82.
The composition further comprises a therapeutic comprising (i) a therapeutic double stranded nucleic acid, and (ii) a targeting ligand bound to the therapeutic double stranded nucleic acid, wherein the dsNABD of the chimeric protein is bound to the therapeutic double stranded nucleic acid (such as by base pairing). The therapeutic double stranded nucleic acid can comprise DNA (in which case the dsNABD is a dsDBD) or RNA (in which case the dsNABD is a dsRBD). Any suitable double stranded nucleic acid can be used that is appropriate for a given use of the composition.
In one embodiment, the therapeutic double stranded nucleic acid comprises or consists of a small interfering RNA (siRNA), a small hairpin RNA (shRNA), or a micro-RNA (miRNA). Any suitable siRNA, shRNA, or miRNA can be used that is appropriate for a therapeutic target of interest. As is known by those of skill in the art, large numbers of such sequences exist. For example, Ambion (now Life Technology) sells more than 200,000 siRNAs.
In embodiments where the therapeutic double stranded nucleic acid is an siRNA. Such siRNAs are well known in the art and are in various stages of clinical development. For example, see Kanasty et al., Nature Materials Volume 12:967-977 (2013); and Burnett and Rossi, Chemistry & Biology 19:60-71, Jan. 27, 2012.
The targeting ligand of the compositions of the invention can be any molecule that can be used to target the composition for delivery to a site of interest on or in a target cell/tissue. In various non-limiting embodiments, the targeting ligand may be a single stranded nucleic acid aptamer, an antibody, affibody, scFv molecule, or small molecule that selectively bind to a target cell/tissue. In all embodiments, the targeting ligand is attached to the double stranded therapeutic nucleic acid: in one embodiment, the targeting ligand is chemically linked to the double stranded therapeutic nucleic acid. In embodiments where the targeting ligand is an RNA aptamer and the double stranded therapeutic nucleic acid is an siRNA, shRNA, or miRNA, the two may be recombinantly expressed from an appropriate expression vector. Similarly, where the targeting ligand is a DNA aptamer and the double stranded therapeutic nucleic acid is double stranded DNA the two may be recombinantly expressed from an appropriate expression vector.
In one embodiment, the targeting ligand comprises a single stranded aptamer. As will be understood by those of skill in the art, a large number of aptamers have been developed to target different cells/tissues in vivo (see, for example, Front. Genet., 2 Nov. 2012|doi: 10.3389/fgene.2012.00234) and Burnett and Rossi, Chemistry & Biology 19:60-71, Jan. 27, 2012. In one embodiment, the aptamer may comprise or consist of a PSMA aptamer selected from the group consisting of SEQ ID NO:85, 86, and 87.
The compositions may further comprise (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (e) a stabilizer; (f) a preservative and/or (g) a buffer. In some embodiments, the buffer in the pharmaceutical composition is a Tris buffer, a histidine buffer, a phosphate buffer, a citrate buffer or an acetate buffer. The pharmaceutical composition may also include a lyoprotectant, e.g. sucrose, sorbitol or trehalose. In certain embodiments, the pharmaceutical composition includes a preservative e.g. benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol, benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol, p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoic acid, and various mixtures thereof. In other embodiments, the pharmaceutical composition includes a bulking agent, like glycine. In yet other embodiments, the pharmaceutical composition includes a surfactant e.g., polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitan monolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitan monooleate, sorbitan trilaurate, sorbitan tristearate, sorbitan trioleaste, or a combination thereof. The pharmaceutical composition may also include a tonicity adjusting agent, e.g., a compound that renders the formulation substantially isotonic or isoosmotic with human blood. Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine, methionine, mannitol, dextrose, inositol, sodium chloride, arginine and arginine hydrochloride. In other embodiments, the pharmaceutical composition additionally includes a stabilizer, e.g., a molecule which, when combined with a protein of interest substantially prevents or reduces chemical and/or physical instability of the protein of interest in lyophilized or liquid form. Exemplary stabilizers include sucrose, sorbitol, glycine, inositol, sodium chloride, methionine, arginine, and arginine hydrochloride.
The therapeutic double stranded nucleic acid may be the sole active agent in the composition, or the composition may further comprise one or more other active agents suitable for an intended use.
The compositions described herein may further comprise a pharmaceutically acceptable carrier, diluent, or excipient. Such compositions are substantially free of non-pharmaceutically acceptable components, i.e., contain amounts of non-pharmaceutically acceptable components lower than permitted by US regulatory requirements at the time of filing this application. In some embodiments of this aspect, if the composition is dissolved or suspended in water, the composition further optionally comprises an additional pharmaceutically acceptable carrier, diluent, or excipient. In other embodiments, the compositions described herein are solid pharmaceutical compositions (e.g., tablet, capsules, etc.).
These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by any suitable route. In a preferred embodiment, the pharmaceutical compositions and formulations are designed for oral, subcutaneous, or intravenuous administration. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
In a further aspect, the invention provides uses of the compositions of the invention to deliver the therapeutic double stranded nucleic acid to a subject in need of treatment that can be effected by the therapeutic double stranded nucleic acid. In embodiments where the therapeutic double stranded nucleic acid is an siRNA, the uses/methods are to interfere with translation from mRNA of the siRNA targets. Thus, for example, the methods may comprise contacting a cell or tissue comprising the target mRNA with the composition of any appropriate embodiment of the invention for a time and under conditions to promote delivery of the siRNA into the cell or tissue to interfere with translation from the mRNA target of the siRNA. As used herein, “contacting cell or tissue” can be in vitro or in vivo, including administering to a patient with a disorder to be treated by reducing translation from a target mRNA. Appropriate dosages of the compositions can be determined by an attending physician based on specifics of the composition, the disorder to be treated, and all other factors.
siRNA-aptamer chimeras have emerged as one of the most promising approaches for targeted delivery of siRNA due to the modularity of their diblock RNA structure, relatively lower cost over other targeted delivery approaches, and, most importantly, the outstanding potential for clinical translation. However, additional challenges must be addressed for efficient RNA interference (RNAi), in particular, endosomal escape. Currently, vast majority of siRNA delivery vehicles are based on cationic materials, which form complexes with negatively charged siRNA. Unfortunately, these approaches complicate the formulations again by forming large complexes with heterogeneous sizes, unfavorable surface charges, colloidal instability, and poor targeting ligand orientation. Here, we report the development of a small and simple protein tag that complements the therapeutic and targeting functionalities of chimera with two functional domains: a dsRNA binding domain (dsRBD) for siRNA docking and a pH-dependent polyhistidine to disrupt endosomal membrane. The protein selectively tags along the siRNA block of individual chimera, rendering the overall size of the complex small, desirable for deep tissue penetration, and the aptamer block accessible for target recognition.
Introduction
siRNA is of considerable current interest because it can elicit potent, target-specific knockdown of virtually any mRNA, creating new opportunities for personalized medicine and for addressing a broad range of traditionally undruggable disease targets using small molecules.1-3 Similar to other antisense approaches, however, cell-specific delivery of siRNA technology in vivo still represents a major technical hurdle.4 Therefore, it is of value to design a delivery system that is simple for potential regulatory approval and mass production, universal for all siRNA-aptamer chimera, neutral and siRNA-binding specific to ensure aptamer targeting, and small to avoid major alteration of chimera's biodistribution profile. A system simultaneously achieving these features could expedite clinically translation of the highly promising siRNA-aptamer chimera technology.
Here, we report the development of a small protein tag for efficient delivery of siRNA-aptamer chimeras. As shown in
To add endosomal escape functionality, a short histidine (His) oligomer is added to the C-terminus of the dsRBD. His molecules have a pKa value of approximately 6. At neutral pH (such as in circulation), they are mainly deprotonated (uncharged), which is desirable over positively charged counterparts due to reduced accumulation within the RES (reticuloendothelial system). Overall, this protein tag is equally small, simple, and biodegradable as siRNA-aptamer chimera, while perfectly complementing chimera's functionalities. When complexed together, they remain small in size, discrete and stable in solution, low positive charge for circulation, and simultaneously achieve therapeutic, targeting, and endosomal escaping capabilities.
Results
Expression and Characterization of dsBRD-His18 Protein Tag. To add endosomal escape capability, a short polyhistidine peptide was added to dsRBD. The dsRBD domain comes from the first 172 amino acids of human protein kinase R (hPKR), and has two double-strand RNA binding motifs (dsRBM1 and dsRBM2) for cooperative and dsRNA-specific binding.31 Because dsRBM1 towards the N terminal dominates the binding with dsRNA,32 we introduced the histidine peptide towards the C terminal (
Post expression and purification, the resulted protein tags were analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE,
More interestingly, the gel electrophoresis experiments also reveal that extending the C-terminal His by another 6 or 12 amino acids completely abolish dsRBD's binding activity. Therefore, for the following gene expression regulation studies we chose the dsRBD with a total of 18 His due to its balanced dsRNA binding and endosomal escape functionalities, in comparison with the original dsRBD with no C-terminus His as a control.
Design, Synthesis, and Characterization of siRNA-Aptamer Chimera. To evaluate the universal protein tag for siRNA-aptamer chimera, we first designed and made a chimera based on the protocols described by Dassie and coworkers, taking advantage of the shortened aptamer sequence for specific targeting of prostate specific antigen (PSMA) as well as the optimized siRNA strands with enhanced therapeutic potency.15 PSMA has been identified as one of the most attractive cell surface markers for both prostate epithelial cells and neovascular endothelial cells.36 Accumulation and retention of PSMA targeting probes at the site of tumor growth is the basis of radioimmunoscintigraphic scanning (e.g., ProstaScint scan) and targeted therapy for human prostate cancer metastasis. We replaced their siRNA sequence with a siRNA silencing GFP expression, because GFP is the best model for quantitative assessment of the silencing effect using optical imaging and flow cytometry.
The long ssRNA composed of PSMA aptamer and siRNA antisense strand (
Targeting Delivery and Silencing in Cells. With the biological activities of our protein tag and siRNA-aptamer chimera separately characterized, we proceeded to evaluate the gene silencing effect of this simple yet functionally highly complementary protein tag in siRNA-aptamer chimera delivery. GFP-expressing C4-2 cell line was used as a model because of the advantages of fluorescence imaging techniques such as microscopy and quantitative flow cytometry.
Quantitative flow cytometry studies further confirm this result (
To further confirm the difference in endosomal escape capability between the two protein tags (dsRBD-His6 and dsRBD-His18), we performed a dual color imaging assay using non-fluorescence LNCaP cells. In this experiment, chimera was labeled with Cy3 and endosome/lysosome was marked with a LysoTracker (spectrally distinguishable green fluorescence). Direct contrast in chimera distribution and intracellular density of endosome/lysosome was observed between the two protein tags. As shown in
Cytotoxicity. Lastly, we probed the cytotoxicity of the best performing protein tag dsRBD-His18 using a standard cell viability assay (CellTiter-Blue®). The assay is based on the ability of living cells to convert a redox dye (resazurin) into a fluorescent end product (resorufin). Nonviable cells lose metabolic capacity and thus do not generate fluorescent signals. As illustrated in
Discussion
siRNA-aptamer chimera is one of the most promising approaches for cell type-specific RNAi, owing to its low immunogenicity, ease of chemical synthesis and modification, small size, and the modularity of both the targeting aptamer block and the therapeutic siRNA segment. Almost all current targeted siRNA delivery formulations involve cationic nanocarriers such as polymers, inorganic nanoparticles, peptides, and proteins.7, 19, 20, 27, 28, 38- 44 Unfortunately, these conventional siRNA nanocarriers are unsuitable for chimera delivery, and, in fact, reverse the signature property of chimera, simple formulation for regulatory approval and clinical translation.15, 16 This is because the charge induced complex formation is basically an aggregation process, which lacks control over aggregate size, shape, stoichiometry, chimera orientation, aptamer functionality, and reproducibility during scale-up production. In addition, the final complexes often carriers positive charges as well, which is unfavorable for systemic uses.23
Our protein tag does not rely on high positive charge to interact with RNA molecules. In fact, it only recognizes relatively long dsRNAs (>16 bp) such as the siRNA segment and the short stem region of the aptamer in our chimera molecule. Extensive biochemistry investigations have shown that for the current length of the chimera, maximum two copies of dsRBD can bind to it with differential affinity (the first copy binds much stronger than the second copy). The gene silencing experiments conducted here reflect this effect since mixing chimera with 1× or 2× protein tags does not affect the silencing efficiency. Considering the molecular weights of the chimera (28.8 kDa) and the protein tag (24.8 kDa), molecular weight of the final complex at 1:1 binding will become 53.6 kDa. Based on well-documented size effect for in vivo drug delivery,49 this size is sufficiently large to reduce premature renal clearance while still small enough for deep tissue penetration. For example, by tagging siRNA-aptamer chimera with a 20 kDa PEG, its in vivo circulating half-life has been shown to increase from approximately 30 min to 30 hours;15 whereas large nanoparticles (>30 nm) have been shown to be ineffective in tumor treatment except for some hyperpermeable tumors.50
In conclusion, to solve the endosome escape problem of the highly promising siRNA-aptamer chimera based therapy, we have designed a dual-block small protein by combining dsRBD and polyhistidine and identified the optimal length of polyhistidine. The resulting protein tag shares the simplicity feature of siRNA-aptamer chimera, yet offers exactly complementary functionalities. The dsRBD selectively binds to the siRNA block, leaving the targeting aptamer accessible. In terms of size, different from conventional cationic delivery vehicles, the dsRBD-His18 tagged chimera remains discrete in solution rather than forming large aggregates. In terms of functionalities, chimera and dsRBD-His18 are highly complementary to each other, and thus offer the complete set of features necessary for targeted siRNA delivery (e.g., targeting, therapeutic, siRNA protection, and endosomal escape). This platform is also universal, able to chaperone any chimera sequences for cell type-specific delivery. Largely based on natural proteins, dsRBD-His18 is an excellent candidate for potential clinical translation because of its simple structure and biodegradability. Further development of this small protein tag with in vivo testing should raise exciting opportunities for siRNA clinical translation and personalized medicine.
Methods
Materials. Vendors for specific chemicals are listed below. In general, restriction enzymes were obtained from New England BioLabs, and cell culture products were purchased from Gibco/Invitrogen.
Chimera Composed of Aptamer Targeting PSMA and siRNA Targeting GFP. ssDNA of the PSMA aptamer (39 nucleotides, 5′-GGGAGGACGATGCGGATCAGCCATGTTTACGTCACTCCT-3′)(SEQ ID NO: 85) was chemically synthesized by Integrated DNA Technologies (IDT) and used as the template to generate one strand of the siRNA-aptamer chimera. For amplification, PCR was performed with 3′ primer containing the anti-sense strand of GFP siRNA (underlined) and 5′ primer containing T7 RNA polymerase promoter site (bolded). The PCR primer sequences are:
The 81 bp PCR product was put into T-A cloning pCR 2.1 vector (Invitrogen). After sequencing, positive plasmids were selected and used as the template for PCR. The resulting PCR product was separated with 2% agarose gel and recovered with QIAEX 11 Gel Extraction Kit (Qiagen). The purified PCR product was used as the template for in vitro transcription with MEGAscriptT7 Kit (Ambion) according to manufacturer's instruction. 2′fluoro-modified pyrimidines (TriLink, San Diego) were added to replace CTP and UTP. RNA molecules generated by the transcription reaction were annealed with the sense strand of GFP siRNA (chemically synthesized with or without 5′-Cy3 or FAM by IDT). The sequence is 5′-(Cy3 or FAM)-CAAGCUGACCCUGAAGUUCUU-3′ (SEQ ID NO: 90). For annealing, the transcripted RNA and the synthetic siRNA sense strand were mixed at molar ratio 1:1 in duplex buffer (IDT) and incubated at 94° C. for 3 min followed by slow cooling to 25° C. in 1 hour. The final chimera was store at −80° C.
Construction of dsRBD with Varying Lengths of Polyhistidine. Full-length PKR gene (clone ID 8068981, BC_101475, Homo sapiens) was ordered from Open Biosystems. The DNA sequence for dsRBD is composed of the first 172 amino acids of PKR. To add polyhistidine of varying lengths to the C-terminus, four constructs were developed by PCR. 5′ primer: 5′-AAA GGA TCC ATG GCT GGT GAT CTT TCA GCA-3′ (SEQ ID NO: 91), containing BamH1 site (underlined), was applied to all four constructs. The 3′ primers containing Xho1 site (bolded) are:
TCATTACACTGAGGTTTCTTCTGATAA-3′
GTGGTGGTGGTGGTGGTGCACTGAGGTTTC
GTGGTGGTGGTGGTGGTGGTGGTGGTGGTG
GTGGTGGTGGTGGTGGTGGTGGTGGTGGTG
The constructs were cloned into PET28a (+) expression vector (Novagen). The constructs for dsRBD-His6 and dsRBD-His18 were obtained using full-length PKR gene (clone ID 8068981) as PCR template, and the dsRBD-His24 and dsRBD-His30 constructs were made by grafting additional histidines to the dsRBD-His18 plasmid using PCR. The restriction enzyme sites for BamH1 and Xho1 were introduced in the PCR primers for cloning. dsRBD-His6 construct was introduced with two stop codons (TAA and TGA) before the Xho1 site. For the other three constructs, the reading frames cover the His6 sequence in the vector at the C-terminal end before the stop codon. The PCR products and PET28a (+) expression vector were digested with BamH1 and Xho1 enzymes. Ligation was performed with Quick Ligation Kit (BioLabs) for 5 min at room temperature. Ligates were transformed into E. coli BL21 (DE3) competent cells for expression. The plasmids were verified with DNA sequencing.
The sequences for the protein tags are
Single colonies were selected and grown at 37° C. for 12 h in Circlegrow medium containing 30 μg/ml kanamycin. Overnight cultures were diluted at 1:100 (v/v) into fresh medium and incubated at 37° C. until the OD600 values reach 0.5-1.0. Expression was induced by addition of isopropyl-β-D-thiogalactopyranoside (IPTG, 1 mM), and cell growth was continued for another 4-5 hour at 30° C. Cells were harvested by centrifugation (Beckman JA-10 rotor) at 10,000 g for 10 min and stored at −20° C.
Cells were suspended in Bug-Buster Mix (Novagen) with 5 ml reagent per gram of wet cell paste. Bug Buster Mix was added with protease inhibitor EDTA-free cocktail (Pierce), 10% glycerol, and 1.0 mM THP (Novagen). The cell suspensions were incubated on a shaker platform for 30 min at room temperature. Insoluble cell debris was removed by centrifugation (Beckman TL 120) at 20,000×g for 20 min at 4° C. The soluble extracts were loaded onto affinity columns with Ni-charged His Bind Resin (Novagen). Following washing with binding buffer and washing buffer, the desired proteins were eluted with 6 volume elution buffer (Novagen). The eluted proteins were dialyzed with PBS containing 10% glycerol and 0.1% (v/v) β-mercaptoethanol for 24 hours.
Purified proteins were probed using 12% SDS-PAGE and stained with Coomassie Brilliant Blue G-250 (Bio-Rad). Protein concentrations were determined with the Bio-Rad Protein Assay with bovine serum albumin as the standard.
Functional Characterization of siRNA-Aptamer Chimera. To test the functionality of the siRNA block, the chimera described above and GFP siRNA control (Qiagen) at a final concentration of 50 nM were transfected into C4-2 prostate cancer cells stably expressing GFP using Lipofectamine RNAi MAX (Invitrogen) following the instructions provided by the manufacturer. To evaluate the targeting specificity of the aptamer block, PSMA-positive LNCaP cells and PSMA-negative PC3 cells were treated with complex of chimera and dsRBD-His18 (chimera/protein tag molar ratio at 1:2, 100 nM chimera) in serum free medium for 2 hours, followed by incubation in complete medium for another 12 h. DAPI (30 nM) was added to stain cell nuclei. Fluorescent images were captured on an Olympus IX-71 inverted microscope equipped with 5 long-pass filters and a colored CCD camera.
Characterization of RNA Binding Capability of the Four Protein Tags. The binding capabilities of the four polyhistidine modified dsRBD proteins were evaluated by native agarose gel. The chimera was labeled with FAM at the 5′ end of siRNA's sense strand (IDT). To prepare chimera/dsRBD complex, chimera (5 μM, 10 μl) was incubated with the protein tags at protein/chimera molar ratios of 1, 2, or 4 for 1 h at 4° C. Bound chimera and unbound chimera were quantified on 1% agarose gel using a Macro imaging system (Lightools Research, CA).
Evaluation of Endosomal Escape. PSMA-expressing LNCaP cells were seeded on 35 mm glass-bottom petri dishes (MatTeck Corp) at a density of 5×104 cells/well for 24 hours in RPMI 1640 supplemented with 10% FCS. Complexes of chimera labeled with Cy3 (IDT) and protein tags (His6 and His18) were added to LNCaP cells in serum-free medium for 2 hours, followed by incubation in complete medium for 12 hours. LysoTracker® Green DND-26 (80 nM, Invitrogen) was then added for 4 hours at 37° C. Images were captured on a confocal laser scanning microscope (LSM 510, Carl Zeiss, Germany).
Microscopy and Flow Cytometry Studies of Gene Knockdown Efficacy. C4-2 prostate cancer cells expressing GFP were seeded into 35 mM glass-bottom petri dishes for confocal imaging or 6-well plates for flow cytometry. Cells were treated with chimera & dsRBD-His18 and compared with five control groups including no treatment, treated with GFP-siRNA alone, chimera alone, a random sequenced siRNA with the protein tag (His18), and chimera with protein tag (His6) for 2 h in serum free media and then incubated in complete media for 60 h. Confocal images were again obtained with LSM 510 confocal microscope equipped with argon (488 nm) and HeNe (543 nm) lasers; and quantitative flow cytometry investigation was done on a BD FACSCantoII flow cytometer.
Cytotoxicity Assay. LNCaP cells were seeded in 96-well plate at 4×103/well for 24 hours, and then treated with different concentrations of dsRBD-His18 protein tag for 72 hours. CellTiter-Blue reagent (20 μl) was added into each well. After 4 h incubation at 37° C., cell viability was assessed by fluorescence intensity at 590 nm (excitation 570 nm) on a TECAN infinite M200 microplate reader.
This application is a U.S. national phase of International Application No. PCT/US 2014/061714, filed Oct. 22, 2014, which claims priority to U.S. Provisional Application No. 61/894,806, filed Oct. 23, 2013, the disclosures of which are hereby incorporated by reference in their entirety.
This invention was made with government support under grant nos. CA 140295 and CA 150301, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2014/061714 | 10/22/2014 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2015/061409 | 4/30/2015 | WO | A |
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| 7772201 | Mixson et al. | Aug 2010 | B2 |
| 8273867 | Dowdy et al. | Sep 2012 | B2 |
| 20120064599 | Jayasinghe | Mar 2012 | A1 |
| 20120101045 | Dowdy | Apr 2012 | A1 |
| 20130102654 | Rossi et al. | Apr 2013 | A1 |
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| Number | Date | Country | |
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| Number | Date | Country | |
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| 61894806 | Oct 2013 | US |
