Patent Application
20230095192
A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on Mar. 1, 2021, having the file name “19-335-PCT_Sequence-Listing_ST25.txt” and is 55 kb in size.
Detection of intracellular DNA by innate immune sensors activates potent antiviral and inflammatory responses. The DNA sensor cGAS and its signaling adaptor STING comprise one important pathway that triggers DNA-activated innate immunity. We recently discovered that human cells have a second, potent, DNA-activated antiviral pathway that is independent of cGAS-STING. The sensor of this STING-independent DNA sensing pathway (SIDSP) is the DNA-dependent protein kinase (DNA-PK). This raises the possibility of designing synthetic agonists that trigger the SIDSP and stimulate potent responses. We describe the unique features of these agonists in this application.
In one aspect, the disclosure provides polynucleotide constructs, comprising:
(a) a first polynucleotide at least 35 nucleotides in length, wherein the first polynucleotide has a 5′ end and a 3′ end;
(b) a second polynucleotide at least 35 nucleotides in length, wherein the first polynucleotide has a 5′ end and a 3′ end;
wherein the first polynucleotide and the second polynucleotide are base-paired to form a double-stranded region of the polynucleotide construct, wherein the double-stranded region is at least 25 contiguous base pairs in length. In one embodiment, one or more of the first polynucleotide, and the second polynucleotide comprise a single stranded region at the 5′ end and/or the 3′ end.
In another aspect, the disclosure provides polynucleotides comprising a nucleic acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NOS:1-30, 33-36, 41-42, 44-45, 51, 53, 55, 59, 61, 81, 83, 85, 87, 89, 92, 105-108, 125-132, 135-140, 145-147, 149, 151, 154-165, 168-169, 174-181, 184-185, 190-191, 196-199, 206-207, 214-217, 220-221, and 224-22S, wherein (a) underlined residues are unpaired overhangs, (b) asterisks between bases denote individual phosphorothioate (PT) linkages, and (c) abasic residues are noted in brackets.
In a further aspect, the disclosure provides methods for treating autoimmune disorders, infections (such as viral infections), and tumors, comprising administering to a subject in need thereof an amount effective to treat the autoimmune disorders, infections (such as viral infections), and tumors, of the polynucleotide construct or pharmaceutical composition of any embodiment or combination of embodiments herein.
In another aspect, the disclosure provides methods for stimulating an immune response, comprising administering to a subject in need thereof an amount effective to stimulate an immune response in a subject in need thereof of the polynucleotide construct or pharmaceutical composition of any embodiment or combination of embodiments herein.
In one aspect, the disclosure provides kits comprising
(a) a first polynucleotide at least 35 nucleotides in length, wherein the first polynucleotide has a 5′ end and a 3′ end;
(b) a second polynucleotide at least 35 nucleotides in length, wherein the first polynucleotide has a 5′ end and a 3′ end;
wherein the first polynucleotide and the second polynucleotide are capable of base-pairing to form a double-stranded region of at least 25 contiguous base pairs in length; and
wherein optionally one or both of the first polynucleotide and the second polynucleotide comprise a single stranded region at the 5′ end and/or the 3′ end.
All references cited are herein incorporated by reference in their entirety. Within this application, unless otherwise stated, the techniques utilized may be found in any of several well-known references such as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), Gene Expression Technology (Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego, Calif.), “Guide to Protein Purification” in Methods in Enzymology (M. P. Deutsheer, ed., (1990) Academic Press, Inc.); PCR Protocols: A Guide to Methods and Applications (Innis, et al. 1990. Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual of Basic Technique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.), 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.).
As used herein, the singular forms “a”, “an” and “the” include plural referents unless the context clearly dictates otherwise.
As used herein. “about” means+/−5% of the recited parameter.
All embodiments of any aspect of the disclosure can be used in combination, unless the context clearly dictates otherwise.
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.
In a first aspect, the disclosure provides polynucleotide constructs, comprising:
(a) a first polynucleotide at least 35 nucleotides in length, wherein the first polynucleotide has a 5′ end and a 3′ end;
(b) a second polynucleotide at least 35 nucleotides in length, wherein the first polynucleotide has a 5′ end and a 3′ end;
wherein the first polynucleotide and the second polynucleotide are base-paired to form a double-stranded region of the polynucleotide construct, wherein the double-stranded region is at least 25 contiguous base pairs in length.
In one embodiment, one or both of the first polynucleotide and the second polynucleotide comprise a single stranded region at the 5′ end and/or the 3′ end.
As disclosed herein, the inventors have discovered that the polynucleotide constructs of the disclosure are potent activators of a STING-independent DNA sensing pathway (SIDSP) they recently identified, in which the DNA damage response protein DNA-PK is the sensor of the SIDSP, along with the heat shock protein HSPA8 as a unique SIDSP target. The polynucleotide constructs of the disclosure are also potent activators of the cGAS-STING DNA sensing pathway. These pathways are key components of the innate immune response that is important for antiviral immunity, contributes to specific autoimmune diseases, and mediates important aspects of antitumor immunity. Thus, the polynucleotide constructs of the disclosure can be used, for example, to treat autoimmune disorders, infections (such as viral infections), and tumors.
In one embodiment, the polynucleotide construct comprises the first and second polynucleotides (two prong embodiment). In other embodiments, the construct may comprise a third polynucleotide (three-prong embodiment) or a third polynucleotide and a fourth polynucleotide (four prong embodiment).
In the three prong embodiment, the construct further comprises:
(c) a third polynucleotide at least 35 nucleotides in length, wherein the third polynucleotide has a 5′ end and a 3′ end; wherein
In one embodiment, the third polynucleotide comprises a single stranded region at the 5′ end and/or the 3′ end.
In the four prong embodiment, the construct further comprises
(c) a third polynucleotide at least 35 nucleotides in length, wherein the third polynucleotide has a 5′ end and a 3′ end; and
(d) a fourth polynucleotide at least 35 nucleotides in length, wherein the fourth polynucleotide has a 5′ end and a 3′ end, wherein
In one embodiment, the third polynucleotide and/or the fourth polynucleotide may comprise a single stranded region at the 5′ end and/or the 3′ end.
The polynucleotides may comprise any residues capable of base-pairing as recited, including but not limited to deoxyadenosine (A), deoxythymidine (T), deoxycytosine (C), deoxyguanine (G), and deoxyuracil (U). Polynucleotides are each at least 35 nucleotides in length and can be of any suitable length. In one non-limiting embodiment, the first and second polynucleotides are polynucleotides and are independently between 35-250 nucleotides in length. The polynucleotides may be the same length, or may differ in length. In a specific embodiment, the polynucleotides are the same length.
The polynucleotides are base-paired to form one or more double-stranded regions of the polynucleotide construct, wherein the double-stranded region is at least 25 contiguous base pairs in length. In one embodiment, the double-stranded regions are perfectly double-stranded (i.e. no mismatches); in another embodiment, the double-stranded regions may include 1 or 2 mismatches.
The double-stranded regions may be of any suitable length. In one embodiment, the double stranded regions are between 25 contiguous base pairs in length and 200 contiguous base pairs in length. In various further embodiments, the double stranded regions are between 25 contiguous base pairs in length and 175 contiguous base pairs in length, 25 contiguous base pairs in length and 150 contiguous base pairs in length, 25 contiguous base pairs in length and 125 contiguous base pairs in length, 25 contiguous base pairs in length and 100 contiguous base pairs in length, 25 contiguous base pairs in length and 80 contiguous base pairs in length, 35 contiguous base pairs in length and 200 contiguous base pairs in length, 35 contiguous base pairs in length and 175 contiguous base pairs in length, 35 contiguous base pairs in length and 150 contiguous base pairs in length, 35 contiguous base pairs in length and 125 contiguous base pairs in length, 35 contiguous base pairs in length and 100 contiguous base pairs in length, 35 contiguous base pairs in length and 80 contiguous base pairs in length, 45 contiguous base pairs in length and 200 contiguous base pairs in length, 45 contiguous base pairs in length and 175 contiguous base pairs in length, 45 contiguous base pairs in length and 150 contiguous base pairs in length, 45 contiguous base pairs in length and 125 contiguous base pairs in length, 45 contiguous base pairs in length and 100 contiguous base pairs in length, 45 contiguous base pairs in length and 80 contiguous base pairs in length. In specific embodiments, the double stranded regions are at least 55 contiguous base pairs, at least 60 contiguous base pairs, or at least 65 contiguous base pairs. In other specific embodiments, the double stranded regions are between 55 contiguous base pairs in length and 75 contiguous base pairs in length, between 60 contiguous base pairs in length and 75 contiguous base pairs in length, between 55 contiguous base pairs in length and 70 contiguous base pairs in length, between 60 contiguous base pairs in length and 70 contiguous base pairs in length, or about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 contiguous base pairs in length. In other embodiments, the double stranded region is between 30 contiguous base pairs in length and 50 contiguous base pairs in length for three prong and four prong embodiments.
The double stranded regions may comprise any sequence of residues suitable for an intended purpose. In one embodiment, the double stranded regions have a GC nucleotide content of at least 10%, 20%, 30%, 40%, 50%, or more. In another embodiment, the double stranded regions have a GC nucleotide content of 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0%. As shown in the examples that follow, constructs with a low GC content in the double stranded region exhibit particularly good SIDSP stimulatory activity.
In the polynucleotide constructs of the disclosure, one or both of the first polynucleotide and the second polynucleotide may comprise a single stranded region at the 5′ end and/or the 3′ end. In various embodiments:
Similarly, the third and fourth polynucleotides, when present, may comprise a single stranded region at their 5′ ends and/or their 3′ ends.
In a specific embodiment, both the first polynucleotide and the second polynucleotide comprise a single stranded region at each of the 5′ end and the 3′ end, and wherein, when present, both the third polynucleotide and the fourth polynucleotide comprise a single stranded region at each of the 5′ end and the 3′ end.
Any suitable length of single stranded region may be present in the polynucleotide constructs. In various embodiments, each single stranded region may independently be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 nucleotides. In various embodiments, each single stranded region may be between 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-12, 7-11, 7-10, 7-9, 7-8, 8-12, 8-11, 8-10, 8-9, 9-12, 9-11, 9-10, 10-12, 10-11, or 11-12 nucleotides in length. Each single stranded region may be the same length, or the lengths of different single stranded regions may differ. In one embodiment, each single stranded region is the same length. In one specific embodiment, each single stranded region is between 3 nucleotides and 7 nucleotides in length, or 3, 4, 5, 6, or 7 nucleotides in length. In another specific embodiment, each single stranded region is between 3 nucleotides and 5 nucleotides in length, or 3, 4, or 5 nucleotides in length.
Any suitable nucleotide composition of the single stranded regions may be present in the polynucleotide constructs. In one embodiment, each single stranded region comprises at least one pyrimidine. In another embodiment, the single stranded regions include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 pyrimidines. In a further embodiment, each single stranded region comprises at least one cytosine. In another embodiment, each single stranded region includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 deoxycytosines.
In one specific embodiment, each single stranded region comprises all pyrimidines. In another specific embodiment, each single stranded region comprises all deoxycytosines.
In another embodiment, one or more of the single stranded regions one or more deoxyuracil residues. As described in the examples that follow, such deoxyuracil-containing polynucleotides can be treated with, for example, uracil DNA glycosylase (UDG), to produce abasic sites where deoxyuracil residues had been present. Thus, such deoxyuracil-containing polynucleotides may be used, for example, as intermediates to generate polynucleotides containing one or more abasic sites. Such abasic-site containing polynucleotides are shown in the examples as potent activators of a SIDSP. In one embodiment, 2, 3, 4, or more of each single stranded region comprise one or more deoxyuracil residue, or each single stranded region comprise one or more deoxyuracil residue. In another embodiment, the deoxyuracil residues are present at the residue in the single stranded region that abuts the double stranded region (also referred to herein as the “inner residue” of the single stranded region). In a further embodiment, a deoxyuracil residue is present in the middle of 1, 2, 3, 4, or more of the single stranded regions.
In further specific embodiments, 1, 2, 3, 4, or more of each single stranded region comprise one or more abasic site, or each single stranded region comprise one or more abasic site. As used herein, an “abasic site” is a residue in which the DNA sugar-phosphate backbone is intact, but the sugar is not linked to a DNA base. Specifically, the N-glycosidic bond between the 1′ position of the sugar and the base is cleaved, releasing the base and leaving an abasic site. The resulting sugar can exist in a furanose ring conformation or can undergo a ring-opening reaction to exist in an open-chain free aldehyde or free alcohol conformation. In one embodiment, the abasic site is generated by uracil DNA glycosylase. However, any suitable procedure for generating abasic sites may be used, including but not limited to those disclosed in published PCT application WO 2016/164363 and U.S. Pat. No. 6,586,586, each incorporated by reference herein in its entirety. Non-limiting examples of such procedures are detailed in Table 1.
In one embodiment the abasic site has the structure shown in
In another embodiment, the one or more abasic sites are present at the residue in the single stranded region that abuts the double stranded region (also referred to herein as the “inner residue” of the single stranded region). In another embodiment, an abasic site is present in the middle of 1, 2, 3, 4, or more of the single stranded regions. As described in the examples that follow, abasic residue-containing constructs in which the abasic residues are present at the single stranded/double stranded boundary or in the middle of the single stranded regions provide the greatest effect on SIDSP activity.
The polynucleotide constructs may include phosphorothioate linkages between nucleotides at one or more boundaries of the single stranded region and the double stranded region. The “boundaries” are the linkage between the terminal residue in the double-stranded region, and the first residue in the single stranded region of the polynucleotides. Thus, there are four such boundaries in the two prong embodiment, six such boundaries in the three prong embodiment, and eight such boundaries in the four prong embodiment, in various embodiments, the polynucleotide constructs include phosphorothioate linkages between nucleotides at 1, 2, 3, 4, 5, 6, 7, or all 8 boundaries of the single stranded region and the double stranded region. In other embodiments, the polynucleotide constructs may include phosphorothioate linkages between nucleotides in 1, 2, 3, 4, 5, 6, 7, or all 8 of the single stranded regions. In one specific embodiment, the construct comprises one or more phosphorothioate linkages between nucleotides at all boundaries of the single stranded region and the double stranded region. In a further specific embodiment, the construct comprises one or more phosphorothioate linkages between nucleotides within one or more of the single stranded regions. In another specific embodiment, the polynucleotide constructs do not include phosphorothioate-linkages between residues that are each in the double stranded regions. In another specific embodiment, the construct does not include phosphorothioate-linkages between nucleotides in the double-stranded regions other than at the one or more boundaries of the single stranded region and the double stranded region.
In one specific embodiment of the polynucleotide constructs,
(i) the first polynucleotide and the second polynucleotide are of equal length and are base-paired to form a perfectly double stranded region of between 55 contiguous base pairs in length and 75 contiguous base pairs, or between 55 contiguous base pairs in length and 70 contiguous base pairs, or between 60 contiguous base pairs in length and 75 contiguous base pairs, or between 60 contiguous base pairs in length and 70 contiguous base pairs, or 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 contiguous base pairs;
(ii) GC content in the double stranded region is 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0%;
(iii) the first polynucleotide comprises a single stranded region at the 5′ end and the 3′ end, wherein each single stranded region is between 3 nucleotides and 7 nucleotides in length or between 3 nucleotides and 5 nucleotides in length;
(iv) the second polynucleotide comprises a single stranded region at the 5′ end and the 3′ end, wherein each single stranded region is between 3 nucleotides and 7 nucleotides in length or between 3 nucleotides and 5 nucleotides in length;
(v) there are no phosphorothioate-linkages between residues that are each in the double stranded regions;
(v) wherein one or more of the following is true:
In various further embodiments, the polynucleotide constructs comprise a first polynucleotide and a second polynucleotide comprising nucleic acid sequences at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence selected from the following combinations, wherein (a) underlined residues are unpaired overhangs, (b) asterisks between bases denote individual phosphorothioate (PT) linkages, and (c) abasic residues are noted in brackets:
In other embodiments, the polynucleotide constructs comprise a first polynucleotide and a second polynucleotide comprising a nucleic acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence selected from the following combinations, wherein (a) underlined residues are unpaired overhangs, (b) asterisks between bases denote individual phosphorothioate (PT) linkages, and (c) abasic residues are noted in brackets:
In further embodiments, the polynucleotide constructs comprise a first polynucleotide and a second polynucleotide comprising a nucleic acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence selected from the following pairs, wherein (a) underlined residues are unpaired overhangs, (b) asterisks between bases denote individual phosphorothioate (PT) linkages, and (c) abasic residues are noted in brackets:
In another embodiment, the disclosure provides individual polynucleotides of the disclosure (i.e.: first, second, third, or fourth polynucleotides), which can be used, for example, to prepare the polynucleotide constructs of the present disclosure. In one embodiment, the polynucleotides comprise a nucleic acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NOS:1-30, 33-36, 41-42, 44-45, 51, 53, 55, 59, 61, 81, 83, 85, 87, 89, 92, 105-108, 125-132, 135-140, 145-147, 149, 151, 154-165, 168-169, 174-181, 184-185, 190-191, 196-199, 206-207, 214-217, 220-221, and 224-225, wherein (a) underlined residues are unpaired overhangs, (b) asterisks between bases denote individual phosphorothioate (PT) linkages, and (c) abasic residues are noted in brackets.
In a further aspect, the disclosure provides expression vectors comprising the polynucleotides of the disclosure operatively linked to a suitable control sequence. “Expression vector” includes vectors that operatively link a nucleic acid coding region or gene to any control sequences capable of effecting expression of the polynucleotides of the disclosure. “Control sequences” operably linked to the polynucleotides of the disclosure are nucleic acid sequences capable of effecting the expression of the polynucleoides. The control sequences need not be contiguous with the polynuclcotides, 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 polynucleotides and the promoter sequence can still be considered “operably linked” to the polynucleotides. Other such control sequences include, but are not limited to, polyadenylation signals, termination signals, etc. Such expression vectors can be of any type, including but not limited plasmid and viral-based expression vectors. The control sequence used to drive expression of the disclosed polynucleotides 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). In various embodiments, the expression vector may comprise a plasmid, viral-based vector, or any other suitable expression vector.
In another aspect, the disclosure provides host cells that comprise the polynucleotides or expression vectors (i.e.: episomal or chromosomally integrated) disclosed herein, wherein the host cells can be either prokaryotic or eukaryotic. The cells can be transiently or stably engineered to incorporate the expression vector of the disclosure, using techniques including but not limited to bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, polycationic mediated-, or viral mediated transfection.
In another embodiment, the disclosure provides pharmaceutical composition comprising the polynucleotide constructs, polynucleotides, kits, expression vectors, and/or cells of any embodiment or combination of embodiments herein.
The pharmaceutical compositions of the disclosure can be used, for example, in the methods of the disclosure described below. The pharmaceutical composition may comprise in addition to the polynucleotide construct of the disclosure (a) a lyoprotectant; (b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent; (c) 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 isosmotic 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 polynucleotide construct may be the sole active agent in the pharmaceutical composition, or the composition may further comprise one or more other active agents suitable for an intended use.
The polynucleotide construct may be used for any suitable purpose, as described in detail herein. In various non-limiting embodiments, the purpose may include treating autoimmune disorders, infections (such as viral infections), and tumors.
In another aspect, the disclosure provides methods for treating autoimmune disorders, infections (such as bacterial or viral infections), and tumors, comprising administering to a subject in need thereof an amount effective to treat the autoimmune disorders, infections (such as viral infections), and tumors, of the polynucleotide construct or pharmaceutical composition of any embodiment or combination of embodiments of the disclosure.
In another aspect, the disclosure provides methods for treating autoimmune disorders, infections (such as bacterial or viral infections), and tumors, comprising administering to a subject in need thereof an amount effective to stimulate an immune response in a subject in need thereof of the polynucleotide construct or pharmaceutical composition of any embodiment or combination of embodiments of the disclosure. In this embodiment, the polynucleotide construct or pharmaceutical composition may be used, for example, as an adjuvant to stimulate an immune response in a subject in need thereof, such as a subject in need of treatment for autoimmune disorders, infections (such as bacterial or viral infections), and tumors. Such methods may further comprise treating the subject with one or more additional therapeutics as appropriate for the subject, including but not limited to other antiviral therapeutics, anti-tumor therapeutics (including but not limited to checkpoint inhibitors such as PD1, PD-L1, CTLA4 inhibitors, etc.)
As used herein, “treat” or “treating” means accomplishing one or more of the following: (a) reducing the severity of the disease; (b) limiting or preventing development of symptoms, including flares, characteristic of the disease; (c) inhibiting worsening of symptoms characteristic of the disease; (d) limiting or preventing recurrence of the disease or symptoms in subjects that were previously symptomatic for.
The amount of therapeutics(s) administered will depend upon a variety of factors, including, for example, the particular indication being treated, the mode of administration, whether the desired benefit is prophylactic or therapeutic, the severity of the indication being treated and the age and weight of the patient, the bioavailability of the particular therapeutics(s), etc. Dosage amounts will typically be in the range of from about 0.001 mg/kg/day, 0.001 mg/kg/day or 0.01 mg/kg/day to about 100 mg/kg/day, but may be higher or lower, depending upon, among other factors, the activity of the active therapeutic, the bioavailability of the therapeutic, its metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors. Dosage amount and interval may be adjusted individually to provide plasma levels of the therapeutic(s) and/or active metabolite compound(s) which are sufficient to maintain therapeutic or prophylactic effect. For example, the therapeutics may be administered once per week, several times per week (e.g. every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing medical personnel.
In another aspect, the disclosure provides kits comprising (a) a first polynucleotide of any embodiment the disclosure, and (b) a second polynucleotide of any embodiment of the disclosure. In this embodiment, the first and second polynucleotides may be base paired or not base paired. Such kits may optionally comprise (c) a third polynucleotide of any embodiment of the disclosure, and (d) a fourth polynucleotide of any embodiment of the disclosure. In one such embodiment, the kits comprise
(a) a first polynucleotide at least 35 nucleotides in length, wherein the first polynucleotide has a 5′ end and a 3′ end;
(b) a second polynucleotide at least 35 nucleotides in length, wherein the first polynucleotide has a 5′ end and a 3′ end;
wherein the first polynucleotide and the second polynucleotide are capable of base-pairing to form a double-stranded region of at least 25 contiguous base pairs in length; and
wherein optionally one or both of the first polynucleotide and the second polynucleotide comprise a single stranded region at the 5′ end and/or the 3′ end; and
(c) optionally a third polynucleotide at least 35 nucleotides in length, wherein the third polynucleotide has a 5′ end and a 3′ end; wherein
wherein optionally the third polynucleotide may comprise a single stranded region at the 5′ end and/or the 3′; or
(d) optionally, (i) a third polynucleotide at least 35 nucleotides in length, wherein the third polynucleotide has a 5′ end and a 3′ end; and (ii) a fourth polynucleotide at least 35 nucleotides in length, wherein the fourth polynucleotide has a 5′ end and a 3′ end, wherein
wherein the third polynucleotide and/or the fourth polynucleotide may optionally comprise a single stranded region at the 5′ end and/or the 3′ end.
All embodiments of the polynucleotide constructs disclosed above are suitable for use in the kits of the disclosure. As will be understood by those of skill in the art, the first and second polynucleotides in the kit may each be single stranded (i.e.: not base-paired), which can then be combined under appropriate condition to promote base pairing and formation of constructs of the disclosure prior to an intended use. In these embodiments, reference is made to double-stranded regions “when formed” or “capable of being formed”, and will be understood to refer to those portions of the polynucleotides that will base pair to form the constructs of the disclosure. Thus, for example, in one embodiment each double-stranded region when formed is perfectly double-stranded. In another embodiment, each double stranded region when formed is between 25 contiguous base pairs in length and 200 contiguous base pairs in length. In further embodiments, the double stranded region when formed is between 35 contiguous base pairs in length and 110 contiguous base pairs in length, or between 45 contiguous base pairs in length and 80 contiguous base pairs in length for two prong embodiments, or between 30 contiguous base pairs in length and 50 contiguous base pairs in length for three prong and 4 prong embodiments. In one embodiment, the double stranded region when formed is between 45 contiguous base pairs in length and 110 contiguous base pairs in length, or between 45 contiguous base pairs in length and 80 contiguous base pairs in length for two prong embodiments.
In one embodiment, both the first polynucleotide and the second polynucleotide comprise a single stranded region at the 5′ end and/or the 3′ end, and wherein, when present, both the third polynucleotide and the fourth polynucleotide comprise a single stranded region at the 5′ end and/or the 3′ end. In another embodiment, both the first polynucleotide and the second polynucleotide comprise a single stranded region at each of the 5′ end and the 3′ end, and wherein, when present, both the third polynucleotide and the fourth polynucleotide comprise a single stranded region at each of the 5′ end and the 3′ end. In a further embodiment, each single stranded region is between 3 nucleotides and 12 nucleotides in length, or between 3 nucleotides and 7 nucleotides in length, or between 3 nucleotides and 5 nucleotides in length. In other embodiments each single stranded region comprises at least one pyrimidine; each single stranded region comprises all pyrimidines; each single stranded region comprises at least one deoxycytosine; and/or each single stranded region comprises all deoxycytosines.
In various further embodiments, one or more phosphorothioate linkages between nucleotides at 1, 2, 3, 4, 5, 6, 7, or 8 boundaries of the single stranded region and the double stranded region when formed; one or more phosphorothioate linkages between nucleotides at all boundaries of the single stranded region and the double stranded region when formed; one or more phosphorothioate linkages between nucleotides within one or more of the single stranded regions; and/or there are phosphorothioate linkages in the double-stranded regions when formed other than at the one or more boundaries of the single stranded region and the double stranded region, when formed.
In one embodiment, the double stranded regions, when formed, have a GC nucleotide content of at least 10%, 20%, 30%, 40%, 50%, or more. In another embodiment, the double stranded regions when formed have a GC nucleotide content of 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0%.
In various other embodiments, 1, 2, 3, 4, or more of each single stranded region comprise one or more deoxyuracil residue; each single stranded region comprise one or more deoxyuracil residue; the one or more deoxyuracil residues are present at the residue in the single stranded region that abuts the double stranded region; and/or a deoxyuracil residues is present in the middle of 1, 2, 3, 4, or more of the single stranded region.
In other embodiments, 1, 2, 3, 4, or more of each single stranded region comprise one or more abasic site; each single stranded region comprise one or more abasic site; the one or more abasic sites are present at the residue in the single stranded region that abuts the double stranded region, when formed, and/or an abasic site is present in the middle of 1, 2, 3, 4, or more of the single stranded regions.
In a specific embodiment of the kits of the disclosure,
(i) the first polynucleotide and the second polynucleotide are of equal length and, when base-paired, form a perfectly double stranded region of between 55 contiguous base pairs in length and 75 contiguous base pairs, or between 55 contiguous base pairs in length and 70 contiguous base pairs, or between 60 contiguous base pairs in length and 75 contiguous base pairs, or between 60 contiguous base pairs in length and 70 contiguous base pairs, or 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 contiguous base pairs;
(ii) GC; content in the double stranded region, when formed, is 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0%;
(iii) the first polynucleotide comprises a single stranded region at the 5′ end and the 3′ end, wherein each single stranded region is between 3 nucleotides and 7 nucleotides in length or between 3 nucleotides and 5 nucleotides in length;
(iv) the second polynucleotide comprises a single stranded region at the 5′ end and the 3′ end, wherein each single stranded region is between 3 nucleotides and 7 nucleotides in length or between 3 nucleotides and 5 nucleotides in length;
(v) there are no phosphorothioate-linkages between residues that are each in the double stranded regions, when formed;
(v) wherein one or more of the following is true:
In further embodiments, the polynucleotides of the kits comprise a combination or pair of polynucleotides as described above for the constructs of the disclosure.
Phosphorylated HSPA8 (Ser638) is a readout for DNA-PK/SIDSP activation. Phosphorylated IRF3 (Ser386) is a readout for both cGAS/STING and DNA-PK/SIDSP activation. Phosphorylated STING (Ser366) is a readout for cGAS/STING activation.
TTTTTAGGTCCCACCAGGGTCCACCTGGGGTCACCTGGGATTTTT/
TTTTTTCCCAGGTGACCCCAGGTGGACCCTGGTGGGACCTTTTTT
GGGGGAGGTCCCACCAGGGTCCACCTGGGGTCACCTGGGAGGGGG/
GGGGGTCCCAGGTGACCCCAGGTGGACCCTGGTGGGACCTGGGGG
TTTAGGTCCCACCAGGGTCCACCTGGGGTCACCTGGGATTT/
TTTTCCCAGGTGACCCCAGGTGGACCCTGGTGGGACCTTTT
GGGAGGTCCCACCAGGGTCCACCTGGGGTCACCTGGGAGGG/
GGGTCCCAGGTGACCCCAGGTGGACCCTGGTGGGACCTGGG
AAAAAAGGTCCCACCAGGGTCCACCTGGGGTCACCTGGGAAAAAA/
AAAAATCCCAGGTGACCCCAGGTGGACCCTGGTGGGACCTAAAAA
CCCCCAGGTCCCACCAGGGTCCACCTGGGGTCACCTGGGACCCCC/
CCCCCTCCCAGGTGACCCCAGGTGGACCCTGGTGGGACCTCCCCC
AAAAGGTCCCACCAGGGTCCACCTGGGGTCACCTGGGAAAA/
AAATCCCAGGTGACCCCAGGTGGACCCTGGTGGGACCTAAA
CCCAGGTCCCACCAGGGTCCACCTGGGGTCACCTGGGACCC/
CCCTCCCAGGTGACCCCAGGTGGACCCTGGTGGGACCTCCC
From this experiment, we conclude that Y-form DNA substrates activate the SIDSP, with pyrimidine-containing overhangs activating DNA-PK more potently than purine-containing overhangs.
Phosphorylated HSPA8 (Ser638) is a readout for DNA-PK/SIDSP activation. Phosphorylated IRF3 (Ser386) is a readout for both cGAS % STING and DNA-PK/SIDSP activation. Phosphorylated STING (Ser366) is a readout for cGAS/STING activation.
CCCCCAGGTCCCACCAGGGTCCACCTGGGGTCACCTGGGACCCCC/
CCCCCTCCCAGGTGACCCCAGGTGGACCCTGGTGGGACCTCCCCC
CCCCCACACCAGGGTCCACCTGGGGTCACCTGGGACCCCC/
CCCCCTCCCAGGTGACCCCAGGTGGACCCTGGTGTCCCCC
CCCCCAGGGTCCACCTGGGGTCACCTGGGACCCCC/
CCCCCTCCCAGGTGACCCCAGGTGGACCCTCCCCC
CCCCCACACCTGGGGTCACCTGGGACCCCC/
CCCCCTCCCAGGTGACCCCAGGTGTCCCCC
CCCCCTGGGGTCACCTGGGACCCCC/
CCCCCTCCCAGGTGACCCCACCCCC
GGGGGAGGTCCCACCAGGGTCCACCTGGGGTCACCTGGGAGGGGG/
GGGGGTCCCAGGTGACCCCAGGTGGACCCTGGTGGGACCTGGGGG
GGGGGACACCAGGGTCCACCTGGGGTCACCTGGGAGGGGG/
GGGGGTCCCAGGTGACCCCAGGTGGACCCTGGTGTGGGGG
GGGGGAGGGTCCACCTGGGGTCACCTGGGAGGGGG/
GGGGGTCCCAGGTGACCCCAGGTGGACCCTGGGGG
GGGGGACACCTGGGGTCACCTGGGAGGGGG/
GGGGGTCCCAGGTGACCCCCAGGTGTGGGGG
GGGGGTGGGGTCACCTGGGAGGGGG/
GGGGGTCCCAGGTGACCCCAGGGGG
From this experiment, we conclude that Y-form DNA substrates need to be at least 30 nucleotides to activate both cGAS/STING and the DNA-PK/SIDSP.
Phosphorylated HSPA8 (Ser638) is a readout for DNA-PK/SIDSP activation. Phosphorylated IRF3 (Ser386) is a readout for both cGAS/STING and DNA-PK/SIDSP activation. Phosphorylated STING (Ser366) is a readout for cGAS/STING activation. B) Duplex DNAs were annealed and transfected into STING deficient U937 cells at 4 ug/mL final concentration using lipofectamine reagent. After 16 hours, cells were harvested in Trizol and RNA was purified using the Zymo Directzol™ RNA purification kit. cDNA was generated via reverse transcription using the RNA to cDNA EcoDry™ Premix (Takara Biosciences), and quantitative PCR (qPCR) was performed using primers to amplify the IFNB and GAPDH loci. IFNB transcript levels were normalized to those of GAPDH for each condition. Experiment was performed in both biological and technical duplicate.
Sequences tested (
CCCCCAGGTCCCACCAGGGTCCACCTGGGGTCACCTGGGACCCCC/
CCCCCTCCCAGGTGACCCCAGGTGGACCCTGGTGGGACCTCCCCC
CCCCCA*G*G*T*C*C*C*A*C*C*A*G*G*G*T*C*C*A*C*C*T*G*G*G*G*T*C*A*C
CCCCCT*C*C*C*A*G*G*T*G*A*C*C*C*C*A*G*G*T*G*G*A*C*C*C*T*G*G*T*G
CCCCCAGGTCCCACCAG*G*G*T*C*C*A*C*C*T*G*G*G*GTCACCTGGGACCCCC
CCCCCTCCCAGGTGAC*C*C*C*C*A*G*G*T*G*G*A*C*C*CTGGTGGGACCTCCCCC
From this experiment, we conclude that phosphorothioate-containing substrates are potent activators of DNA-PK (as read out by HSPA8 phosphorylation); however, these 35 bp sequences (both phosphodiester-(5C-35) and phosphorothioate (PT)-containing) are insufficient to stimulate the production of IFNB.
CCCCCAGGTCCCACCAGGCTCCACCTGGGGTCACCTGGGACCCCC/
CCCCCTCCCAGGTGACCCCAGGTGGACCCTGGTGGGACCTCCCCC
CCCCCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGTATACCCCC/
CCCCCTATACTGACTAGACATGTACTAGATGTATGTCTAGATAATCACTACCCCC
CCCCCATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGTATCTACCCCC/
CCCCCTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAATCACTAGATCCCCC
CCCCCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGTATCTAGTCC
CCC/
CCCCCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAATCACTAGATACTCC
CCC
CCCCCTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGTATCTAGT
CCCCCATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAATCACTAGATA
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGTA
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAATCACTAGA
CCCCCAATGTCTAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCA
CCCCCATGTCTAGATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAA
From this experiment, we conclude that SIDSP activation requires >=45 bp double-stranded DNA, with the optimal length at 65 base pairs. Also, both blunt and Y-form DNA can activate the SIDSP, though Y-form is a more potent agonist, especially for shorter ligands.
Duplex oligos were designed as Y-form (Y) 65 bp substrates with 5 bp C-containing overhangs, containing either phosphodiester linkages or phosphorothioate (PT) linkages across the double-stranded portion of the ligand. A) Duplex DNAs were annealed and transfected into STING deficient U937 cells at 4 ug/m final concentration using lipofectamine reagent. After 16 hours, cells were harvested in Trizol and RNA was purified using the Zymo Directzol™ RNA purification kit. cDNA was generated via reverse transcription using the RNA to cDNA EcoDry™ Premix (Takara Biosciences), and quantitative PCR (qPCR) was performed using primers to amplify the IFNB and GAPDH loci. IFNB transcript levels were normalized to those of GAPDH for each condition. Experiment was performed in both biological and technical duplicate. B) Duplex DNAs were annealed and transfected into tert-immortalized human foreskin fibroblasts (pretreated with either DMSO or the DNA-PK inhibitor Nu-7441 at 2 uM) at 4 ug/mL final concentration using lipofectamine reagent. After 6 hours, cells were harvested in RIPA buffer and lysates were run through gel electrophoresis. Proteins were transferred to nitrocellulose membranes, blocked with 5% BSA in TBST, washed, and blotted with antibodies to recognize the following phosphorylation events:
Phosphorylated HSPA8 (Ser638) is a readout for DNA-PK/SIDSP activation. Phosphorylated IRF3 (Ser386) is a readout for both cGAS/STING and DNA-PK/SIDSP activation. Phosphorylated STING (Ser366) is a readout for cGAS/STING activation.
Sequences tested (
CCCCCAGGTCCCACCAGGGTCCACCTGGGGTCACCTGGGACCCCC/
CCCCCTCCCAGGTGACCCCAGGTGGACCCTGGTGGGACCTCCCCC
CCCCCA*G*G*T*C*C*C*A*C*C*A*G*G*G*T*C*C*A*C*C*T*G*G*G*G*T*C*A*C*
CCCCCT*C*C*C*A*G*G**G*A*C*C*C*C*A*G*GT*G*G*A*C*C*C*T
CCCCCA*G*T*C*A*G*T*A*T*C*T*A*G*T*G*A*T*T*A*T*C*T*A*G*A*C*A*T*A
CCCCCA*T*A*A*T*C*A*C*T*A*G*A*T*A*C*T*G*A*C*T*A*G*A*C*A*T*G*T*A
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGTA
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
From this experiment, we conclude that while 65 bp Y-form DNA activates the SIDSP, including phosphorothioate linkages across the double-stranded portion of the substrate fails to promote IFNB production via the SIDSP. However, both phosphodiester- and phosphorothioate-containing 65Y substrates activate DNA-PK (strong pHSPA8 that is sensitive to DNA-PK inhibitor).
*in figure, PT linkages are schematized in red, solid lines, while phosphodiester linkages are schematized in black, dashed lines. In the sequences below, PT linkages are denoted by asterisks.
CCCCCAGTCAG*T*A*T*C*T*A*G*T*G*A*T*T*A*T*C*T*A*G*A*C*A*T*A*
CCCCCATAATC*A*C*T*A*G*A*T*A*C*T*G*A*C*T*A*G*A*C*A*T*G*
CCCCCA*G*T*C*A*G*T*A*T*C*T*A*G*T*G*A*T*T*A*T*C*T*A*G*A*C*A
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAA
C*C*C*C*C*A*G*T*C*A*G*T*A*T*C*T*A*G*T*G*A*T*T*A*T*C*T*A*G*
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAA
C*C*C*C*C*A*G*T*C*A*GTATCTAGTGATTATCTAGACATACATCTAGTACATGT
CCCCCATAATC*A*C*T*A*G*A*T*A*C*T*G*A*C*T*A*G*A*C*A*T*G*T
C*C*C*C*C*A*G*T*C*A*GTATCTAGTGATTATCTAGACATACATCTAGTACATGT
C*C*C*C*C*A*T*A*A*T*C*A*C*T*A*G*A*T*A*C*T*G*A*C*P*A*G*A
CCCCCAGTCAG*T*A*T*C*T*A*G*T*G*A*T*T*A*T*C*T*A*G*A*C*A*T*A*
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAA
CCCCCAGTCAG*T*A*T*C*T*A*G*T*G*A*T*T*A*T*C*T*A*G*A*C*A*T*A
C*C*C*C*C*A*T*A*A*T*C*A*C*T*A*G*A*T*A*C*T*G*A*C*T*A*G*
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGTA
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
C*C*C*C*C*A*G*T*C*A*GTATCTAGTGATTATCTAGACATACATCTAGTACATGT
C*C*C*C*C*A*T*A*A*T*CACTAGATACTGACTAGACATGTACTAGATGTATG
C*C*C*C*C*A*G*T*C*A*GTATCTAGTGATTATCTAGACATACATCTAGTACATGT
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
From this experiment, we conclude that the presence of PT linkages in the middle of one or both strands of the substrate inhibits activation of the SIDSP/production of IFNB. The two conditions in which we see “intermediate” phenotypes (IFNB signal between lipo and (1)) are (2) and (8), which include only phosphodiester linkages in the middle of both strands of the substrate.
(where underlined residues represent unpaired overhang regions)
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGTA
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGTA
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGTA
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
CCCCCAAATCACAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
From this experiment, we conclude that potent SIDSP activation can be achieved with either 5′ or 3′ overhangs, or with overhangs only on one side, however, the most potent SIDSP activation is achieved with Y-form DNA ((1)), in which both 5′ and 3′ unpaired overhangs are present on both sides of the substrate.
Substrates Tested:
*in figure, PT linkages are schematized in red, solid lines (or dots), while phosphodiester linkages are schematized in black, dashed lines. In the sequences below, PT linkages are denoted by asterisks.
CCCCCAGTCAG*T*A*T*C*T*A*G*T*G*A*T*T*A*T*C*T*A*G *A*C*A*T*A*
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAA
CCCCCAGTCAGTATCT*A*G*T*G*A*T*T*A*T*C*T*A*G*A*C*A*T*A*C*A*T
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAA
CCCCCAGTCAGTATCTAGTGATTATC*T*A*G*A*C*A*T*A*C*A*T*C*T*A*G*T
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAA
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGTA
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACA*T*C*T*A*GTACATGTCTAGT
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
C*CCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGT
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAG
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGTA
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
C*C*C*C*C*AGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGT
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
C*C*C*CCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCA
CCCCCAAATCACAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
C*CCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGT
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAG
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
C*C*C*C*C*A*G*T*C*A*GTATCTAGTGATTATCTAGACATACATCTAGTACATGT
C*C*C*C*C*A*T*A*A*T*CACTAGATACTGACTAGACATGTACTAGATGTATG
C*C*C*C*C*AGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGT
C*C*C*C*C*A*T*A*A*T*CACTAGATACTGACTAGACATGTACTAGATGTATG
C*C*C*CCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCA
C*C*C*C*C*A*T*A*A*T*CACTAGATACTGACTAGACATGTACTAGATGTATG
C*CCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGT
C*C*C*C*C*A*T*A*A*T*CACTAGATACTGACTAGACATGTACTAGATGTATG
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCA
C*C*C*C*C*A*T*A*A*T*CACTAGATACTGACTAGACATGTACTAGATGTATG
From this experiment, we conclude that PT linkages in the middle of Y-form substrates inhibit the SIDSP, even with just 5 PT linkages (8A:(5)). However, adding PT linkages at the very end (8B:(6)) or at the single-stranded/double-stranded boundary region (8B:(7)) potentiates the SIDSP, even more so than a fully phosphodiester substrate ((I)). Boundary PT linkages also overcome limitations of less ideal substrates on the opposite strand (C:(10)).
Substrates Tested:
*in figure, PT linkages are schematized in red, solid lines (or stars), while phosphodiester linkages are schematized in black, dashed lines. Numbers denote the number of PT linkages in the middle of the substrate, if applicable. In the sequences below, PT linkages are denoted by asterisks.
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAGTA
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACA*T*C*T*A*GTACATGTCTAGT
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACAT*C*T*AGTACATGTCTAGTCA
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATC*TAGTACATGTCTAGTCAGT
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACA*T*C*T*A*GTACATGTCTA
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACAT*C*T*AGTACATGTCTAGT
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATC*TAGTACATGTCTAGTCA
CCCCCATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATAAT
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACA*T*C*T*A*GTACATGTCTAGT
CCCCC*A*TAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATA
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACAT*C*T*AGTACATGTCTAGTCA
CCCCC*A*TAATCACTAGATACTGACTAGACATGTACTAGTACATGTCTAGTCAG
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATC*TAGTACATGTCTAGTCAG
CCCCC*A*TAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATA
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACA*T*C*T*A*GTACATGTCT
CCCCC*A*TAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATA
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACAT*C*T*AGTACATGTCTAG
CCCCC*A*TAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATA
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATC*TAGTACATGTCTAGTC
CCCCC*A*TAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATA
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCA
CCCCC*A*TAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATA
CCCCC*AGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAG
CCCCC*ATAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATA
CCCCCA*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTCAG
CCCCCA*TAATCACTAGATACTGACTAGACATGTACTAGATGTATGTCTAGATA
From this experiment, we conclude that adding boundary PT linkages on either strand increases SIDSP activation, and that having boundary PT linkages on both strands ((14), (15), (16)) gives rise to the most potent ligands, stimulating IFNB production via the SIDSP at levels 25-100×above the same substrate without PT linkages ((1)). Also, having single PT linkages at the boundaries ((15), (16)) is just as potent as having two PT linkages at each boundary ((14)).
CCCCCAGTCAG*T*A*T*C*T*A*G*T*G*A*T*T*A*T*C*T*A*G*A*
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACA*T*C*T*A*GTACA
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGT
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGT
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACA*T*C*T*A*GTACA
From this experiment, we conclude that the potent SIDSP ligands from
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
C*CCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTC
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGT
C*CCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATG
From this experiment, we conclude that the addition of a terminal PT linkage in combination with boundary PT linkages does not enhance SIDSP activation over boundary PT linkages alone.
CAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAGTC
CCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCTAG
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
CCCCCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATG
CCCCCCCCCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTA
From this experiment, we conclude that overhangs of any length increase IFNB production above the corresponding blunt oligo duplex, with 5 bp overhangs being optimal.
B) Duplex oligos were designed as Y-form 65 bp substrates with 5 bp overhangs containing As, Ts, Gs, or Cs. Duplex DNAs were annealed and transfected into STING deficient U937 cells at 4 ug/mL final concentration using lipofectamine reagent. After 16 hours, cells were harvested in Trizol and RNA was purified using the Zymo Directzol™ RNA purification kit. cDNA was generated via reverse transcription using the RNA to cDNA EcoDry™ Premix (Takara Biosciences), and quantitative PCR (qPCR) was performed using primers to amplify the IFNB and GAPDH loci. IFNB transcript levels were normalized to those of GAPDH for each condition. Experiment was performed in biological triplicate and technical duplicate.
TTTTTAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
GGGGGAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
AAAAAAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
From this experiment, we confirm that cytosine-containing overhangs on 65 bp duplexes activate the SIDSP most potently, with some activation resulting from 65 bp duplexes with adenine-containing overhangs.
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGT
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGT
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGT
From this experiment, we conclude that 3- or 4-pronged Y-form substrates do not increase SIDSP activation over that of traditional Y-form duplexes.
CCCCCTGGGTGAACCAGCAGGTGGGCAAAGATGCAGTCCTAGCAATGTAA
/CCCCCAACGGCATAAAGCTTGACGATTACATTGCTAGGACTGCATCTTT
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
CCCCCAGTCAGCCTCCAGTGAGCATCTAGACATGCATCTAGTGCCTGTCC
CCCCCTGAATGAACAATCATCTATAGACAGATACAGTATTATCAATGTAA
From this experiment, we conclude that low GC content in the double-stranded region of our agonist designs more potently stimulates the SIDSP, whereas high GC content decreases their potency.
CCCCCTGGGTGAACCAGCAGGTGGGCAAAGATGCAGTCCTAGCAATGTAA
/CCCCCAACGGCATAAAGCTTGACGATTACATTGCTAGGACTGCATCTTT
CCCCCATATATATATATATATATATATATATATATATATATATATATATA
CCCCCATATATATATATATATATATACATATATATATATATATATATAGA
/CCCCCTATATATATATATATATATATCTATATATATATATATATATATG
CCCCCATATACTATATGATATAGTATATCATATACTATATGATATAGTAT
/CCCCCTATATCATATAGTATATGATATACTATATCATATAGTATATGAT
From this experiment, we conclude that GC content as low as 0% in the double-stranded region of our agonist designs more potently stimulates the SIDSP.
TTTTTAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
UUUUUAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
From this experiment, we conclude that abasic sites in the overhangs, generated from UDG treatment of deoxyuracil-containing DNA, effect highly stimulatory SIDSP agonists. However, deoxyuracil in the overhangs alone does not activate the SIDSP.
UUUUUAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
UCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
CCCCUAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
CCCC[abasic]AGTCAGTATCTAGTGATTATCTAGACATACATCTAGTA
From this experiment, we conclude that abasic sites in the overhangs, generated from UDG treatment of deoxyuracil-containing DNA, effect highly stimulatory SIDSP agonists. Specifically, abasic sites at the single-stranded/double-stranded boundary region effect the most stimulatory activity.
CCCC[abasic]AGTCAGTATCTAGTGATTATCTAGACATACATCTAGTA
CCCC[dSpacer]AGTCAGTATCTAGTGATTATCTAGACATACATCTAGT
From this experiment, we conclude that “natural” abasic sites, generated by Uracil DNA glycosylase (UDG) treatment of deoxyuracils in DNA, are potent agonists of the SIDSP. However, “synthetic” abasic sites, lacking the 1′ OH on the sugar ring, are not potent SIDSP agonists.
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGT
CCCC[abasic]AGTCAGTATCTAGTGATTATCTAGACATACATCTAGTA
TTTT[abasic]AGTCAGTATCTAGTGATTATCTAGACATACATCTAGTA
From this experiment, we conclude that the presence of abasic sites (specifically, those at the single-stranded/double-stranded boundary position) is sufficient to make previously inert 65 bp Y-overhang DNA (e.g. ST overhangs,
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGT
CCCC[abasic]AGTCAGTATCTAGTGATTATCTAGACATACATCTAGTA
CCCC[abasic]AGTCAGTATCTAGTGATTATCTAGACATACATCTAGTA
CCCCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
From this experiment, we conclude having one abasic site in each strand is intermediately potent, but having two abasic sites per strand (one in each overhang—CCCCU) is the most potent.
CCCCUAGTCAGTATCTAGTGATTATAGTCAGTATCTAGTGATTATUCCCC
C
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGT
CCCCUAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
CCCC[abasic]AGTCAGTATCTAGTGATTATCTAGACATACATCTAGTA
CCCC[abasic]AGTCAGTATCTAGTGATTATAGTCAGTATCTAGTGATT
From this experiment, we conclude that having abasic sites in the overhangs of shorter (>=35 bp) SIDSP agonists increase the potency of these agonists to generate an interferon response.
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGT
CCCCUAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
CCCC[abasic]AGTCAGTATCTAGTGATTATCTAGACATACATCTAGTA
CCUCCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGTCT
CC[abasic]CCAGTCAGTATCTAGTGATTATCTAGACATACATCTAGTA
From this experiment, we concluded that abasic sites either at the single-stranded/double-stranded boundary position (“inner”) or in the middle of the single-stranded overhang effect potent SIDSP agonism.
CCCCC*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAGTACATGT
CCCC[abasic]AGTCAGTATCTAGTGATTATCTAGACATACATCTAGTA
CCCC[abasic]*A*GTCAGTATCTAGTGATTATCTAGACATACATCTAG
From this experiment, we concluded that combining phosphorothioate linkages at the single-stranded/double-stranded boundary regions with abasic sites in the boundary does not significantly improve potency to stimulate the SIDSP over individual factors.
This application claims priority to U.S. Provisional Patent Ser. No. 62/984,462 filed Mar. 3, 2020, incorporated by reference herein in its entirety.
This invention was made with government support under Grant No. R21 AI130940, awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2021/020254 | 3/1/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62984462 | Mar 2020 | US |
