Patent Application
20220010302
The present disclosure provides methods for the rapid synthesis of large libraries of spherical nucleic acid (SNA) nanoparticles, their screening for activity, and a machine learning algorithm to analyze the data.
Nanotechnology is beginning to play a major role in developing new therapeutic modalities. Currently, over 100 drugs based upon nanomaterials are currently in clinical trials or approved for therapeutic use1. These structures are promising because of their multifunctionality, which directly relates to their relatively large size and often complex architectures when compared with conventional small molecules or biologics. However, due to this complexity, little attention has been paid to how structural changes inform biological activity. Consider, for example, spherical nucleic acids (SNAs), structures made by arranging short sequences of DNA or RNA around a nanoparticle core. (
Because of these properties, SNAs have shown promise in cancer immunotherapy, where structures with dual functionality can be rapidly prepared from lipids, oligonucleotide adjuvants, and peptide antigens. When delivered to antigen presenting cells (APCs), SNAs activate the immune system and, in a lymphoma model, have shown superior activity compared to the same free antigen and linear oligonucleotides5. However, the modularity of an SNA allows for a large number of possible designs, and the best nanoparticle architectures for maximizing potency and efficacy are unclear.
The present disclosure provides a high throughput method for making different forms of SNAs that are qualitatively similar but structurally distinct, and a mass spectrometry-based screening protocol that allows one to rapidly determine activity for enzyme activation. Collectively, these insights are useful in designing SNA-based therapeutics. Further, in light of the fact that the methodology can be extended to other nanotherapeutics, the present disclosure provides a new way of designing and optimizing nanomedicines for a wide variety of uses.
Accordingly, in some aspects the disclosure provides a method of screening activity of a library of oligonucleotide-functionalized spherical nucleic acids (SNAs) comprising: (a) individually contacting each SNA of the library with a cell, wherein upon contact with the SNA, the cell modulates expression of an enzyme and the amount of enzyme expressed is in proportion to the activity of the SNA; (b) contacting the enzyme expressed in step (a) with a substrate under conditions to transform the substrate to a product, wherein the product has a mass different from the substrate; (c) immobilizing the product and the substrate on a self-assembled monolayer (SAM) on a surface; (d) subjecting the immobilized substrate and product to mass spectrometry to produce a mass spectrum having a product signal and a substrate signal; and (e) correlating the product signal intensity to the substrate signal intensity to determine the extent of product formation and thereby assay the activity of each SNA.
In some embodiments, at least one SNA in the library further comprises an antigen. In further embodiments, the SNAs of the library differ in at least one structural parameter, and the structural parameter is a SNA core property, an antigen property, an oligonucleotide property, or a combination thereof. In still further embodiments, the SNA core property is core diameter, core composition, or a combination thereof. In some embodiments, the core diameter is from about 30 nanometers (nm) to about 150 nm in mean diameter. In further embodiments, the core composition is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), or a combination thereof. In still further embodiments, the antigen property is antigen composition, antigen location, antigen density, or a combination thereof.
In some embodiments, the antigen composition comprises human papillomavirus (HPV) E7 protein or ovalbumin (OVA). In further embodiments, the antigen location is encapsulated within the core or associated with the outer surface of the core. In some embodiments, the antigen is associated with the oligonucleotide that is functionalized on the outer surface of the core.
In some embodiments, the at least two SNAs differ from each other in that one SNA comprises 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× of encapsulated antigen relative to a second SNA. In some embodiments, the at least two SNAs differ from each other in that one SNA has 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the oligonucleotide on the outer surface of the core associated with antigen relative to a second SNA.
In some embodiments, the oligonucleotide property is oligonucleotide sequence, oligonucleotide conjugation chemistry, oligonucleotide conjugation terminus, oligonucleotide backbone, oligonucleotide density, complement density, or a combination thereof. In some embodiments, the at least two SNAs differ from each other in that one SNA comprises a density of oligonucleotide on its outer surface that is 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× that of a density of oligonucleotide on the outer surface of a second SNA. In further embodiments, the at least two SNAs differ from each other in that one SNA has 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the oligonucleotide on the outer surface of the core associated with a complementary oligonucleotide relative to a second SNA.
In further embodiments, the oligonucleotide sequence activates a Toll-like receptor (TLR). In some embodiments, the TLR is TLR-9. In still further embodiments, the oligonucleotide sequence comprises a CpG motif.
In some embodiments, the oligonucleotide conjugation chemistry is a cholesterol-modified oligonucleotide or a 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE)-modified oligonucleotide. In further embodiments, the oligonucleotide conjugation terminus is a 5′ terminus of the oligonucleotide or a 3′ terminus of the oligonucleotide.
In some embodiments, the oligonucleotide backbone is a phosphodiester (PO) backbone or phosphorothioate (PS) backbone.
In some embodiments, each SNA in the library of oligonucleotide-functionalized SNAs is in a separate well of a multiwell plate. In further embodiments, the SAM comprises an immobilizing moiety that interacts with and immobilizes the substrate and the product. In still further embodiments, the immobilizing moiety comprises a maleimide, a thiol, an alkyne, an azide, an amine, or a carboxyl group. In some embodiments, (i) the immobilizing moiety comprises a maleimide and the substrate and the product each comprise an alkane thiol; (ii) the immobilizing moiety comprises an alkane thiol and the substrate and the product each comprise a maleimide; (iii) the immobilizing moiety comprises an alkyne and the substrate and the product each comprise an azide; (iv) the immobilizing moiety comprises an azide and the substrate and the product each comprise an alkyne; (v) the immobilizing moiety comprises an amine and the substrate and the product each comprise a carboxyl group; or (vi) the immobilizing moiety comprises a carboxyl group and the substrate and the product each comprise an amine, so as to form a chemical bond between the immobilizing moiety and the substrate.
In some embodiments, the enzyme is a deacetylase, acetyltransferase, esterase, phosphorylase/kinase, phosphatase, protease, methylase, demethylase, or a DNA or RNA modifying enzyme. In further embodiments, the phosphatase is secreted embryonic alkaline phosphatase (SEAP). In further embodiments, the deacetylase is KDAC8. In still further embodiments, the esterase is cutinase or acetylcholine esterase. In some embodiments, the protease is TEV. In some embodiments, the substrate comprises an acylated peptide and the product comprises a deacylated peptide. In further embodiments, the substrate comprises a deacylated peptide and the product comprises an acylated peptide In some embodiments, the substrate comprises a phosphorylated peptide and the product comprises a dephosphorylated peptide. In further embodiments, the substrate comprises a dephosphorylated peptide and the product comprises a phosphorylated peptide. In some embodiments, the substrate comprises a methylated peptide and the product comprises a demethylated peptide. In some embodiments, the substrate comprises a demethylated peptide and the product comprises a methylated peptide.
A tiny fraction of the nanomedicine design space has been explored, in large part, due to the complexities of such structures and the lack of high-throughput methods to make and analyze them. To address this challenge, the present disclosure provides methods for testing spherical nucleic acids (SNAs), which have over ten different parameters that can be systematically and independently changed to optimize performance. In some embodiments, the performance of the SNAs is optimized in the context of immune cell activation.
By focusing on reasonable parameter ranges, thousands of therapeutic candidate structures have been identified herein that are qualitatively similar but could have significant differences in activity, thereby creating both a synthesis and an analysis challenge. To overcome this daunting task, a high-throughput method for making such structures at picomole (pmol) scale in 384-well format, and a self-assembled monolayer matrix desorption ionization (SAMDI) mass spectrometry assay to rapidly measure innate activation of an enzyme by quantitatively determining enzyme activity. Traditionally, cell-based optical assays are used for measuring such activity, but they are susceptible to optical artifacts due to the absorption and scattering of light associated with the nanostructures that define the SNAs. Through the methods described herein, structure-activity relationships between SNAs and enzyme activation (e.g., immune activation) are identified, which provide new design rules for SNA-based therapeutics (e.g., cancer vaccine candidates). Finally, machine learning was utilized to quantitatively model the enzyme activation of SNAs, and then applied to identify the minimum number of SNAs needed to capture optimum structure-activity relationships for a given library. By doing so, one can reduce the number of nanoparticles to be tested by an order of magnitude, and still get the same information from screening the entire library. These insights and techniques can be generalized to include many other types of nanomedicines and provide a next generation screening tool for therapeutic development.
In some aspects and embodiments of the disclosure, methods are provided for creating immunostimulatory SNAs. Immunostimulatory SNAs consist of three components: the nanoparticle core, the oligonucleotide shell, and the peptide antigen, all of which can be arranged in a variety of configurations5. To establish an appropriate library for high-throughput assessment, focus was placed on eleven properties across these components—core diameter, lipid composition, antigen, antigen location (in core or on complement), antigen density, oligonucleotide sequence, oligonucleotide conjugation chemistry, oligonucleotide conjugation terminus (e.g., 3′ or 5′), oligonucleotide backbone, oligonucleotide density, and complement density (
The oligonucleotide shell serves two roles. It facilitates cellular uptake and serves as the adjuvant, which activates the innate immune system in a sequence-specific and orientation-dependent manner5. In embodiments wherein the SNA is an immunostimulatory SNA, a CpG DNA oligonucleotide (ODN1826), known to activate mouse Toll-like receptor 9 (TLR9), may be utilized. In further embodiments, an inactive control SNA where the CpG motif is inverted to GpC11,12 is also produced. TLR9 is an endosomal protein that recognizes unmethylated CpG oligonucleotides associated with bacteria and viruses13. To explore the importance of backbone composition, linear oligonucleotides with phosphodiester (PO) or phosphorothioate (PS) backbones were synthesized because PS oligonucleotides are known to induce higher immune activation, but SNAs yield PO structures with activities comparable to PS, structures5,14. Thus, in various embodiments, SNAs are contemplated that comprise oligonucleotides having PO or PS backbones. Oligonucleotide-nanoparticle conjugation was studied by investigating structures conjugated with cholesterol or DOPE, which insert into the liposomes and can be chemically attached to the 3′- or 5′ ends of the oligonucleotides. Finally, since oligonucleotide density is known to influence cellular uptake and protein binding of SNAs, various oligonucleotide surface densities are contemplated. These properties and variations thereof are discussed further herein below.
The antigen is not particularly limiting and can be any antigen of interest for a protein of interest. In some embodiments, the peptide antigen is the OVA257-264 peptide from ovalbumin, a well-studied model antigen. In further embodiments, the peptide antigen is Glycoprotein 100 (Gp100), human papillomavirus antigens (including, without limitation, E6 and E7), prostate-specific antigen (PSA), prostate-specific membrane antigen (PSMA), or transmembrane AMPA receptor Regulatory Proteins (TARP). Since peptide properties can vary dramatically as a function of amino acid composition, a peptide antigen from the E7 protein of the human papillomavirus17 is also described and tested herein. To study how the release rate of the antigen influences NF-κB activation, variations in which the antigen is encapsulated within the SNA architecture or hybridized to the oligonucleotide shell through a complementary oligonucleotide are contemplated as well as tested herein. In further embodiments, addition of a complementary oligonucleotide is contemplated for its effects on TLR (e.g., TLR9) stimulation.
To enable screening SNA libraries, a high-throughput assay for rapid and quantitative measurement of cellular responses to SNAs was developed (
Self-Assembled Monolayer (SAM) Surfaces. The present disclosure contemplates the use of self-assembled monolayers as surfaces for assay applications (Mrksich et al., Annu Rev Biophys Biomol Struct 25: 55-78 (1996); Hodneland et al., Langmuir 13: 6001-6003 (1997); Houseman et al., FASEB J 11: A1095-A1095 (1997); Mrksich, Curr Opin Colloid In 2: 83-88 (1997); Mrksich et al., Acs Sym Ser 680: 361-373 (1997); Houseman et al., Mol Biol Cell 9: 430a-430a (1998); Mrksich, Cell Mol Life Sci 54: 653-662 (1998); Houseman et al., Angew Chem Int Ed 38: 782-785 (1999); Li et al., Langmuir 15: 4957-4959 (1999); Yousaf et al., J Am Chem Soc 121: 4286-4287 (1999); Houseman et al., Mol Biol Cell 11: 45a-45a (2000); Luk et al., Langmuir 16: 9604-9608. (2000); Mrksich, Chem Soc Rev 29: 267-273 (2000); Yousaf et al., Angew Chem Int Ed Engl 39: 1943-1946 (2000); Yousaf et al., Biochemistry 39: 1580-1580 (2000); Houseman et al., Biomaterials 22: 943-955 (2001); Kato et al., Biochemistry 40: 8608-8608 (2001); Yeo et al., Chembiochem 2: 590-593 (2001); Yousaf et al., Proc Natl Acad Sci USA 98: 5992-5996. (2001); Yousaf et al., Angew Chem Int Ed Engl 40: 1093-1096 (2001); Hodneland et al., Proc Natl Acad Sci USA 99: 5048-5052 (2002); Houseman et al., Nat Biotechnol 20: 270-274 (2002); Houseman et al., Top Curr Chem 218: 1-44 (2002); Houseman et al., Trends Biotechnol 20: 279-281 (2002); Houseman et al., Chem Biol 9: 443-454 (2002); Kwon et al., J Am Chem Soc 124: 806-812 (2002); Lee et al., Science 295: 1702-1705 (2002); Mrksich, Curr Opin Chem Biol 6: 794-797 (2002); Houseman et al., Langmuir 19: 1522-1531 (2003); Luk et al., Biochemistry 42: 8647-8647 (2003); Yeo et al., Angew Chem Int Ed Engl 42: 3121-3124 (2003); Dillmore et al., Langmuir 20: 7223-7231 (2004); Feng et al., Biochemistry 43: 15811-15821 (2004); Kato et al., J Am Chem Soc 126: 6504-6505 (2004); Min et al., Curr Opin Chem Biol 8: 554-558 (2004); Murphy et al., Langmuir 20: 1026-1030 (2004); Yeo et al., Adv Mater 16: 1352-1356 (2004); Yonzon et al., J Am Chem Soc 126: 12669-12676 (2004); Mrksich, MRS Bull 30: 180-184 (2005); James et al., Cell Motil Cytoskeleton 65: 841-852 (2008)). Previous work utilized a monolayer that presented a peptide against a background of tri(ethylene glycol) groups (Houseman et al., Nat Biotechnol 20: 270-274 (2002)). The peptide was a substrate for Src kinase and the glycol groups prevented non-specific adsorption of protein to the monolayer. Treatment of the monolayer with enzyme and ATP resulted in phosphorylation of the peptide, which was detected by measuring radioactivity from a 32P label or by using an anti-phosphotyrosine antibody with detection by fluorescence scanning or surface plasmon resonance spectroscopy. This example showed that the use of monolayers gave solid-phase assay with exceptional performance. It further indicated that blocking procedures were unnecessary; the signal was 80-fold above background; and that enzyme constants and inhibitor dissociation constants could be measured quantitatively. The monolayers offer the benefits that immobilized ligands are presented in a homogeneous environment and the density of the immobilized ligands can be controlled and made uniform across the entire array (Gawalt et al., J Am Chem Soc 126: 15613-7 (2004)). The monolayers are also compatible with a range of immobilization chemistries (Montavon et al., Nat Chem 4: 45-51 (2012); Ban et al., Nat Chem Biol 8: 769-773 (2012); Li et al., Langmuir 23, 11826-11835 (2007)). In these respects, the monolayers are more effective as substrates in assay applications than is the nitrocellulose material, or even the common use of glass. A significant additional benefit of the monolayer substrates is that they can be analyzed by matrix-assisted laser desorption-ionization mass spectrometry (i.e., SAMDI mass spectrometry) and therefore provide a route to label-free assays of biochemical activities (Su et al., Langmuir 19: 4867-4870 (2003)).
SAMDI mass spectrometry (MS) can be used to detect the mass of a substrate or product. The monolayer is reacted with the substrate and product formed by the enzyme to form a covalent bond with each of the substrate and product on the monolayer. Then, the monolayer is subjected to mass spectrometry, and due to the mass difference between the substrate and product, the MS analysis can assess how much substrate and product are present based upon a single MS analysis. SAMDI can be performed in high throughput using plates having a number of distinct reaction zones (e.g., 1536 or 384) offering a throughput of about 50,000 assays per day, and is quantitative with Z-factors greater than 0.8. The assay can also be used to screen the activity of the antigens of interest in the assays described herein to identify inhibitors or activators of enzymes of interest.
In SAMDI, the monolayer is irradiated with a laser, which results in desorption of the products and substrates through dissociation of a thiolate-gold bond, but with little fragmentation of these molecules. Hence, the resulting spectra are straightforward to interpret. Assays using this SAMDI technique can be used on a range of enzyme activities, and are quantitative, compatible with complex lysates, and adaptable to high throughput formats (Ban et al., Nat Chem Biol 8: 769-773 (2012); Li et al., Langmuir 23: 11826-11835 (2007); Su et al., Langmuir 19: 4867-4870 (2003); Su et al., Angew Chem Int Ed Eng. 41: 4715-4718 (2002); Min et al., Angewandte Chemie 43: 5973-5977 (2004); Min et al., Anal Chem 76: 3923-3929 (2004); Yeo et al., Angew Chem Int Ed Engl 44: 5480-5483 (2005); Marin et al., Angew Chem Int Ed Engl 46: 8796-8798 (2007); Patrie et al., Anal Chem 79: 5878-5887 (2007); Ban et al., Angew Chem Int Ed Eng 47: 3396-3399 (2008); Gurard-Levin et al., Annu Rev Anal Chem (Palo Alto Calif.) 1: 767-800 (2008); Gurard-Levin et al., Biochemistry 47: 6242-6250 (2008); Mrksich, ACS Nano 2: 7-18 (2008); Tsubery et al., Langmuir 24: 5433-5438 (2008); Gurard-Levin et al., Chembiochem 10: 2159-2161 (2009); Liao et al., Chemistry 15, 12303-12309 (2009); Gurard-Levin et al., ACS Chem Biol 5: 863-873 (2010); Kim et al., Nucleic Acids Res 38: e2 (2010); Cai et al., Carbohydr Res 346: 1576-1580 (2011); Gurard-Levin et al., ACS Comb Sci 13: 347-350 (2011); Liao et al., Angew Chem Int Ed Engl 50: 706-708 (2011); Prats-Alfonso et al., Small 8: 2106-2115 (2012); Li et al., Langmuir 29: 294-298 (2013)).
In general, the disclosure provides methods of screening activity of a library of oligonucleotide-functionalized spherical nucleic acids (SNAs) comprising: (a) individually contacting each SNA of the library with a cell, wherein upon contact with the SNA, the cell modulates expression of an enzyme and the amount of enzyme expressed is in proportion to the activity of the SNA; (b) contacting the enzyme expressed in step (a) with a substrate under conditions to transform the substrate to a product, wherein the product has a mass different from the substrate; (c) immobilizing the product and the substrate on a self-assembled monolayer (SAM) on a surface; (d) subjecting the immobilized substrate and product to mass spectrometry to produce a mass spectrum having a product signal and a substrate signal; and (e) correlating the product signal intensity to the substrate signal intensity to determine the extent of product formation and thereby assay the activity of each SNA.
The methods described herein offer several advantages. First, the technology to synthesize SNA libraries did not exist prior to the instant disclosure. The methods disclosed herein enable the use of large numbers of SNAs to understand their behavior and screen libraries to find the best SNAs for a given purpose. Second, current technologies based on optical technologies to detect enzyme activation suffer from artifacts in nanoparticle testing due to the ability of nanoparticles to interact with light. Although it is possible to correct for these artifacts, they introduce many steps that make the screen infeasible. The methods of the disclosure use mass spectrometry to detect enzyme activation (e.g., immune activation) and is not susceptible to these types of artifacts. Finally, the assay can measure activities from enzymes, such as phosphatases, which are impractical to measure in high-throughput from cell lysates using other assay technologies.
Cell-based screening is an increasingly popular tool used in drug discovery. This technology opens up the potential of conducting cell-based screens that use enzyme activity measurements as the readout. This is of significant value because cell-based screens provide more physiologically relevant information about the activity of compounds, potentially leading to better lead compounds in drug discovery efforts.
Surface. The surface can be any material capable of forming a monolayer, e.g., a monolayer of alkanethiols. Particularly, the substrate may be a metal, such as Au, Ag, Pd, Pt, Cu, Zn, Fe, In, Si, Fe2O3, SiO2 or ITO (indium tin oxide) glass. In various embodiments, the disclosure contemplates that a surface useful in the methods described herein comprises Au, Ag, or Cu. In some cases, the surface comprises Au.
In some embodiments, the disclosure contemplates that the substrate and the product comprise a moiety capable of reacting with the SAM so as to be immobilized on the SAM for SAMDI analysis, e.g., a maleimide, a thiol, an alkyne, an azide, an amine, or a carboxyl group. This immobilizing moiety can react with the SAM to form a covalent bond, for example the substrate and product or SAM comprises a maleimide and the other comprises an alkane thiol; the substrate and product or SAM comprises an alkyne and the other comprises an azide; the substrate and product or SAM comprises an amine and the other comprises a carboxyl group.
Intracellular enzyme. The disclosure generally provides methods of assaying activity of an intracellular enzyme. It is contemplated that, in some aspects, the enzyme to be assayed is secreted from the cell. Any enzyme is contemplated for use according to the methods provided herein, including but not limited to a deacetylase, acetyltransferase, esterase, phosphorylase/kinase, phosphatase, protease, methylase, demethylase, or a DNA or RNA modifying enzyme. In some embodiments, the enzyme is a secreted enzyme. Thus, in some embodiments the enzyme is secreted embryonic alkaline phosphatase.
High Throughput Formats for SAMDI. SAMDI for use in the disclosed methods uses a high throughput format based on standard 384 and 1536 microtiter plate formats. This format uses a stainless steel plate in the size of a microtiter plate and having an array of gold-coated islands modified with a monolayer presenting a reactive group that can react with an immobilizing moiety on the substrate and product to form a covalent bond (e.g., maleimide groups) against a background of tri(ethylene glycol) groups. Substrates and products are then immobilized to each of the islands; in various embodiments, in a high throughput screen each island has the same substrate and product produced in response to the antigen of interest for a single SNA, to identify active SNAs for an enzyme. Standard robotic liquid handling equipment can be used to prepare arrays of reactions and to transfer those reaction mixtures to the array plates. The treated plates are incubated (e.g., between 30-60 minutes), washed, and a solution of matrix is applied to the surface. The plate is then loaded into a MALDI-ToF instrument, and each spot is analyzed in an automated fashion in approximately 30 minutes. Resulting data is analyzed using custom written software that can compare the location and magnitude of the peaks in the SAMDI spectra to a reference mass file unique to each set of peptides to look for specific reaction profiles based on characteristic mass shifts (i.e., −42 for deacetylation, +80 for phosphorylation, +14 for methylation). The software presents the data in a manner that can be further analyzed with standard commercial packages (such as Excel) to identify hits in a high throughput screen, or to generate heatmaps of activities. Recent work has demonstrated the screening of 100,000 molecules against the KDAC8 deacetylase (Gurard-Levin et al., ACS Comb Sci 13: 347-350 (2011)).
Modulators/Activators. As described herein, various aspects of the disclosure provide methods for screening activity of a library of oligonucleotide-functionalized spherical nucleic acids (SNAs) comprising: (a) individually contacting each SNA of the library with a cell, wherein upon contact with the SNA, the cell modulates expression of an enzyme and the amount of enzyme expressed is in proportion to the activity of the SNA; (b) contacting the enzyme expressed in step (a) with a substrate under conditions to transform the substrate to a product, wherein the product has a mass different from the substrate; (c) immobilizing the product and the substrate on a self-assembled monolayer (SAM) on a surface; (d) subjecting the immobilized substrate and product to mass spectrometry to produce a mass spectrum having a product signal and a substrate signal; and (e) correlating the product signal intensity to the substrate signal intensity to determine the extent of product formation and thereby assay the activity of each SNA. In some embodiments, the assay is performed in the presence of one or more potential modulators of the enzyme-substrate binding; subjecting the substrate and product to mass spectrometry to produce a mass spectrum having a product signal and a substrate signal; and binding of the enzyme and the substrate is detected by correlating a signal intensity of the product to a signal intensity of the substrate to determine the extent of product formation and thereby detecting the binding of the enzyme and the substrate in the presence of the one or more potential modulators.
In some embodiments, the modulator is an inhibitor of the enzyme and substrate binding. In further embodiments, the modulator is an activator of the enzyme and substrate binding.
According to the methods of the disclosure, there are several variables that are available for modification when creating a SNA library. In various embodiments, the variables include but are not limited to structural properties (e.g., a SNA core property, an antigen property, an oligonucleotide property, or a combination thereof).
SNA core property. In some embodiments SNAs in a library vary by a property including but not limited to core diameter, core composition, or a combination thereof. In further embodiments, the core diameter is from about 30 nanometers (nm) to about 150 nm in mean diameter. In some embodiments, a plurality of SNAs is produced and the SNAs in the plurality have a mean diameter of less than or equal to about 50 nanometers (e.g., about 5 nanometers to about 50 nanometers, or about 5 nanometers to about 40 nanometers, or about 5 nanometers to about 30 nanometers, or about 5 nanometers to about 20 nanometers, or about 10 nanometers to about 50 nanometers, or about 10 nanometers to about 40 nanometers, or about 10 nanometers to about 30 nanometers, or about 10 nanometers to about 20 nanometers). In further embodiments, the SNAs in the plurality have a mean diameter of less than or equal to about 20 nanometers, or less than or equal to about 25 nanometers, or less than or equal to about 30 nanometers, or less than or equal to about 35 nanometers, or less than or equal to about 40 nanometers, or less than or equal to about 45 nanometers.
In some embodiments, the core composition is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dimyristoyl-sn-phosphatidylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (DOPG), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DPPE), or a combination thereof.
Antigens. In some embodiments, the disclosure contemplates that at least one SNA in the library further comprises an antigen. Variations in the antigen contemplated by the disclosure include antigen composition, antigen location, antigen density, or a combination thereof. In some embodiments, the antigen composition comprises human papillomavirus (HPV) E7 protein or ovalbumin (OVA). In further embodiments, the antigen location is encapsulated within the core or associated with the outer surface of the core. When the antigen is associated with the outer surface of the core, it is also contemplated that in some embodiments the antigen is associated with the oligonucleotide that is functionalized on the outer surface of the core, either through hybridization to an oligonucleotide attached to the core, or through direct attachment to the core.
The disclosure contemplates that, when making a library of SNAs, variation may be introduced in the relative amount of antigen that is encapsulated in one SNA in the library versus another SNA in the library. Thus, in various embodiments, the disclosure contemplates that at least two SNAs differ from each other in that one SNA comprises 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× of encapsulated antigen relative to a second SNA. As used herein, the terms “2×” and “3×” etc. simply mean that one SNA comprises twice as much, or three times as much, etc., antigen relative to a second SNA.
The disclosure further contemplates that, when making a library of SNAs, variation may be introduced in the relative amount of antigen that is present on the outer surface of the SNA. As described herein, antigen may be associated with an oligonucleotide that is attached or associated with a SNA. Accordingly, in further embodiments, the disclosure contemplates that at least two SNAs differ from each other in that one SNA has 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the oligonucleotide on the outer surface of the core associated with antigen relative to a second SNA.
Oligonucleotide properties. In some embodiments, the disclosure contemplates that SNAs in a library vary by a property including but not limited to oligonucleotide sequence, oligonucleotide conjugation chemistry, oligonucleotide conjugation terminus, oligonucleotide backbone, oligonucleotide density, complement density, or a combination thereof.
In some embodiments, the oligonucleotide sequence activates a Toll-like receptor (TLR). In further embodiments, the toll-like receptor is chosen from the group consisting of toll-like receptor 1, toll-like receptor 2, toll-like receptor 3, toll-like receptor 4, toll-like receptor 5, toll-like receptor 6, toll-like receptor 7, toll-like receptor 8, toll-like receptor 9, toll-like receptor 10, toll-like receptor 11, toll-like receptor 12, and toll-like receptor 13. In some embodiments, the TLR is TLR-9. In further embodiments, the oligonucleotide sequence comprises a CpG motif. Synthetic immunostimulatory oligonucleotides that contain CpG motifs that are similar to those found in bacterial DNA stimulate a similar response of the TLR receptors. Therefore immunomodulatory oligonucleotides have various potential therapeutic uses, including treatment of immune deficiency and cancer. Employment of liposomal nanoparticles functionalized with immunomodulatory oligonucleotides will allow for increased preferential uptake and therefore increased therapeutic efficacy. Thus, SNAs of the disclosure, functionalized with stabilized with functional CpG motif-containing nucleic acid, would provide enhanced therapeutic effect.
In some embodiments, the oligonucleotide conjugation chemistry is a cholesterol moiety. In further embodiments, the oligonucleotide conjugation chemistry is a cholesterol-modified oligonucleotide or a 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE)-modified oligonucleotide. In still further embodiments, the oligonucleotide conjugation chemistry is a tocopherol moiety. In additional embodiments, the tocopherol is chosen from the group consisting of alpha-tocopherol, beta-tocopherol, gamma-tocopherol and delta-tocopherol.
Conjugation of an oligonucleotide with, e.g., a cholesterol or tocopherol moiety, is a further variable when creating a library of SNAs. In some embodiments, the oligonucleotide conjugation terminus is a 5′ terminus of the oligonucleotide or a 3′ terminus of the oligonucleotide.
In some embodiments, the oligonucleotide backbone affects the immunostimulatory activity of the SNA. Thus, in some embodiments the disclosure contemplates that the oligonucleotide backbone is a phosphodiester (PO) backbone or phosphorothioate (PS) backbone.
Density of oligonucleotide on the surface of the SNA is another variable that may be utilized when creating a library of SNAs. There are a variety of ways that oligonucleotide may be described. In some embodiments, the oligonucleotide surface density is about 0.5, about 1, or about 2 pmol/cm2 (alternatively referred to herein as 1×, 2× and 4×, respectively)15,16. In some embodiments, the oligonucleotide surface density is at least about 1 pmol/cm2, or at least about 2 pmol/cm2. In further embodiments, the oligonucleotide surface density is approximately 5 pmol/cm2, 10 pmol/cm2,11 pmol/cm2, 12 pmol/cm2, 13 pmol/cm2, 14 pmol/cm2, 15 pmol/cm2,16 pmol/cm2, 17 pmol/cm2, 18 pmol/cm2, 19 pmol/cm2, 20 pmol/cm2, or higher. In further embodiments, the oligonucleotide surface density is at least 2 pmol/cm2, at least 3 pmol/cm2, at least 4 pmol/cm2, at least 5 pmol/cm2, at least 6 pmol/cm2, at least 7 pmol/cm2, at least 8 pmol/cm2, at least 9 pmol/cm2, at least 10 pmol/cm2, at least about 15 pmol/cm2, at least about 19 pmol/cm2, at least about 20 pmol/cm2, at least about 25 pmol/cm2, at least about 30 pmol/cm2, at least about 35 pmol/cm2, at least about 40 pmol/cm2, at least about 45 pmol/cm2, at least about 50 pmol/cm2, at least about 55 pmol/cm2, at least about 60 pmol/cm2, at least about 65 pmol/cm2, at least about 70 pmol/cm2, at least about 75 pmol/cm2, at least about 80 pmol/cm2, at least about 85 pmol/cm2, at least about 90 pmol/cm2, at least about 95 pmol/cm2, at least about 100 pmol/cm2, at least about 125 pmol/cm2, at least about 150 pmol/cm2, at least about 175 pmol/cm2, at least about 200 pmol/cm2, at least about 250 pmol/cm2, at least about 300 pmol/cm2, at least about 350 pmol/cm2, at least about 400 pmol/cm2, at least about 450 pmol/cm2, at least about 500 pmol/cm2, at least about 550 pmol/cm2, at least about 600 pmol/cm2, at least about 650 pmol/cm2, at least about 700 pmol/cm2, at least about 750 pmol/cm2, at least about 800 pmol/cm2, at least about 850 pmol/cm2, at least about 900 pmol/cm2, at least about 950 pmol/cm2, at least about 1000 pmol/cm2 or more. Alternatively, the density of oligonucleotide on the surface of a SNA is measured by the number of oligonucleotides on the surface of the SNA. With respect to the oligonucleotide surface density on the surface of a SNA of the disclosure, it is contemplated that a SNA as described herein comprises from about 1 to about 100 oligonucleotides on its surface. In various embodiments, a SNA comprises from about 10 to about 100, or from 10 to about 90, or from about 10 to about 80, or from about 10 to about 70, or from about 10 to about 60, or from about 10 to about 50, or from about 10 to about 40, or from about 10 to about 30, or from about 10 to about 20 oligonucleotides on its surface. In further embodiments, a SNA comprises at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 oligonucleotides on its surface. In still further alternatives, and as discussed above, density may be referred to herein as 1×, 2×, etc. Thus, in some embodiments, at least two SNAs in the library differ from each other in that one SNA comprises a density of oligonucleotide on its outer surface that is 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, or 10× that of a density of oligonucleotide on the outer surface of a second SNA. In still further alternatives, density is referred to in terms of how much of the oligonucleotide on the surface of the SNA is associated with a complementary oligonucleotide strand. Thus, in some embodiments, the disclosure contemplates that at least two SNAs differ from each other in that one SNA has 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the oligonucleotide on the outer surface of the core associated with a complementary oligonucleotide relative to a second SNA.
The following examples show how the methodology described herein was used to make and screen approximately 1000 (800 of which were unique) SNA architectures. In addition, it is described how machine learning models can be trained to predict immune activation from SNA structural considerations. Significantly, these models provide a ranking of the order of importance of eleven structural parameters and SNA drug concentration.
Materials. DOPE and DOPC were purchased from Avanti Lipids (Alabaster, Ala.).
Phosphoramidites for DNA synthesis were purchased from Glen Research (Sterling, Va.). Peptides were custom ordered from Genscript (Piscataway, N.J.). 2,2′-dipyridyldisulfide was purchased from Sigma Aldrich.
DNA Synthesis. DNA was synthesized with a MerMaid 12 synthesizer. Cholesterol modification was done on the column in the synthesizer. For DOPE-modified oligonucleotides, a thiol modified oligonucleotide was synthesized. DNA sequences are shown in Table 1, below.
X-SP18-SP18-TCC ATG
X-SP18-SP18-TCC ATG
Synthesis of DOPE-SMPB. One mol equivalent of succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB, Thermo-Fisher Scientific, Waltham, Mass.) and 1 mol equivalent of N,N-Diisopropylethylamine was added 1 mL of DOPE as received from Avanti Lipids (25 mg/mL in chloroform). The reaction was incubated for 24 hours at room temperature. The reaction was checked for completion with TLC using 20% methanol in dichloromethane as the mobile phase. Upon disappearance of the DOPE band in TLC, the reaction was washed three times with water and the organic phase was dried under a N2 stream.
DOPE Modification of Oligonucleotides. The thiol modified oligonucleotides was reduced with 200 mM DTT in 100 mM phosphate buffer (pH 8.0) for 2 hours at 40° C. The oligonucleotide was purified away from DTT with NAP-10 columns using water as the mobile phase (GE Healthcare, Chicago, Ill.). The reduced oligonucleotide was immediately reacted with DOPE-SMPB as follows. 50 equivalent of DOPE-SMPB was dissolved in ethanol in the same volume as the oligonucleotide. The two solutions were mixed together and incubated at room temperature for 24-48 hours. The reaction mixture was washed with chloroform three times to remove excess lipid. The interface and the aqueous phase was lyophilized. The reaction yield was determined by denaturing PAGE gels. Typically, yields were greater than 90% and no further cleanup was performed.
Synthesis of liposomes. 25 mg of DOPC in chloroform transferred to a glass vial and dried overnight into a thin film, first under a N2 stream followed by high vacuum. For DOPC-DOPE mixture liposomes, 20% by mol DOPE was added to the 25 mg of DOPC before drying. The lipid film was rehydrated with 1 mL of 1×PBS and vortexed until no more clumps were visible. For encapsulated peptides, the peptide was dissolved into the PBS at 0.1 and 1 mg/mL. The lipid suspensions were frozen in liquid nitrogen and thawed in a bath sonicator with sonication. The freeze thaw was repeated three times. The solution was then extruded through 200, 100, 80 and 50 nm filters. Two filters were used for each extrusion and the solution was passed through these filters 11 times. The liposomes were split into two after the 80 nm extrusion. Half of the solution was saved as the 80 nm liposomes, and the remainder was extruded through the 50 nm filter. The liposomes were dialyzed against 1×PBS overnight to remove non-encapsulated peptide. The liposomes were characterized by DLS for size (Z-average reported) and phosphatidylcholine assay for concentration (Millipore-Sigma, St. Louis, Mo.). DOPE did not interfere with the phosphatidylcholine assay, so it was assumed that the DOPE:DOPC ratio remained the same. The liposome concentrations were calculated from the diameter and the lipid concentration as described in Banga et a133.
Synthesis of complementary oligonucleotides with peptide. The complementary oligonucleotides were reduced with DTT as described above and mixed with 55 equivalents of 2,2′-dipyridyldisulfide in 100 mM phosphate buffer (pH 8.0). The reaction was incubated at 40° C. for 24 hours. The reaction process was monitored by absorption of pyridinethione at 343 nm. Upon completion, the modified oligonucleotide was washed three times with water in a 3K MWCO spin filter. The oligonucleotide was then mixed with 1 equivalent of C-OVA and incubated at 40° C. overnight again. The process was again monitored at 343 nm, and washed with a spin filter as described above.
Duplex formation. The purified peptide-oligonucleotide conjugate and 1 equivalent of the lipid-conjugated oligonucleotide was mixed in duplex buffer (30 mM HEPES (pH 7.4), 100 mM potassium acetate and 2 mM magnesium acetate). The mixture was heated to 65° C. for 10 minutes and slow cooled to room temperature.
SNA Synthesis. Lipid modified oligonucleotides or duplexed were mixed with liposomes in a 384 well plate in 40 μL final volume. The final concentration of lipid-modified oligonucleotide or duplex in each well was 10 μM. The concentration of liposomes was adjusted to accommodate SNAs of various oligonucleotide densities. After mixing, the plate was sealed and incubated at room temperature for 24 hours.
Synthesis of peptide substrate. The CRpY-NH2 peptide substrate was synthesized using standard fluorenylmethoxycarbonyl (Fmoc) solid phase peptide synthesis methods on a Rink-Amide resin. The N-terminus was acetylated. The peptide was purified by reverse phase HPLC on a C-18 column, in a gradient from water to acetonitrile and fractions were checked for the correct mass by MALDI-MS. The peptide was lyophilized and stored as a solid until use.
SAMDI plate and monolayer preparation. Stainless steel plates custom-designed for use in MALDI instruments were cleaned and used to evaporate a 1536-spot pattern of 5 nm Ti (0.02 nm/s), then 35 nm Au (0.05 nm/s), using an aluminum mask. The gold array plates were incubated overnight at 4° C. in an ethanolic solution containing a 1:4 ratio of an asymmetric disulfide terminated with a maleimide group and a tri(ethylene glycol) group and a symmetric disulfide terminated with tri(ethylene glycol) groups, with a 0.5 mM total disulfide concentration. The plates were then rinsed with ethanol, dried, and placed in a solution of 10 mM hexadecyl phosphonic acid in ethanol for 10 min at room temperature. Plates were then rinsed with ethanol and dried and used for the SEAP assay.
SEAP assay. RAW-Blue cells (Invivogen) were cultured as described by the manufacturer. The cells were collected and suspended at 550,000 cells/mL, and 17,000 cells were distributed into 384-well plate culture plates with a Thermo Scientific Multidrop Combi. 10×SNA solutions were added to the cell culture plates with a Tecan liquid handler, then cultured at 37° C. and 5% CO2. After approximately 16 hours, the cell culture plates were centrifuged at 300 crf for 1 minute, then 10 μL of media was transferred to a 384-well reaction plate. Recombinant SEAP (0-1,600 ng/mL) was prepared in media from untreated cells and was added to empty wells, and was used as the standard curve. To minimize free thiols in the media, which compete with the substrate immobilization, 1 μL of 11 mM TCEP in water was added and incubated for 15 minutes at 60° C. to the plates to first reduce cystine to cysteine. The 60° C. incubation also inactivates any potential phosphatases other than SEAP, which is stable at 60° C. Next, 1 μL of 12 mM maleimide was added to react with free cysteines for 1 hour, 37° C. 8 μL of 75 μM CRpY peptide substrate in reaction buffer (300 mM Tris, pH 8.5, 2.5 mM MgCl2) was added to the reaction plate, then incubated for 1 hour at 37° C. 2 μL of 11 mM pridoxal 5′-phosphate hydrate in reaction buffer was added. The 0.75 μL of the reaction solutions were transferred to 1536-spot SAMDI array plates and incubated for 1 hour at 37° C. The plates were rinsed with water and ethanol, then dried with air. Matrix (15 mg/mL 2,4,6-trihydroxyacetophenone in acetone) was applied to the SAMDI plates, where were analyzed by MALDI using an AB Sciex 5800 MALDI TOF/TOF instrument in positive reflector mode. The spectra were analyzed by calculating the area under the curves for the [M+H]+ and [M+Na]+ disulfide peaks corresponding to the substrate and product masses, using custom software. Each SNA subset was tested in two wells (biological replicates) and each sample was tested on two SAMDI spots (technical replicates). Technical replicates with sub-threshold signal-to-noise were excluded from analysis.
Quantitative structure-activity relationship (QSAR) model. QSAR models were trained to predict immune activation from SNA properties. The training data contained 336 SNA rows with 9 property columns for datasets 1 and 2 and 288 SNA rows with 8 property columns for dataset 3. The response vector, also called predicted variable, is the immune activation measured via SEAP concentration. Cross-validation was used, where a sample of data is left out for model testing, to calculate the predictive power Q2 metric:
In this formulation, yi is the immune activation for test SNA i, ŷι is the predicted immune activation,
Statistics. Multi-way ANOVA was performed on each SNA subset, using MATLAB software. Statistical comparisons of paired data were made using the two-tailed Wilcoxon test; unpaired data was compared with a two-tailed t-test. All error bars in figures represent standard error of the mean.
Three subsets of SNAs (OVA encapsulated SNAs, E7 encapsulated SNAs and surface-presented OVA SNAs) are tested herein, representing the key possible combinations of the parameters, with a few synthesis-limited exceptions noted below regarding lipid composition, oligonucleotide surface density, and surface conjugated peptide antigen (see below and
Here, media containing SEAP was mixed with a phosphorylated peptide substrate, captured the substrate and dephosphorylated product on monolayers and analyzed the samples by SAMDI. This platform was chosen for its ability to quantify enzyme activities at high-throughput, without dependence on optical methods of measurement. Optical measurement techniques can be negatively affected by the light scattering and absorbance of the nanoparticles, which are difficult to correct for because of their dependence on nanoparticle properties such as size and concentration. Furthermore, SAMDI requires small sample volumes for analysis, thereby reducing the amount of SNAs, cells, and reagents necessary for evaluation.
First, the immune activation of SNAs was compared to linear PO and PS oligonucleotides. The SNAs induced a broad range of immune activation (
To understand which properties had a significant impact on immune activation, a multifactor analysis of variance (ANOVA) (Table 2) was performed on the encapsulated antigen SNA data subsets, which revealed that after oligonucleotide concentration and oligonucleotide sequence (i.e., active or control), oligonucleotide conjugation chemistry had the greatest impact on immune activation. Cholesterol conjugation resulted in higher levels of immune activation than DOPE conjugation (P=4.8×10−16). However, SNAs with cholesterol-conjugated control oligonucleotides also induced similarly high levels of activation at 1000 nM oligonucleotide concentration (798 and 747 ng/mL SEAP for active and inactive, respectively—
In contrast, SNAs with control oligonucleotides conjugated to DOPE lead to dramatically lower SEAP secretion compared to their cholesterol-conjugated counterparts (P<1×10−16,
<1E−220
Due to the dominant effects of conjugation chemistry, the remaining SNA properties were analyzed separately for SNAs with cholesterol- and DOPE-conjugated oligonucleotides. Interestingly, differences in conjugation terminus were observed with different conjugation chemistries (
Oligonucleotide backbone also influenced immunostimulatory activity of SNAs (Table 2 and
In contrast, SNAs with PO oligonucleotides outperformed their PS counterparts at the highest concentration of 1000 nM. Notably, the activity induced by DOPE-conjugated SNAs with PS oligonucleotides consistently decreased when the oligonucleotide concentration was increased from 100 nM to 1000 nM. The DOPE-modified PS linear oligonucleotide, but not the unmodified version, showed a similar reduction in activity at 1000 nM (
These results lead to the conclusion that DOPE-conjugated oligonucleotides with PS backbones provide an advantage if greater potency is desired. PS backbones have the added benefit of resistance to nuclease degradation in vivo27. However, these results also show that SNAs with oligonucleotides composed of PO backbones can achieve similar levels of activation, though at higher concentrations. While class B CpG oligonucleotides are less effective with PO backbones, using SNAs with PO oligonucleotides may be worth the loss in potency because of the reduction in both toxicity and side effects, since the SNA structure may provide sufficient resistance to nuclease activity.
Surprisingly, there was not a strong or consistent trend showing either the lowest or highest oligonucleotide densities as the most effective designs. In previous studies, SNAs with higher oligonucleotide densities led to higher biological activity, such as uptake and RNAse H mediated degradation of mRNA; however, the nanoparticle designs in these studies were limited to a small parameter space with multiple differences (e.g., gold cores, different core sizes and oligonucleotide densities)15,16. From these observations, it was concluded that the selection of oligonucleotide density based on other considerations, such as stability in vivo, is essential since all densities tested here show approximately equal efficacy.
In both encapsulated subsets, lipid composition generally did not have a significant impact (Table 2), except for in a particular context discussed below. Core diameter was not significant in the encapsulated OVA subset, whereas it had a significant impact with encapsulated E7.
To isolate the effects of encapsulating the peptides, the differences of each SNA variant with and without peptide was investigated. SNAs with identical properties except for the amount of peptide encapsulation were paired, and then the SEAP concentration of the SNA without peptide was subtracted from the SNA with 10× peptide (
These results illustrated that peptide encapsulation can impact the efficacy of SNAs, positively or negatively, and that the impact is dependent on other SNA design properties as well. Unlike oligonucleotides, the physicochemical properties of peptides vary dramatically with sequence, which can affect their interaction with the rest of the SNA structure. For example, one possibility is that the differences in isoelectric points of the peptides, which are 5.7 and 8.8 for the E7 and OVA peptides, respectively, result in different net charges for the peptides, which could affect their interaction with the positively charged liposome core. It was concluded that the interactions between liposomes and peptides have to be taken into account when synthesizing nanomedicines, as they can lead to large shifts in the immune activation of SNAs, especially at high concentrations of peptide encapsulation.
Next, an alternative method for incorporating the antigen into the SNA design was investigated. A cysteine-modified OVA peptide was conjugated to a thiol-modified oligonucleotide complementary to the SNA-conjugated oligonucleotide, the two oligonucleotides were hybridized to form a duplex and SNAs were synthesized that present this duplex on the surface. To separate the impact of the hybridized complement from the conjugated peptide, SNAs with the complement but without the peptide were also synthesized. In this conjugated OVA subset, DOPE-conjugated oligonucleotides were used to prevent the non-specific NF-κB activation by cholesterol-conjugated SNAs described above.
Results showed that SNAs synthesized with this strategy shared some trends with their single-stranded counterparts. It was observed that after oligonucleotide sequence, the most influential property on immune activation was backbone chemistry, with PS backbones outperforming PO versions (
Interestingly, for the SNAs with PS oligonucleotides, the addition of the complement oligonucleotide, either to half or to all of the anchored oligonucleotides, did not change immune activation at concentrations of 100 or 1000 nM, respectively (
Three supervised learning models (linear regression, logistic regression, and non-linear xgboost) were trained to predict immune activation and to evaluate the relationships between SNA properties and confirm their relative impact28,29. The predictive power of the models was quantified with the Q2 statistic, which describes how close the predicted SEAP concentrations are to the measured values. Q2 ranges from −∞ to 1, where 0 indicates no predictive power, equivalent to predicting the mean, and 1 indicates perfect prediction30.
Each model was trained with all combinations of properties (e.g., all pairs, all triplets, etc.) and their Q2 performance was analyzed. As additional properties were added to the models, the Q2 performance increased, plateauing for most models and decreasing in the xgboost model for the surface-presented OVA subset (
For the encapsulated OVA and surface-presented OVA subsets, the Q2 value stops increasing beyond five and four properties, respectively (
Next, it was investigated whether a similar Q2 level is attainable with fewer randomly selected SNA designs. This question is particularly relevant when synthesis and evaluation of full libraries are impractical, but where exploration of a large design space is desired. In that case, one could synthesize a random subset, while capturing most of the trends. To this end, this process was simulated by training an xgboost model on a random selection of SNAs and testing predictions on the remaining, unselected SNAs within the three subsets (
The above examples, when combined with other high throughput approaches pioneered by Anderson and Langer, makes the case for the need to consider the vast possibility of structure-property relationships in designing nanomedicines31,32. Moreover, it shows that properties can be strongly interrelated, and it emphasizes the danger in making global conclusions about one structural consideration being more critical than others. This interdependence and non-linearity are underscored when applying the non-linear machine-learning models, as opposed to linear ones, in predicting the biological response of SNAs. Indeed, to realize rational approaches to vaccinology, this work makes a strong case for the combination of high-throughput experimentation and computational analysis, in determining the structure-activity relationships of nanomedicines in general and SNAs in particular.
This application claims the priority benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application No. 62/657,441, filed Apr. 13, 2018, the disclosure of which is incorporated herein by reference in its entirety.
This invention was made with government support under U54CA199091-01 and U54CA151880-01, each awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2019/027229 | 4/12/2019 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62657441 | Apr 2018 | US |
