Patent Application
20230019867
The present invention relates to a macromolecule-based nanostructure, such as a DNA-based nanostructure, for encapsulating a virus or viral particle, to a composition comprising a virus or viral particle encapsulated by such a macromolecule-based nanostructure according to the present invention, and to a method for encapsulating a virus or viral particle by using such a macromolecule-based nanostructure.
Viral infections cause millions of deaths per year globally, enormous suffering and morbidity, and impose huge drains on societies and economies in health care costs, lost work time, and other less easily measured burdens such as mental health issues associated with loss of parents, children, and care givers or stigmatization. Climate change and global migration are projected to increase the threat of viral outbreaks because vectors spread to regions that so far were too cold for them to survive. The burden of virus infections will further increase due to habitat encroachment by humans, urbanization and megacities with increasing population density, increasing travel not only locally but also far distance, and numerous other drivers of disease emergence (1). Viruses are the pathogen class most likely to adapt to new environmental conditions because of their short generation time and genetic variability allowing rapid evolution (2). For the majority of viral diseases (˜70% of current WHO-listed viruses), no effective treatment is available. The few existing antiviral therapies are almost exclusively targeted to a specific virus and do not allow application against a newly emerging pathogen. In addition, antiviral therapy typically faces the challenge that it must be started very soon after infection to be effective, before the viral load gets too high and caused disease symptoms. Emerging virus threats require a rapid response, but broadly applicable ready-to-use antivirals do not exist.
In this context, it is useful to first consider how current antiviral therapies work. Existing antiviral drugs target either virus-specific proteins, mostly polymerases, or essential virus or cellular structures that enable virus replication and spread. The major targetable steps in a virus replication cycle are (1) virus particles docking to the cell membrane of host cells; (2) uptake into the host cell; (3) release of the virus capsid into the cytoplasm and transport of the viral genome to the replication spot; (4) synthesis of viral nucleic acids and proteins and posttranslational processing of viral proteins; (5) assembly of virus components into new viral particles; (6) release of the newly formed viruses from the infected cell. Most clinically available antivirals are polymerase-inhibitors that are specific for a given viral enzyme. Examples include acyclovir (3), active against herpes simplex and varizella zoster virus; tenofovir, active against hepatitis B virus (HBV) and HIV and sofosbuvir, active against hepatitis C virus (HCV). Examples for drugs targeting different stages of the virus life cycle are: enfuvirtide (4), which inhibits HIV fusion (stage 2); amantadine (5), which inhibits influenza A virus uncoating (stage 3); or the neuraminidase inhibitor oseltamivir (6), which interferes with influenza virus release from host cells (stage 6) (6). These drugs, however, can only act when a virus is replicating or spreading but cannot kill or neutralize it. None of these antivirals is broadly applicable.
Recently, a star-shaped designer DNA nanostructure has been presented as a template to display multiple binding motifs with spatial pattern-recognition properties, which confers sensing and viral inhibitory capabilities for a dengue (DENV) viral surface (Kwon et al.; (56)). While it is stated that the molecular-platform design strategy could be adapted to detect and combat other disease-causing pathogens by generating the requisite ligand patterns on customized DNA nanoarchitectures, it is also stated that a defined DNA nanoarchitecture with precise, multivalent spatial pattern-recognizing properties is required, since without the optimal shape identity to keep the required hairpin interactions from forming, sensing and inhibitory abilities suffer. Thus, this proposal does not appear to provide a generically applicable approach for targeting different or yet uncharacterized virus particles, such as needed in case of the emergence of a new virus, as seen in these days with the Coronavirus. Additionally, the rather open structure of the nanostructures bears the danger that viral capsid proteins may still be able to protrude, to be immunologically active and/or to trigger the viral infection process.
A number of additional oligonucleotide-based nanostructures that contain certain cavities have been developed in the past. CN 110 272 980 describes a core shell structure made from DNA monomers having a trigeminal structure, wherein the generated spherical shaped structure is used for detecting miRNA molecules. US 2018/016569 describes a DNA based nanostructure comprising a cavity, a so called nano cage. WO 2017/049136 describes a cylindrical nucleic acid nano structure, having spherical shape with a cavity comprising a binding sequence for nucleic acids or peptides. WO 2013/148186 describes a wholly nucleic acid core nanostructure with a lipid coating which is used for encapsulating a therapeutic agent. WO 2017/189870 describes nucleic acid nano-structures having different geometrical shapes. Li et al. (57) describe spherical nano structures of DNA. In all these cases, the cavities comprised in these structures are way too small to enable the successful encapsulation of full viruses and/or the structures are too open so that surface proteins of the viruses may still be able to interact with the host's target cell structures.
The past couple of weeks have shown that the genomes of viruses, such as those of Covid-19 frequently present mutations, which may lead to a diminished, or even potentially abolished, success of treatment options, such as vaccinations. Thus, there is a great need for therapeutic interventions that permit the fast adaptation to new emerging developments with respect to, for example, the infectivity of a given virus. None of the approaches mentioned above are modular and flexible enough to enable a fast adaptation of the structures to mutational changes of the viruses.
Thus, while different strategies for the treatment of viral infections have been developed or suggested up to date, there is still a need for the development of a concept of a generic antiviral drug platform for targeting a variety of viral pathogens. In particular, a concept would be highly desirous that does not rely on prior detailed knowledge about genetics and properties of the target virus.
It is an object of the present invention to provide constructs that enable the encapsulation of a virus or viral particle. The solution to that problem, i.e. the use of macromolecular building blocks, such as DNA-based nanostructures, has not yet been taught or suggested by the prior art.
Therefore, in one aspect, the disclosure provides a macromolecule-based nanostructure encasing a cavity with a diameter of at least 20 nm for encapsulating a virus or viral particle.
In a particular embodiment, the disclosure provides a macromolecule-based nanostructure, which is a DNA-based nanostructure.
In another aspect, the disclosure provides a composition comprising a virus or viral particle encapsulated by a macromolecule-based nanostructure according to the present invention.
In yet another aspect, the disclosure provides a method for encapsulating a virus or viral particle, comprising the steps of: providing a macromolecule-based nanostructure according to the present invention, and contacting said macromolecule-based nanostructure with a medium comprising, or suspected to comprise, said virus or viral particle.
The disclosure contemplates all combinations of any one or more of the foregoing aspects and/or embodiments, as well as combinations with any one or more of the embodiments set forth in the detailed description and examples.
Other features, objects, and advantages of the compositions and methods herein will be apparent from the description and drawings, and from the claims.
The small triangles represent the triangular building blocks. (h,k) indicates the location of pentamers within a shell. (Aa) Icosahedral shell encapsulating a virus capsid. (B) Natural icosahedral virus shells. Left, T=1 shell built out of identical subunits (MVM) (22). Right, T=3 shell built out of multiple copies of three different subunits (CCMV) (23). (C) ENRG-MD simulation (38) of an exemplary shell subunit design prototype at the start (left) and end (right) of the simulation. (D and E) Cylindrical model of DNA-origami triangles and the corresponding shells. The sides of the triangles are modified with protrusions (light) and recesses (dark). The arrows indicate shape-complementary sides. For each shell design, one of its 20 icosahedral faces has been displaced (see (A)). a is the bevel angle of the sides, #the number of DNA-origami triangles building the shell (design details see
In this context, it should be noted that with respect to
The present disclosure provides constructs that enable the encapsulation of a virus or viral particle.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains.
The terms “comprising” and “including” are used herein in their open-ended and non-limiting sense unless otherwise noted. With respect to such latter embodiments, the term “comprising” thus includes the narrower term “consisting of”.
The terms “a” and “an” and “the” and similar references in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. For example, the term “a cell” includes a plurality of cells, including mixtures thereof. Where the plural form is used for compounds, salts, and the like, this is taken to mean also a single compound, salt, or the like.
Therefore, in one aspect, the disclosure provides a macromolecule-based nanostructure encasing a cavity with a diameter of at least 20 nm for encapsulating a virus or viral particle.
In the context of the present disclosure, the term “macromolecule-based nanostructure” refers to a nanostructure that is formed by the a set of macromolecules, wherein the macromolecules are selected from DNA, RNA, proteins and hybrids thereof, in particular hybrids selected from DNA-RNA hybrids and DNA-protein hybrids, including DNA-(protein linker)-DNA hybrids.
In the context of the present disclosure, the term “DNA” refers to deoxyribonucleic acid composed of a single-strand of monomeric units called nucleotides, wherein each nucleotide is composed of a nitrogen-containing nucleobase, a 2-deoxyribose sugar moiety, and a phosphate group, wherein the individual nucleotides are linked in the single-strand by a phosphate group linking the OH group in position 5′ of a 2-deoxyribose sugar moiety to the OH group in 3′ of a neighboring 2-deoxyribose sugar moiety. In particular embodiments, the nitrogen-containing nucleobases are independently selected from cytosine [C], guanine [G], adenine [A] and thymine [T]. In particular embodiments, one or more of the nucleobases are non-canonical bases, in particular a non-canonical base selected from the list of: a modified adenosine, in particular N6-carbamoyl-methyladenine or N6-methyadenine; a modified guanine, in particular 7-deazaguanine or 7-methylguanine; a modified cytosine, N4-methylcytosine, 5-carboxylcytosine, 5-formylcytosine, 5-glycosylhydroxymethylcytosine, 5-hydroxycytosine, or 5-methylcytosine; a modified thymidine, in particular α-glutamyl thymidine or α-putrescinyl thymine; a uracil or a modification thereof, in particular uracil, base J, 5-dihydroxypentauracil; or 5-hydroxymethyldeoxyuracil; deoxyarchaeosine and 2,6-diaminopurine. A stretch of a single-strand of DNA may interact with a complementary stretch of DNA by interaction of complementary nucleobases, wherein cytosine and guanine, and adenine and thymine, are complementary to each other, respectively by forming two (NT) and three (G/C) hydrogen bonds between the nucleobases. Two single-strands of DNA may be fully complementary to each other, as in the case of genomic DNA, or may be partially complementary to each other, including situations, where one single-strand of DNA is partially complementary to two or more other single-stranded DNA strands. The interaction of two complementary single-stranded DNA sequences results in the formation of a double-stranded DNA double helix.
As is well known, DNA has evolved in nature as carrier of the genetic information encoding proteins. DNA further includes non-coding regions that include regions having regulatory functions. Thus, any DNA-based application usually critically depends on the specific DNA sequence and is almost always only enabled by naming the specific DNA sequence. In contrast, in the context of the present invention, such coding and/or regulatory functions do not play any role and may or may not be present, since the underlying DNA sequences are solely designed and selected in a way that the desired arrangement of double-helical subunits is formed. Thus, in one embodiment any form of a long single-stranded DNA sequence, whether naturally occurring DNA (such as the DNA of a bacteriophage) or synthetically produced DNA may be selected as template, and a set of short single-stranded DNA sequences may be designed, wherein each sequence is complementary to one or more different parts of the template and thus forms one or more double-helical sections. Collectively, all such double-helical sections created by interaction of the full set of short single-stranded DNA sequences with the template, then form the desired three-dimensional arrangement. Starting from a given single-stranded template sequence, the design of a set of complementary can be set up using known techniques, such as, for example, the methods described for the synthesis of megadalton-scale discrete objects with structurally well-defined 3D shapes (18, 24-35). In particular, iterative design with caDNAno (37) paired with elastic-network-guided molecular dynamics simulations (38) can be used.
In addition to the interaction of complementary nucleobases of different stretches of single-stranded DNA via hydrogen bonds, additional interactions between different DNA strands are possible, including the stacking interactions between the blunt ends of two double-stranded DNA helices (36), thus enabling the design and the formation of complex DNA-based nanostructures via the shape-complementarity of double-helical subunits. Thus, two three-dimensional arrangements formed in accordance with the previous paragraph, may interact with each other by stacking interactions between double-helical subunits present on the two three-dimensional arrangements, including specific interactions between two three-dimensional arrangements having complementary protrusions and recessions (or knobs and holes), as shown, for example, in
In the context of the present disclosure, the term “RNA” refers to ribonucleic acid composed of a single-strand of monomeric units called nucleotides, wherein each nucleotide is composed of a nitrogen-containing nucleobase, a ribose sugar moiety, and a phosphate group, wherein the individual nucleotides are linked in the single-strand by a phosphate group linking the OH group in position 5′ of a ribose sugar moiety to the OH group in 3′ of a neighboring ribose sugar moiety. In particular embodiments, the nitrogen-containing nucleobases are independently selected from cytosine [C], guanine [G], adenine [A] and uracil [U]. In particular embodiments, one or more of the nucleobases are non-canonical bases, in particular a non-canonical base selected from the list of: pseudouridine, ribothymidine, and inosine. Unlike DNA, RNA is most often in a single-stranded form, but the formation of double-stranded forms is possible by interaction of complementary nucleobases, wherein cytosine and guanine, and adenine and uracil, are complementary to each other, respectively by forming two (NU) and three (G/C) hydrogen bonds between the nucleobases. In a particular embodiment, the disclosure provides a macromolecule-based nanostructure, which is a RNA-based nanostructure.
In a particular embodiment, said cavity has a diameter of at least 50 nm, at least 100 nm, at least 150 nm, at least 200 nm or at least 250 nm.
In particular embodiments, said cavity has a diameter of at most 1,000 nm.
In the context of the present invention, the term “diameter” refers to the diameter of the smallest circle that is encompassed by the surface of the macromolecule-based nanostructure. For the sake of clarity, in the case of a macromolecule-based nanostructure in the form of a capsule (or spherocylinder), the diameter is the diameter of the hemispherical ends and/or the diameter of the cylindrical central part.
In a particular embodiment, the macromolecule-based nanostructure has a molecular mass of at least 1 MDa, particularly at least 10 MDa, particularly at least 20 MDa, more particularly at least 30 MDa. In other particular embodiments, the DNA-based nanostructure has a molecular mass of at least 50 MDa, at least 80 MDa, at least 100 MDa, at least 200 MDa, or at least 500 MDa. In particular embodiments, the DNA-based nanostructure has a molecular mass of at most 1,500 MDa.
In particular embodiments, the ratio between the numerical value of the molecular mass of the macromolecule-based nanostructure (in MDa) and the numerical value of the volume of the cavity encased by said macromolecule-based nanostructure (in nm3) is less than 10,000, particularly less than 9,000. In particular embodiments said ratio has a value of between 1,000 and 10,000, particularly between 2,000 and 9,000. For example, in the case of certain octahedral nanostructures, where the molecular mass is about 40 MDa, and where the encased volume is about 113,000 nm3, said ratio is about 2,800.
In particular embodiments, the ratio between the outer surface area of the macromolecule-based nanostructure covered by the macromolecules forming said macromolecule-based nanostructure and the outer surface area not covered by said macromolecules (excluding the area of the opening of a macromolecule-based nanostructure in the form of a shell) is at least 1, in particular at least 2, in particular at least 4, in particular at least 6, in particular at least 8. In other particular embodiments, the ratio is at least 10. In particular embodiments, the ratio is between 1 and 20, in particular between 2 and 18, between 4 and 16, between 6 and 14, and more particularly between 8 and 12. For example, in a case, where the macromolecule-based nanostructure is a shell in the form of a half sphere, only the area of the curved surface, but not that of the opening, i.e. the area of the flat face of the half sphere, is used for calculating said ratio.
In a particular embodiment, the present invention relates to a DNA-based nanostructure.
In a particular embodiment, the DNA-based nanostructure is formed by self-assembling DNA-based building blocks.
In a particular embodiment, each of said self-assembling DNA-based building blocks is formed by a single-stranded DNA template strand and a set of oligonucleotides complementary to said single-stranded DNA template, wherein each of said oligonucleotides is either complementary to one contiguous DNA sequence stretch or to at least two non-contiguous DNA sequence stretches on said single-stranded DNA template.
In a particular embodiment, the molecular weight of each self-assembling DNA-based building block is between 4.5 and 5.5 MDa.
In a particular embodiment, the each self-assembling DNA-based building block comprises between 7,500 and 8,500 base pairs.
In a particular embodiment, the DNA-based nanostructure consists of between 4 and 180 of such self-assembling DNA-based building blocks.
In particular embodiments, said single-stranded DNA template is single-stranded DNA of filamentous bacteriophage, or is derived from single-stranded DNA of filamentous bacteriophage.
In the context of the present invention, the term “filamentous bacteriophage” refers to a type of bacteriophage, or virus of bacteria, which is characterized by its filament-like shape that usually contains a genome of circular single-stranded DNA and infects Gram-negative bacteria. Filamentous phage includes Ff phage, such as M13, f1 and fd1 phage, and Pf1 phage.
In particular embodiments, said single-stranded DNA template has a sequence selected from SEQ ID NO: 1 (M13 8064) and 2 (M13 7249) (see Table 1). In particular embodiments, said single-stranded DNA is circular.
In the context of the present invention, a single-stranded DNA template that is “derived from single-stranded DNA of filamentous bacteriophage” refers to a DNA construct that is derived from a naturally occurring of published DNA sequence of a filamentous bacteriophage by one or more of: (i) opening of the circular structure to a linear sequence; (ii) deletion of one or more nucleotides; (iii) insertion of one or more nucleotides; (iii) substitution of one or more nucleotides; (iv) addition of one or more nucleotides; and (v) modification of one or more nucleotides. While any such variation might have detrimental, or at least rather unpredictable, effects on bacteriophage biology, its infectivity and its ability to propagate, such effects do not play any role in the context of the present invention, since, as already mentioned above, said single-stranded DNA template is only used as naked template without any requirement for having any functional property, and all structural aspects, such as the correct formation the three-dimensional shape of said self-assembling DNA-based building blocks, are implemented by the proper choice of said set of complementary oligonucleotides.
In particular embodiments, said single-stranded DNA template has at least 80%, particularly at least 90%, more particularly at least 95%, sequence identity to the sequence of a naturally occurring or published sequence of a filamentous bacteriophage, in particular to a M13, f1 or fd1 phage, in particular to a sequence selected from SEQ ID NO: 1 (M13 8064) and 2 (M13 7249).
In a particular embodiment, the DNA-based nanostructure is a closed three-dimensional geometric shape, in particular a closed three-dimensional geometric shape selected from a sphere, a spherocylinder, and a polyhedron, in particular a tetrahedron, an octahedron or an icosahedron, formed in situ from said self-assembling DNA-based building blocks in the presence of said virus or viral particle to be encapsulated.
In another particular embodiment, the DNA-based nanostructure is a shell with an opening for accessing said cavity.
In the context of the present invention, the term “shell” refers to a structure that is a part of a closed three-dimensional geometric shape, in particular a closed three-dimensional geometric shape selected from a sphere, a spherocylinder, and a polyhedron, in particular a tetrahedron or an octahedron,
In yet another particular embodiment, the DNA-based nanostructure is a combination of a first shell and a second shell with an opening to access a first and a second inner cavity, respectively, wherein said first and said second inner cavity together form said cavity.
In a particular embodiment, said first and said second shells are connected by at least one linker.
In particular embodiments, said linker is a linker selected from a DNA linker, an RNA linker, a polypeptide linker, a protein linker and a chemical linker.
In the context of the present invention, the term “DNA linker” refers to a linker formed from DNA, wherein the sequence of said DNA linker is not complementary to the DNA of said single-stranded DNA template or to any of said set of oligonucleotides complementary to said single-stranded DNA template, wherein said DNA linker is linked at one terminus to a DNA sequence forming a self-assembling DNA-based building block of said first shell, and at the other terminus to a DNA sequence forming a self-assembling DNA-based building block of said second shell.
In the context of the present invention, the term “polypeptide linker” refers to a linker formed from at least 2, particularly at least 5, at least 10, or at least 20 amino acid residues linked by peptide bonds, wherein said polypeptide has no tertiary or quaternary structure, and wherein said polypeptide linker is linked at one terminus to a DNA sequence forming a self-assembling DNA-based building block of said first shell, and at the other terminus to a DNA sequence forming a self-assembling DNA-based building block of said second shell.
In the context of the present invention, the term “protein linker” refers to a linker formed from at least 20, particularly at least 50, at least 100, at least 200 amino acid residues, at least 500 amino acid residues, or at least 1,000 amino acid residues, particularly less than 1,500 amino acid residues linked by peptide bonds, wherein said polypeptide has tertiary and/or quaternary structure, and wherein said protein linker is linked at one terminus to a DNA sequence forming a self-assembling DNA-based building block of said first shell, and at the other terminus to a DNA sequence forming a self-assembling DNA-based building block of said second shell. In particular embodiments, said protein linker is covalently attached to said DNA sequences. In particular other embodiments, said protein linker is non-covalently attached to said DNA sequences, in particular wherein said protein linker is an antibody-based protein linker, in particular selected from a diabody and a full antibody, including an IgG antibody.
In the context of the present invention, the term “chemical linker” refers to a continuous chain of between 1 and 30 atoms (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 atoms; thus, in the context of the present invention, the term “between” is used so that the borders mentioned are included) in its backbone, i.e. the length of the linker is defined as the shortest connection as measured by the number of atoms or bonds between the two DNA sequences linked by said chemical linker. In the context of the present invention, a chemical linker preferably is an C1-20-alkylene, C1-20-heteroalkylene, C2-20-alkenylene, C2-20-heteroalkenylene, C2-20-alkynylene, C2-20-heteroalkynylene, cycloalkylene, heterocycloalkylene, arylene, heteroarylene, aralkylene, or a heteroaralkylene group, which may optionally be substituted. The linker may contain one or more structural elements such as carboxamide, ester, ether, thioether, disulfide, urea, thiourea, hydrocarbon moieties and the like. The linker may also contain combinations of two or more of these structural elements. Each one of these structural elements may be present in the linker more than once, e.g. twice, three times, four times, five times, or six times. In some embodiments the linker may comprise a disulfide bond. It is understood that the linker has to be attached either in a single step or in two or more subsequent steps to the two DNA sequences linked by said chemical linker. To that end the linker to be will carry two groups, preferably at a proximal and distal end, which can (i) form a covalent bond to a group present in one of the two DNA sequences to be linked, or (ii) which is or can be activated to form a covalent bond with one of the two DNA sequences.
In a particular embodiment, the DNA-based nanostructure is based on an icosahedral structure.
In a particular embodiment, each of said self-assembling DNA-based building blocks is a prismoid.
In the context of the present invention, the term “prismoid” refers to a polyhedron, wherein all vertices lie in two parallel planes.
In particular embodiments, said prismoid is a triangular prismoid. In other embodiments, said prismoid is a rectangular prismoid.
In particular embodiments, the DNA-based nanostructure is based on a mixture of a triangular and a rectangular prismoid.
In a particular embodiment, the present invention relates to a DNA-based nanostructure,
wherein each said triangular, or said rectangular prismoid, is formed by m triangular, or rectangular, respectively, planes, wherein m is an integer independently selected from 4, 5, 6, 7 and 8, in particular independently selected from 5, 6 and 7, more particularly wherein said integer is 6.
wherein the three, or four, respectively, edges of each of said m planes are formed by n parallel stretches of DNA double helices, wherein n is an integer independently selected from 1, 2, 3, 4, 5 and 6 in particular independently selected from 2, 3, 4 and 5, more particularly independently selected from 3 and 4,
wherein each plane is connected to a plane above and/or a plane beyond said plane (i) by stacking interactions between the DNA double helices forming said planes, and (ii) partially by DNA stretches within said single-stranded DNA template and/or said oligonucleotides forming said DNA-based building block bridging at least two of said planes, and
wherein at least two of the three, or four, respectively, side trapezoids comprises a specific pattern of recesses and/or extrusions formed by missing or additional DNA double helical stretches for specific interaction with a complementary pattern on the side trapezoid of another one of said self-assembling DNA-based building blocks.
In a particular embodiment, the average length of each of the n stretches of DNA double helices in the m planes of a triangular, or rectangular, respectively, prismoid is between 80 and 200 base pairs.
In particular embodiments, said triangular prismoid is a triangular frustum. In particular embodiments, said rectangular prismoid is a rectangular frustum.
In the context of the present invention, the term “triangular frustum” refers to a three-dimensional geometric shape in the form of a triangular pyramid, and the term “rectangular frustum” refers to a three-dimensional geometric shape in the form of a rectangular pyramid, where the tip of the pyramid has been removed resulting in a plane on the top parallel to the basis of the pyramid.
In a particular embodiment, for at least part of said self-assembling DNA-based building blocks the length of at least one edge of each of said m planes is decreasing from the first to the mth plane, so that a bevel angle results between planes perpendicular to said first plane and the trapezoid plane formed by said m edges (see
In a particular embodiment, a bevel angle is between 16° and 26°, particularly between 18° and 24°, more particularly between 20° and 22°, most particularly about 20.9°.
In a particular embodiment, said DNA-based nanostructure comprises at least one set of self-assembling DNA-based building blocks, wherein all three, or four, respectively, side trapezoids comprises a specific pattern of recesses and/or extrusions formed by missing or additional DNA double helical stretches for specific interaction with a complementary pattern on the side trapezoid of another one of said self-assembling DNA-based building blocks.
In a particular embodiment, particularly in the case of a DNA-based nanostructure closed three-dimensional geometric shape, all said self-assembling DNA-based building blocks are identical.
In a particular embodiment, said DNA-based nanostructure comprises two or more sets of self-assembling DNA-based building blocks.
In a particular embodiment, said DNA-based nanostructure is rod-shaped.
In particular embodiments, said DNA-based nanostructure comprises two or more sets of self-assembling DNA-based building blocks.
In particular such embodiment, said rod-shaped DNA-based nanostructure comprises at least a first and a second set of self-assembling DNA-based building blocks, wherein said first and set second set differ at least with respect to the bevel angles. In a particular embodiment, at least one set consists of self-assembling DNA-based building blocks exhibiting only two bevel angles. In a particular embodiment, said at least one set consists of rectangular frusta, which comprise a bevel angle on each of two opposing trapezoids.
In a particular embodiment, the side trapezoids forming the rim of said shell, or of said first and second shell, respectively, do not comprises a specific pattern of recesses and/or extrusions formed by missing or additional DNA double helical stretches for specific interaction with a complementary pattern on the side trapezoid of another one of said self-assembling DNA-based building blocks.
In a particular embodiment, said DNA-based nanostructure is a shell selected from
In a particular embodiment, the present invention relates to a DNA-based nanostructure further comprising one or more types of DNA brick constructs, each type of such DNA brick constructs being characterized by one or more interaction sites for specific interaction by edge-to-edge stacking contacts with one or more complementary interaction sites present on the plane of said triangular, or rectangular, respectively, prismoid on the outer surface of said DNA-based nanostructure, wherein said DNA brick constructs cover the free space between the three, or four, respectively, edges of said plane (see
In a particular embodiment, the present invention relates to a DNA-based nanostructure further comprising one or more cross-linkages within one of said triangular, or rectangular, respectively, prismoids, and/or between two of said triangular, or rectangular, respectively, prismoids.
In the context of the present invention, the term “cross-linkage” refers to any permanent or intermittent linkage within one of said triangular, or rectangular, respectively, prismoids, and/or between two of said triangular, or rectangular, respectively, prismoids. Any such linkage may be achieved a priori by linking two of the oligonucleotides being used for forming the self-assembling DNA-based building blocks prior to the assembly, or a priori, e. g. by chemically or photochemically adding linkages between different parts of the three-dimensional nanostructure. Permanent linkages may, for example, be created by photochemically cross-linking T residues appropriately positioned in the structure under formation of covalent cyclobutane pyrimidine dimer (CPD) bonds (41), and intermittent linkages may, for example, be created by photochemically cross-linking the blunt ends of two double-helical subunits between a 3-cyanovinylcarbazole (cnvK) moiety positioned at a first blunt end and a thymine residue (T) positioned at the other blunt end (40).
In a particular embodiment, the present invention relates to a macromolecule-based nanostructure further comprising at least one moiety specifically interacting with said virus or viral particle.
In particular embodiments, said macromolecule-based nanostructure is a DNA-based nanostructure in accordance with the present invention, wherein said at least one moiety is linked to one of said triangular, or rectangular, respectively, prismoids forming the DNA-based nanostructure in a way that said at least one moiety is located on the inside of said DNA-based nanostructure and is pointing into the cavity formed by said DNA-based nanostructure.
In particular embodiments, said at least one moiety is specifically interacting with said virus or viral particle by being able to specifically bind to said virus or virus particle. In particular embodiments, said at least one moiety is an antibody or a binding moiety based on an antibody comprising at least an antigen-binding site of an antibody, in particular at least a VH domain of an antibody or at least a combination of a VH and a VL domain of an antibody.
In particular embodiments, said at least one moiety is specifically interacting with said virus or viral particle by being able to bind to inactivate said virus or virus particle.
In particular embodiments, said macromolecule-based nanostructure comprises, on average, between one and 10 antibody-based binding sites attached to the interior site of the cavity formed by said macromolecule-based nanostructure, in particular between 4 and 10, in particular four, five, six, seven, eight, nine or ten embodiments, antibody-based binding sites.
In particular such embodiments, said antibody-based binding site is a single-chain Fv (scFv) fragment.
In particular such embodiment, wherein said macromolecule-based nanostructure is a oligonucleotide-based nanostructure, said antibody-based binding site, in particular said scFv fragment, is linked to a single-stranded oligonucleotide having a sequence that is complementary to, or otherwise able enter site-specific interactions with, oligonucleotide stretches present in the interior of the cavity formed by said oligonucleotide-based nanostructure.
In another aspect, the disclosure provides a composition comprising a virus or viral particle encapsulated by a macromolecule-based nanostructure according to the present invention.
In particular embodiments, said composition is formed in a process of removing said virus or viral particle from a medium containing said virus or viral particle. In particular other embodiments, said composition is formed in a process of incorporating said virus or viral particle as cargo in said macromolecule-based nanostructure.
In another aspect, the disclosure provides a composition comprising a cargo different from a virus or viral particle, where said cargo, such as a complex macromolecule, is encapsulated by a macromolecule-based nanostructure according to the present invention
In yet another aspect, the disclosure provides a method for encapsulating a virus or viral particle, comprising the steps of: providing a macromolecule-based nanostructure according to the present invention, and contacting said macromolecule-based nanostructure with a medium comprising, or suspected to comprise, said virus or viral particle.
In particular embodiments, said method is for removing said virus or viral particle from said medium. In particular embodiment, said method is for encapsulating said virus or viral particle in order to transport said virus or viral particle.
In yet another aspect, the disclosure provides a method for encapsulating a cargo different from a virus or viral particle, such as a complex macromolecule, comprising the steps of: providing a macromolecule-based nanostructure according to the present invention, and contacting said macromolecule-based nanostructure with a medium comprising, or suspected to comprise, said cargo.
It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.
The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
To the extent possible under the respective patent law, all patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference.
The following Examples illustrates the invention described above, but is not, however, intended to limit the scope of the invention in any way. Other test models known as such to the person skilled in the pertinent art can also determine the beneficial effects of the claimed invention.
We report the successful design of a suite of molecular building blocks inspired by nature that self-assemble into a wide family of virus-sized icosahedral shells. The blocks, constructed from DNA origami, are designed according to symmetry principles that dictate the minimal number of distinct bonds per icosahedral shell, and have programmable lock-and-key interaction patterns and strengths. These programmable shells could provide a novel route to virus deactivation, premised on the concept of trapping whole viruses in pre-designed macromolecular shells for effectively blocking molecular interactions between virus and host cells. The shells form robustly, with few defects and at high yields, as we validated by cryo electron microscopy and electrophoretic mobility shift assays. We used hepatitis B virus core particles to demonstrate the feasibility of our virus-trapping and interaction-blocking concept. Realizing the promise of our novel methods for fighting viral infections holds potential for reducing disease, health care costs, and lowering economic burden caused by currently untreatable viral diseases. In addition, our methods hold particular potential for treating emerging and as-yet undescribed viruses, because our method is not dependent on any particular viral structure or host-virus interaction. We show examples for further expanding the functionality of the shells to create “smart” antigen-responsive agents. In addition to antiviral agents, potential applications of these shells include building compartmentalized systems to use as antigen-carriers for vaccination, drug delivery vehicles, protective storage containers and synthetic organelles.
To develop our antiviral concept we considered how the mammalian immune defense can protect us against invading viral pathogens. On the one hand, neutralizing antibodies can emerge that bind to specific antigens on viruses and then can block infection, for example by inhibiting interactions of the virus with a host-cell receptor, or by binding to a viral capsid in order to inhibit uncoating of the genome. Unfortunately, most of the antibodies developed in our body in response to a pathogen do not have neutralizing activity. In addition, it takes time for the host to produce effective neutralizing antibodies. During this period the virus is replicating unchecked and can spread or even establish persistence and may be transmitted to other individuals. Some viral invaders have even evolved mechanisms to escape antibody neutralization. For example, HIV shields sensitive surface regions by flexible protein chains, and HCV engulfs itself into lipids (7, 8).
Antibodies are much smaller than viruses and generally only cover small patches on the surface of the virus per antibody. On the other hand, mammalian cells also produce interferons and (retro-)viral restriction factors that can inhibit invading viruses, as exemplified by the natural restriction factor TRIM5α, which assembles into a hexagonal shell surrounding entire HIV-1 retroviral capsids (9, 10). The TRIM5α example shows that virus encapsulation in principle could be an effective therapeutic strategy, but the natural TRIM5α lattice is highly specific and cannot be readily modified and adapted to different viruses.
Our route to virus deactivation is premised on re-creating and integrating synthetically the functional aspects of neutralizing antibodies and those of lattice-forming natural restriction factors such as TRIM5α. We envision trapping the viruses within de novo designed macromolecular shells to cover large areas of viral surfaces to effectively block molecular interactions between virus and host cells. We envision shells whose interiors allow modular, highly multivalent attachment of virus-recognizing moieties in virus-commensurate symmetries. The same type of shell “platform” could then be used modularly to target a variety of viruses. Because affinity increases exponentially with number of bonds formed (11), the multivalent attachment allows exploiting avidity to effectively trap viruses in shells functionalized with individually weakly virus-binding molecules. As virus binding molecules to be attached we envision antibodies, antibody fragments or aptamers, all of which can be made in principle without detailed knowledge about the target virus, and none of which need to have any a priori virus-neutralizing function. This is because in our concept the shell material, rather than the moieties directly contacting the virus, will prevent access to viral surfaces. Hence, with our concept any virus binder, including non-neutralizing antibodies, weakly binding aptamers, or other molecules such as heparin that binds a broad variety of different virus classes (12), can be utilized as an interior coating on the shell to create an effective antiviral agent.
Here, we report design principles for creating thick and robust macromolecular shells with user-defined sizes that are capable of accommodating entire viruses. We accomplished the construction of such artificial virus-sized macromolecular shells with high structural quality and high production yield. We have further modified our designs to build defined partial shells that feature openings for engulfing target viruses. We used hepatitis B virus (HBV) core particles to demonstrate the feasibility of our virus-trapping and interaction-blocking concept. This paper also describes routes for further expanding the functionality of the shells to create “smarter” antigen-responsive agents.
Briefly, construction of our macromolecular shells relies on several theoretical concepts. First, self-assembly of molecular complexes, such as virus capsids, proceeds typically without external guidance or energy input. Therefore, all information about the desired target shell must be encoded by the shape of the shell subunits and by the local interactions between them (13). Protein designers have previously succeeded in creating artificial macromolecular cages (14-17) by combining and modifying natural non-viral protein scaffolds that display suitable oligomerization symmetries. However, these artificially designed protein-cages are much smaller than the vast majority of natural viruses and cannot be easily modified, and thus are not suitable for our purposes. DNA nanoengineers previously created a variety of polyhedral structures based on DNA origami methods by first building vertices, programmed to assemble into different wireframe polyhedra by varying the vertex angle (18). Yields were low, however, because flexibility of the vertices limited specificity for the target structure. Subsequently, we built blocks which we sequentially assembled into more rigid vertices and larger higher-order wireframe polyhedra (19). While the yields for the blocks and the resulting polyhedra were now higher, the sequential stepwise assembly was tedious and the resulting wireframe polyhedra were still far too skeletal-like, that is too full of holes to be effective for covering the surfaces of viruses. In this work, we have finally succeeded building artificial DNA-based shells that demonstrate exquisite controllability, virus-commensurate sizes and symmetries, and high yield of assembly, thus providing a powerful platform to interface with large natural macromolecular assemblies such as viruses.
Caspar and Klug elucidated the geometric principles that govern the structure of natural viral capsids in 1962 (20) by mapping a 2D triangulated net of protein positions to a 3D surface with positive curvature, through systematic replacement of 12 six-fold vertices with five-fold vertices. According to Casper and Klug theory-which has been expanded recently (21)—the number of distinct environments occupied by proteins within an icosahedral capsid is described by its triangulation number (T-number), which can be computed by a triangular net projection of the arrangement of pentamers and hexamers within an icosahedral capsid (T=h2+hk+k2,
To accommodate the required local symmetries for icosahedral shells, we created rigid pseudo-symmetric triangular subunits (
Since in our system each triangular edge represents one protein, the Casper and Klug triangulation number now gives the number of unique triangular edges required to build a particular shell. Hence, T=1 and T=3 shells may both be built with a single triangle, with three identical edges for T=1 (
We encoded different triangle designs in DNA sequences (see methods below), produced the corresponding sets of oligonucleotides, and self-assembled the triangle variants in one-pot mixtures according to previously described procedures (39). Gel-electrophoretic folding quality analysis demanded some design iterations to improve triangular subunit assembly yields (
We then successfully assembled the iteratively improved triangle variants into closed shells having expected dimensions, as confirmed by direct imaging with cryo-EM (
To elucidate effects of orientational specificity of subunit-subunit interactions, we varied the bevel angle of the T=1 subunits from the ideal geometry)(α=20.9°). We designed two additional variants of the T=1 triangle whose bevel angles deviated by +5° or −5° from the icosahedral ideal. The decrease or increase of the bevel angle caused the appearance of larger shell-like structures in addition to T=1 shells or octahedra, respectively (
We also successfully constructed octahedron cryo EM maps with and without imposed symmetry (
Low-density gel electrophoretic mobility analysis (
Subunit-exchange experiments with fluorescently labeled subunits revealed that under shell-favoring conditions triangles that are incorporated in closed shells do not exchange with solution (
We envision multiple strategies for using our shell system to block viral surfaces from potential virus-cell interactions. One strategy consists in self-assembling protective shells from triangular building blocks on the surface of viruses. A second strategy is to coordinate and engulf a single virus by two opposing half-shells, while the virus is in the middle. A third strategy consists in swallowing viruses within pre-made shells featuring sufficiently large entry portals, similar to how a pitcher plant captures its prey. We demonstrate the latter two strategies. To accomplish this task, we designed new variants of the triangle subunits to steer self-assembly toward forming user-defined shell fragments, including half octahedron (
To demonstrate the virus trapping capability, we trapped hepatitis B virus core particles (
For the half-octahedral variant, the majority of particles consisted of two half octahedra coordinating a single HBV core particle (
Finally, as a proof-of-concept of the virus surface interaction-inhibiting capacity of our shells, we performed in vitro virus blocking assays with HBV-binding antibodies immobilized on a solid phase mimicking a cell surface (
Our current shell variants add a ˜15 nm thick DNA layer around a trapped virus particle (not counting the internal spacing created by the layer of virus-binders), and it is hard to imagine how cell-virus interactions could still take place through such thick envelopes. However, our current designs do feature several apertures, notably the cavities in the triangular subunits, which may permit residual interactions. As a demonstration for a route toward sealing the cavities in the triangular subunits we built a DNA brick having a triangular cross-section roughly corresponding to the dimensions of the triangular cavity in the shell subunits. We anchored the brick via multiple attachment points to the outer surface of a T=1 shell triangle (
In our virus-trapping experiments (
Viruses can be trapped in, or coordinated by, pre-assembled icosahedral shell segments featuring sufficiently large apertures (
We determined 3D cryo EM maps of octahedral and T=1 half 203 shells with trapped HBV core particles (
To test the capacity of our shells to prevent a trapped virus to undergo interactions with surfaces, we performed in vitro virus blocking assays with HBV-binding antibodies immobilized on a solid surface (
Residual HBV core particles that are bound to the surface are detected via HRP catalyzed production of a colorimetric signal.
In the presence of our virus-engulfing shells (half T=1 shells), virus interactions with the surface were blocked up to 99% (
HBV core particles directly incubated with antibodies, but without any shells present, were negligibly blocked from binding the surface. This finding indicates that the antibodies by themselves do not fully passivate the HBV capsid surface even though they were added at 400-fold excess over HBV particles. However, in contrast, when using the shells functionalized with on average as few as five antibodies, we achieved a virus blocking efficiency of greater than 80%. The blocking was nearly complete (up to 99%) when using more than five antibodies in the shells. The data thus shows that the shell-trapping method can be highly effective even when only a handful of physical interactions are formed between the virus surface and surrounding shell. Our data indicates that the shells, and not the antibodies used for holding the virus inside the shell, shield the virus from its exterior by steric occlusion.
We tested the neutralization capacity of the DNA-origami half octahedron shells using adeno-associated virus serotype 2 (AAV2) (46) virions carrying an enhanced green fluorescent protein (eGFP) expression cassette (59) using both microscopy and flow cytometry (
We quantified the efficacy of virus neutralization by determining the dose response curves for DNA half-shells functionalized with on average 36 anti-AAV2 antibodies per half-shell and free anti-AAV2 antibodies as reference (
As above with the in vitro HBV blocking experiments in
We also investigated whether the DNA-origami half shells without any conjugated antibody had an effect, and we found a low but non-negligible neutralization activity at the highest origami concentration tested (
We have successfully experimentally demonstrated a symmetry-based approach for self-assembly of virus-like shells of various sizes with high yield and fidelity. This capability is enabled by rigid building blocks that correspond to patchy particles (44) featuring specific lock-and-key interactions whose geometry can be designed and realized with sub-nanometer precision. The exquisite programmability of the shell building blocks furthermore allows building defined-size partial shells, such as half-shells or shells with entry portals, which we propose as virus-neutralizing agents for trapping and inactivating viruses. We tested this concept with HBV core particles as a model virus particle, and in our experiments, we achieved up to 99% inactivation by engulfing HBV in a surrounding shell.
In our concept all recognition elements are integrated on the nanoparticle rather than relying on cascaded reactions between multiple diffusing components. This makes our concept insensitive to dilution, which is a crucial requirement for potential future biomedical applications. Beyond the proposed application as virus-neutralizing agents for trapping and inactivating viruses, variants of our shells could also be used as antigen-carriers for vaccination, as DNA or RNA carriers for gene therapy or gene modification, as drug delivery vehicles, as protective storage containers, or to build compartmentalized systems such as synthetic organelles. Our shells cover a wide range of internal cavity dimensions and may also offer opportunities as alternative gene delivery vectors that can accommodate genomic information much larger as those that can be delivered with e.g. adeno associated viruses (AAV) (45, 46), which are popular gene therapy vectors. The nucleic acids to be delivered could be combined with proteins or protein complexes rendering e.g. CRISPR/Cas based gene silencing or gene modification approaches safer and more efficient.
Whereas natural viruses assemble from protein subunits, we constructed icosahedral shells that consist purely of DNA, thereby taking viral construction principles from protein chemistry into the realm of nucleic acids. Nucleic acids are durable, available commercially, and easily functionalized and modified as we also showed in this work. By using nucleic acids to engulf viral pathogens, we have opened up a new realm for developing drugs to combat viral threats. Using nucleic-acid based agents potentially confers the advantage of avoiding neutralization, phagocytosis and degradation by pathways of the innate and adaptive immune system targeting protein structures. Recently, methods to produce DNA-based objects at scale (47) have emerged. These methods may help transform our concept for fighting viral diseases into candidate therapeutics for clinical use.
Methods
1. Self-Assembly of Shell Subunits
All self-assembly experiments were performed in standardized “folding buffers” containing x mM MgCl2 in addition to 5 mM Tris Base, 1 mM EDTA, 5 mM NaCl at pH 8 (FoBx). Single-scaffold-chain DNA origami objects were self-assembled in one-pot folding reactions containing 50 nM scaffold DNA and 200 nM of each staple strand. DNA origami objects containing multiple scaffolds were self-assembled using each scaffold DNA chain at 10 nm concentration and 200 nM of each staple strand. The individual scaffolds were sequence-orthogonal and designed and produced as described previously (48, 49). Folding buffer (FoB20) was used with x=20 mM MgCl2. All reaction mixtures were subjected to thermal annealing ramps as detailed in Table 2 in Tetrad (Bio-Rad) thermal cycling devices. Staple strands were purchased from IDT (Integrated DNA Technologies).
2. Purification of Shell Subunits and Self-Assembly of Shells
All shell subunits were purified using gel purification and, if necessary, concentrated with ultrafiltration (Amicon Ultra 500 μl with 100 kDa molecular weight cutoff) before self-assembling the subunits into shells. Both procedures were performed as previously described (39) with the following alterations: for gel purification, we used 1.5% agarose gels containing 0.5x TBE and 5.5 mM MgCl2. For ultrafiltration, the same filter was filled with gel-purified sample multiple times (about 2-5 times, ˜400 μl every step) in order to increase the concentration of objects that are recovered from the filter. Before putting the filter upside down in a new filter tube we performed two washing steps with 1xFoB5 (˜400 μl) to achieve well-defined buffer conditions for the shell assembly. To assemble the purified (and concentrated) shell subunits into shells we adjusted the subunit and MgCl2 concentrations by adding 1xFoB5 and 1.735 M MgCl2 in suitable amounts. Typical subunit concentrations were in the range of 5 nM and up to 100 nM (for cryo-EM measurements, see Table 3). Typical MgCl2 concentrations for shell self-assembly were in the range of 10-40 mM. Shell self-assembly was performed at 40° C. Reaction times were varied depending on the shell type (see
3. Half Shells and HBV Core Binding
Nine staples on the inside of the triangles were modified with handles with 26 single-stranded bases at the 5′ ends (seq.: ‘GCAGTAGAGTAGGTAGAGATTAGGCA-oligonucleotide’, for design details see
4. Octahedron Oligolysine Stabilization
The octahedral shells were assembled at 35 mM MgCl2 and UV cross-linked as described in (41) for 1 h using the Asahi Spectra Xenon Light source 300W MAX-303. The shells were incubated in a 0.6:1 ratio with a mixture of K10 oligolysine and K10-PEG5K oligolysine (1:1) for 1h at room temperature as similarly described in (42).
5. T=1 Shell Exterior Modification
The T=1 triangle and the triangular brick (
6. Gel Electrophoresis
The size distribution of folding reactions or shell assemblies was investigated using agarose gel electrophoresis. For solutions including only shell subunits, we used 1.5% agarose gels containing 0.5xTBE Buffer (22.25 mM Tris Base, 22.25 mM Boric Acid, 0.5 mM EDTA) and 5.5 mM MgCl2. For solutions including oligomeric assemblies such as shells, an agarose concentration of 0.5% was used. The gel electrophoresis was performed in 0.5xTBE buffers supplemented with the same MgCl2 concentration as the solutions in which the shells were incubated in. For MgCl2 concentration larger than 15 mM, a surrounding ice-water bath was used for cooling the gel. The gel electrophoresis was performed for 1.5 to 2 hours at 90 V bias voltage. The agarose gels were then scanned with a Typhoon FLA 9500 laser scanner (GE Healthcare) with a pixel size of 50 μm/pix.
7. Negative-Staining Tem
Samples were incubated on glow-discharged collodion-supported carbon-coated Cu400 TEM grids (in-house production) for 30 to 120 s depending on structure and MgCl2 concentration. The grids were stained with 2% aqueous uranyl formiate solution containing 25 mM sodium hydroxide. Imaging was performed with magnifications between 10000× to 42000×. T=3 triangles were imaged on a Phillips CM100 equipped with a AMT 4Mpx CCD camera. All other negative staining data was acquired at a FEI Tecnai T12 microscope operated at 120 kV with a Tietz TEMCAM-F416 camera. TEM micrographs were high-pass filtered to remove long-range staining gradients and the contrast was auto-leveled (Adobe Photoshop CS6). To obtain detailed information on individual particles and investigate successful encapsulation negative stain EM tomography was used as a visualization technique. The grids were prepared as described above, and the tilt series acquired with magnifications between 15000× and 30000× using the FEI Tecnai 120. The stage was tilted from −50° to 50° and micrographs were acquired in 2° increments.
All tilt series were subsequently processed with IMOD (50) to acquire tomograms. The micrographs were aligned to each other by calculating a cross correlation of the consecutive tilt series images. The tomogram is subsequently generated using a filtered back-projection. The Gaussian-Filter used a cutoff between 0.25 and 0.5 and a fall-off of 0.035.
8. Cryo Electron Microscopy
The DNA origami concentrations used for preparing the cryo-EM grids are summarized in Table 3. Samples with concentrations higher than 100 nM were applied to glow-discharged C-flat 1.2/1.3 or 2/1 thick grids (Protochip). Samples containing shells with less than 30 nM monomer concentrations were incubated on glow-discharged grids with an ultrathin carbon film supported by a lacey carbon film on a 400 mesh copper grid (Ted Pella). The concentration of all single triangles was increased above 500 nM with PEG precipitation (39). 1 ml of folding reaction (˜50 nM monomer concentration) was mixed with 1 ml of PEG, centrifuged at 21 k rcf for 25 min and re-suspended in 50 to 100 μl 1 xFoB5. The DNA origami triangles used for assembling the shells were all gel purified and concentrated with ultrafiltration as described above before increasing the MgCl2 concentration. Plunge freezing in liquid ethane was performed with a FEI Vitrobot Mark V with a blot time of 1.5 to 2 s, a blot force of −1 and a drain time of 0 s at 22° C. and 95% humidity. The samples with less than 100 nM monomer concentrations were incubated on the support layer for 60 to 90 s before blotting. All cryo-EM images were acquired with a Cs-corrected Titan Krios G2 electron microscope (Thermo Fisher) operated at 300 kV and equipped with a Falcon III 4 k direct electron detector (Thermo Fisher). We used the EPU software for automated single particle acquisition. See Table 3 for microscope settings for all individual datasets. The defocus for all acquisitions was set to −2 μm. The image processing was done at first in RELION-2 (51) and then later in RELION-3 (52). The recorded movies were subjected to MotionCor2 (53) for movie alignment and CTFFIND4.1 (54) for CTF estimation. After reference-free 2D classification the best 2D class averages, as judged by visual inspection, were selected for further processing. A subset of these particles was used to calculate an initial model. After one to two rounds of 3D classification, the classes showing the most features or completely assembled shells were selected for 3D auto-refinement and post-processing. For the corresponding shells octahedral (O) or icosahedral (I1) symmetry was used for the last two steps.
9. In Vitro Virus Blocking ELISA
Various concentrations of assembled half-T1 shells were incubated overnight at room temperature with 2 nM oligonucleotide-conjugated capture antibody (anti-HBc 17H7, Isotype IgG-2b) in FoB30-T (FoB30+0.05% Tween-20). The next day the pre-incubated mixtures were added to 5 pM HBV core particles and incubated overnight at room temperature, yielding 1 nM capture antibody, 2.5 pM HBV core particle and 0-200 pM half-T=1 shells. A flat-bottom transparent 96 well microplate (Nunc MaxiSorp) was treated overnight at 4° C. with 100 μl/well anti-CAgHB antibody (1 μg/ml in PBS). After washing 4 times with 200 μl/well PBS-T (PBS+0.05% Tween-20) the well surface was blocked by incubating with 200 μl/well 5% bovine serum albumin in PBS for 2 hours at room temperature. After washing 4 times with 200 μl/well FoB30-T, 90 μl of the pre-incubated samples were added to the wells and incubated for 2 hours at room temperature, followed by washing and subsequent incubation for 1 hour with 100 μl/well horseradish peroxidase conjugated detection antibody (anti-CAgHB-HRP in FoB30-T). After washing with FoB30-T, 100 μl/well HRP substrate (3,3′,5,5′-Tetramethylbenzidine, Life Technologies) was added and product formation was monitored in time by measuring the absorbance at 650 nm with a 60 s interval in a plate reader pre-equilibrated to 30° C. (CLARIOstar, BMG LABTECH). HRP activity was calculated by fitting linear regression slopes to the linear regime of the kinetic data (typically the first 5 min). Virus blocking efficiency was calculated relative to a control of HBV core particles only and blank measurements. All experiments were performed in triplicates. Antibodies used for the ELISA were kindly provided by Centro De Ingenieria Genetica y Biotecnologia de sancti spiritus in Cuba.
10. Helium Ion Microscopy (HIM)
Imaging was performed with negative-stained TEM grids coated with a 5 nm layer of AuPd using a Quorum Q150T sputter coater in ORION Nanofab (Zeiss). We used an acceleration voltage of 30 kV and a beam current of 0.3 to 0.4 pA. The images were acquired in scanning mode with an Everhart-Thornley 2 k detector.
11. Production of HBV Core Particles
Hepatitis B virus core particles of genotype D (subtype ayw2) were produced recombinantly in E. coli K802 and BL21 cells (purchased from the Latvian Biomedical Research and Study Centre, Riga, Latvia). Briefly, particles were obtained by sonication and clarification from bacterial protein extracts and purified by ammonium sulphate precipitation and subsequent anion exchange and size exclusion chromatography as described (55). Final preparations were constantly kept at 4° C. in the dark in conventional PBS (including 0.05% NaN3, 1 mM DTT).
12. Production of Anti-HBc Antibody
Anti-HBV core (anti-HBc) antibody 17H7 (Isotype IgG-2b) was produced by the Monoclonal Antibody Core Facility at Helmholtz Zentrum Munchen in Munich (HMGU). Briefly, mouse HBc-recognizing B cells were generated by common hybridoma technology. The mice were challenged with the peptide NLEDPASRDLVVC (aa 75-86 of HBV core). Mouse hybridoma clones were selected, and secreted antibodies were analyzed by immune staining and precipitation of HBcAg and ELISA for native antigen recognition and by Western Blot analysis for detection of denatured antigen. Final 17H7 preparations were purified via standard affinity chromatography using a protein A/G column and concentrated to 0.8 mg/ml (5.33 μM) of protein and kept in conventional PBS (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4) at 4° C. in the dark.
13. Cargo Encapsulation in T=1 Shells
Nine staples of the T=1 shell subunits were modified by adding 16 bases on the 5′ ends. These nine modified staples and unmodified T=1 staples are folded with p8064 scaffold to produce T=1 triangles with nine ssDNA “handles” (
14. Half Shells and HBV Core Binding
Anti-HBV core (anti-HBc) antibody 17H7 (Isotype IgG-2b) was produced by the Monoclonal Antibody Core Facility at Helmholtz Zentrum Munchen in Munich (HMGU). Briefly, mouse HBc-recognizing B cells were generated by common hybridoma technology. The mice were challenged with the peptide NLEDPASRDLVVC (aa 75-86 of HBV core). Mouse hybridoma clones were selected, and secreted antibodies were analyzed by immune staining and precipitation of HBcAg and ELISA for native antigen recognition and by Western Blot analysis for detection of denatured antigen. Final 17H7 preparations were purified via standard affinity chromatography using a protein A/G column and concentrated to 0.8 mg/ml (5.33 μM) of protein and kept in conventional PBS (137 mM NaCl, 10 mM Phosphate, 2.7 mM KCl, pH 7.4) at 4° C. in the dark.
15. Shell Oligolysine Stabilization
The complete octahedral shells were assembled at 35 mM MgCl2 and UV cross-linked as described in (33) for 1 h at 310 nm wavelength using the Asahi Spectra Xenon Light source 300W MAX-303. The shells were incubated in a 0.6:1 ratio of N:P with a mixture of K10 oligolysine and K10-PEG5K oligolysine (1:1) for 1h at room temperature as similarly described in (34). The octahedra were incubated in 55% mouse serum for 1h and 24h at 37° C. To allow imaging with negative stain the samples were diluted with PBS to a final mouse serum concentration of 5%, immediately before application to the negative stain grids.
The partial shells used for virus neutralization experiments in vivo were assembled at 60 mM MgCl2 and UV cross-linked as described in (33) for 30 min using the Asahi Spectra Xenon Light source 300W MAX-303. We introduced three-bases long sticky overhangs at every stacking contact and added one thymidine at the ends of both oligonucleotides (see
16. Cell Culture and Neutralization Assays
HEK293T (human embryonic kidney cell line, DSMZ) cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco, cat. no. 31966047) with 10% heat-inactivated Fetal Bovine Serum (FBS, Sigma-Aldrich, cat. no. F9665). Cells were cultured routinely in a humidified incubator at 37° C. with 5% CO2. AAV2 carrying eGFP (Biocat, cat. no. AA002-GVO-GC) were utilized for transduction experiments, where the concentration of infectious particles was determined by titration as per the manufactures protocol. Briefly, cells were seeded in 24-well plates at 80,000 cells mL-1 16-24 h prior to transduction, and harvested 72 h after transduction for quantification of transduction efficiency by flow cytometry. Samples were acquired and analyzed using Attune™ NxT Flow Cytometer and software (Thermofisher) respectively. 20,000 single cell events, gated on side scatter area vs height were recorded for analysis. eGFP was excited by 488 nm laser, and emission was measured with a 530/30 nm bandpass filter. Untreated cells were used as a negative control. Concentration of infectious particles was determined to be 1.23×109 IFU mL-1. Total number of AAV2 virus particles was measured by ELISA as per manufacture's protocol (Progen, cat. No. PRATV), and determined to be 2.24×1012 VP mL-1. For neutralization experiments, cells were cultured as above. 48-well plates were coated with poly-L-lysine (Sigma Aldrich cat. no. P2636, 0.1 mg mL-1, 10 min r.t. incubation) and then washed 2× with H2O and then PBS. HEK293T cells were seeded at 80,000 cells mL-1 16-24 h prior to transduction. Stock solutions were prepared for the overnight binding of conjugated anti-AAV2 to half shell origamis. Binding occurred in the presence of 0.1 mg mL-1 bovine serum albumin (BSA). Similarly, conjugated anti-AAV2, and half shells without antibody were also prepared in an identical manner. The next day, the half shells were coated with PEG-oligolysine/664 oligolysine by r.t. incubation for 2 h. Next, each of the different titration conditions were prepared and diluted to a total of 33.5 μL per condition with PBS. 4 μL of diluted AAV-2 sample (1/100, in PBS) was added and mixed, and samples were left to incubate (2 h, r.t.). Cells were washed with PBS and 62.5 μL of DMEM with 2% FBS was added to each well. Mixtures (37.5 μL) were then added dropwise to each well. Cells were incubated for 2 h, before 100 μL of DMEM with 18% FBS and 1x antibiotic/antimycotic was added. The cells were incubated for a further 22 h, before media was removed, cells were washed with 1x PBS, and 250 μL of DMEM with 10% FBS and 1x antibiotic/antimycotic was added. At 48 h post-transduction, the cells were trypsinized and prepared for flow cytometry. Transduction efficiency was quantified by flow cytometry as above, representative gates are given in
17. Viability Assay
Cytotoxicity was quantified by cell viability following 24 or 48 h incubation of cells with the half-shell mixtures. HEK293T cells were seeded in poly-L-lysine treated 96-well plates at 80,000 cells mL-1. Cells were allowed to settle overnight, media was removed and cells exposed to half shell mixtures in an identical procedure to the neutralisation assays. Cells were incubated with half shell solutions for a further 24 h or 48 h, and alamarBlue reagent (Invitrogen, 10 μL per well) was added. Plates were mixed and incubated for 4 h before being read on plate reader (CLARIOstar). Absorbance readings at 570 nm and 600 nm were taken, as per manufacturer's protocol. Measured values were normalized to control wells, which were treated identically, but received PBS containing no origami structures. All conditions were measured at least in triplicate.
| Number | Date | Country | Kind |
|---|---|---|---|
| 20158577.5 | Feb 2020 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/054307 | 2/22/2021 | WO |
