Patent Application
20240124892
This document relates to adeno-associated viruses (AAVs) and methods and materials for making and using AAVs. For example, this document provides AAVs containing a modified capsid polypeptide and methods for using such AAVs to produce compositions containing two or more AAVs covalently linked together.
Viruses, such as AAVs, are efficient modes for in vivo gene transfer, and their use in the clinic is expanding. Improved AAV designs and production techniques for making effective AAV preparations should further expand the use of AAVs in the laboratory and clinic.
This document provides AAVs and methods and materials for making and using AAVs. For example, this document provides AAVs containing a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide with the VP3 polypeptide containing a heterologous amino acid segment and the VP1 and VP2 polypeptides not containing that heterologous amino acid segment. That heterologous amino acid segment can be any appropriate amino acid segment. In some cases, the heterologous amino acid segment of a VP3 polypeptide can be an amino acid segment having the ability to bind to (e.g., to covalently bind to) a binding partner. For example, heterologous amino acid segment of a VP3 polypeptide can be a spytag sequence, a snooptag sequence, a spycatcher sequence, a snoopcatcher sequence, a DPIVMIDNDKPIT sequence (SEQ ID NO:1) or a variant SEQ ID NO:1. When using a spytag sequence as a heterologous amino acid segment of a VP3 polypeptide as described herein, then the binding partner can be a spycatcher sequence. When using a spycatcher sequence as a heterologous amino acid segment of a VP3 polypeptide as described herein, then the binding partner can be a spytag sequence. Likewise, when using a snooptag sequence as a heterologous amino acid segment of a VP3 polypeptide as described herein, then the binding partner can be a snoopcatcher sequence. When using a snoopcatcher sequence as a heterologous amino acid segment of a VP3 polypeptide as described herein, then the binding partner can be a snooptag sequence.
Having the ability to design AAVs having (a) a VP3 polypeptide containing a heterologous amino acid sequence (e.g., a tag sequence that can bind covalently to a binding partner), and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid sequence as described herein, can allow for the production and use of high amounts of functional AAVs having the ability to bind to other agents such as other AAVs (e.g., AAVs containing a VP3 polypeptide having a binding partner for the heterologous amino acid sequence) or a polypeptide having a binding partner for the heterologous amino acid sequence (e.g., a Cas9 polypeptide containing a binding partner for the heterologous amino acid sequence or a conjugating polypeptide containing a binding partner for the heterologous amino acid sequence). For example, an AAV having a VP3 polypeptide containing a heterologous amino acid sequence (e.g., a tag sequence that can bind covalently to a binding partner) can have the ability to form a covalent bond with a polypeptide designed to include a binding partner that covalently binds to that heterologous amino acid segment of the VP3 polypeptide. For example, a conjugating polypeptide can be designed as described herein to include a binding partner that covalently binds to a heterologous amino acid segment (e.g., a heterologous amino acid segment having SEQ ID NO:1 or a variant thereof) of a VP3 polypeptide of an AAV provided herein and used to conjugate that AAV to, for example, another AAV to form two AAVs that are covalently attached together (see, e.g.,
This document also provides (a) compositions containing an AAV described herein, (b) nucleic acid molecules encoding the components of an AAV described herein, (c) conjugating polypeptides, (d) nucleic acid molecules encoding a conjugating polypeptide described herein, (e) methods for making a composition that includes two or more different AAVs covalently linked together, and (f) methods for making a composition that includes an AAV covalently linked a polypeptide (e.g., a Cas9 polypeptide containing a binding partner for the heterologous amino acid sequence or a conjugating polypeptide containing a binding partner for the heterologous amino acid sequence).
As described herein, AAVs designed to contain (a) VP3 polypeptides that include a heterologous amino acid segment and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid segment can be used to produce high titer preparations of AAVs. For example, AAVs designed to contain (a) VP3 polypeptides that include a heterologous amino acid segment and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid segment can be used to produce a preparation of functional AAVs with a titer greater than 1011 (e.g., greater than 1×1011, greater than 2×1011, greater than 3×1011, greater than 4×1011, greater than 5×1011, greater than 6×1011, greater than 7×1011, greater than 8×1011, or greater than 9×1011, greater than 1×1012, greater than 2×1012, greater than 3×1012, greater than 4×1012, greater than 5×1012, greater than 6×1012, greater than 7×1012, greater than 8×1012, or greater than 9×1012, greater than 1×1013, greater than 2×1013, greater than 3×1013, greater than 4×1013, greater than 5×1013, greater than 6×1013, greater than 7×1013, greater than 8×1013, or greater than 9×1013, greater than 1×1014, greater than 2×1014, greater than 3×1014, greater than 4×1014, greater than 5×1014, greater than 6×1014, greater than 7×1014, greater than 8×1014, or greater than 9×1014). In some cases, an AAV designed to contain (a) VP3 polypeptides that include a heterologous amino acid segment and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid segment can be used to produce an AAV preparation with a titer that is greater than (e.g., at least 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold greater than) an AAV preparation of a comparable AAV that contains the heterologous amino acid segment as part of the VP1, VP2, and VP3 polypeptides.
Having the ability to produce AAV preparations with such high titers of functional AAVs containing a VP3 polypeptide engineered to include a heterologous amino acid segment as described herein can greatly increases the efficiency of using a preparation of AAVs (e.g., modified AAVs) to produce products that include a functional AAV covalently attached to another moiety of interest such as another AAV and/or a polypeptide.
In general, one aspect of this document features an AAV comprising a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide, wherein the VP3 polypeptide comprises a heterologous amino acid segment having the ability to bind to a binding partner, and wherein the VP1 polypeptide and the VP2 polypeptide lack the heterologous amino acid segment. The AAV can be an AAV2. The amino acid segment can comprise a spytag sequence, a snooptag sequence, a spycatcher sequence, a snoopcather sequence, or the amino acid sequence set forth in SEQ ID NO:1. The VP3 polypeptide can comprise the amino acid segment at an amino acid position within three amino acid residues of an amino acid residue corresponding to position 453 or position 588 of a reference VP1 polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The VP3 polypeptide can comprise a protease cleavage site.
In another aspect, this document features a composition comprising (or consisting essentially of or consisting of) an AAV comprising a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide, wherein the VP3 polypeptide comprises a heterologous amino acid segment having the ability to bind to a binding partner, and wherein the VP1 polypeptide and the VP2 polypeptide lack the heterologous amino acid segment. The AAV can be an AAV2. The amino acid segment can comprise a spytag sequence, a snooptag sequence, a spycatcher sequence, a snoopcather sequence, or the amino acid sequence set forth in SEQ ID NO:1. The VP3 polypeptide can comprise the amino acid segment at an amino acid position within three amino acid residues of an amino acid residue corresponding to position 453 or position 588 of a reference VP1 polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The VP3 polypeptide can comprise a protease cleavage site. The titer of the AAV of the composition can be greater than 1011.
In another aspect, this document features an isolated nucleic acid comprising (or consisting essentially of or consisting of) a nucleic acid sequence encoding a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide except that the nucleic acid sequence lacks the start codon of the VP1 polypeptide, and except that the nucleic acid sequence comprises a stop codon that is (a) within the sequence encoding the VP2 polypeptide and upstream of the sequence encoding the VP3 polypeptide. Expression of the isolated nucleic acid sequence can produce the VP3 polypeptide without producing the VP1 polypeptide or the VP2 polypeptide. The VP3 polypeptide can comprise a heterologous amino acid segment having the ability to bind to a binding partner. The amino acid segment can comprise a spytag sequence, a snooptag sequence, a spycatcher sequence, a snoopcather sequence, or the amino acid sequence set forth in SEQ ID NO:1. The VP3 polypeptide can comprise the amino acid segment at an amino acid position within three amino acid residues of an amino acid residue corresponding to position 453 or position 588 of a reference VP1 polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The VP3 polypeptide can comprise a protease cleavage site.
In another aspect, this document features an isolated nucleic acid comprising (or consisting essentially of or consisting of) a nucleic acid sequence encoding a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide except that the nucleic acid sequence lacks the start codon of the VP3 polypeptide. Expression of the isolated nucleic acid sequence can produce the VP1 polypeptide and the VP2 polypeptide without producing the VP3 polypeptide.
In another aspect, this document features a vector system for making an AAV comprising a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide, wherein the VP3 polypeptide comprises a heterologous amino acid segment having the ability to bind to a binding partner, and wherein the VP1 polypeptide and the VP2 polypeptide lack the heterologous amino acid segment, wherein the vector system comprises: (a) a first vector comprising a nucleic acid sequence encoding a VP1 polypeptide, a VP2 polypeptide, and the VP3 polypeptide comprising the heterologous amino acid segment except that the nucleic acid sequence of the first vector lacks the start codon of the VP1 polypeptide, and except that the nucleic acid sequence of the first vector comprises a stop codon that is (a) within the sequence encoding the VP2 polypeptide and upstream of the sequence encoding the VP3 polypeptide, and (b) a second vector comprising a nucleic acid sequence encoding a VP1 polypeptide lacking the heterologous amino acid segment, a VP2 polypeptide lacking the heterologous amino acid segment, and a VP3 polypeptide except that the nucleic acid sequence of the second vector lacks the start codon of the VP3 polypeptide of the second vector. The AAV can be an AAV2. Expression of the first and second vectors can produce the AAV comprising a VP1 polypeptide expressed from the second vector, a VP2 polypeptide expressed from the second vector, and a VP3 polypeptide expressed from the first vector. The heterologous amino acid segment can comprise a spytag sequence, a snooptag sequence, a spycatcher sequence, a snoopcather sequence, or the amino acid sequence set forth in SEQ ID NO:1. The VP3 polypeptide can comprise the heterologous amino acid segment at an amino acid position within three amino acid residues of an amino acid residue corresponding to position 453 or position 588 of a reference VP1 polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The VP3 polypeptide comprising the heterologous amino acid segment can comprise a protease cleavage site.
In another aspect, this document features a composition comprising (or consisting essentially of or consisting of) (a) a first AAV comprising a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide comprising a heterologous amino acid segment having the ability to bind to a binding partner, wherein the VP1 and VP2 polypeptides of the first AAV lack the heterologous amino acid segment, and (b) a second AAV comprising a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide. The AAV can be an AAV2. The heterologous amino acid segment can comprise a spytag sequence, a snooptag sequence, a spycatcher sequence, a snoopcather sequence, or the amino acid sequence set forth in SEQ ID NO:1. The VP3 polypeptide of the first AAV can comprise the heterologous amino acid segment at an amino acid position within three amino acid residues of an amino acid residue corresponding to position 453 or position 588 of a reference VP1 polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The VP3 polypeptide of the first AAV can comprise a protease cleavage site. The VP3 polypeptide of the second AAV can comprise the binding partner. The VP1 and VP2 polypeptides of the second AAV can lack the binding partner. The first and second AAVs can be covalently attached to each other. The first AAV can be covalently attached to the second AAV via at least one covalent bond between the amino acid segment of the VP3 polypeptide of the first AAV and the binding partner of the VP3 polypeptide of the second AAV. The VP3 polypeptide of the second AAV can comprise a heterologous amino acid segment having the ability to bind to a binding partner. The VP1 and VP2 polypeptides of the second AAV can lack the binding partner. The first and second AAVs can be covalently attached to each other. The first AAV can be covalently attached to the second AAV via a conjugating polypeptide comprising (a) the binding partner of the heterologous amino acid segment of the VP3 polypeptide of the first AAV and (b) the binding partner of the heterologous amino acid segment of the VP3 polypeptide of the second AAV.
In another aspect, this document features a method for producing a composition comprising (or consisting essentially of or consisting of) a first AAV covalently linked to a second AAV, wherein the first AAV comprises a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide comprising a heterologous amino acid segment having the ability to bind to a binding partner, wherein the VP1 and VP2 polypeptides of the first AAV lack the heterologous amino acid segment, and wherein the second AAV comprises a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide comprising the binding partner, wherein the method comprises contacting the first AAV with the second AAV to form a first AAV-second AAV complex, thereby producing the composition. The first AAV can be an AAV2. The second AAV can be an AAV2. The heterologous amino acid segment can comprise a spytag sequence, a snooptag sequence, a spycatcher sequence, a snoopcather sequence, or the amino acid sequence set forth in SEQ ID NO:1. The VP3 polypeptide of the first AAV can comprise the heterologous amino acid segment at an amino acid position within three amino acid residues of an amino acid residue corresponding to position 453 or position 588 of a reference VP1 polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The VP3 polypeptide of the first AAV can comprise a protease cleavage site. The VP1 and VP2 polypeptides of the second AAV can lack the binding partner. The first and second AAVs of the first AAV-second AAV complex can be covalently attached to each other. The first AAV can be covalently attached to the second AAV via at least one covalent bond between the amino acid segment of the VP3 polypeptide of the first AAV and the binding partner of the VP3 polypeptide of the second AAV.
In another aspect, this document features a method for producing a composition comprising (or consisting essentially of or consisting of) a first AAV attached to a second AAV, wherein the first AAV comprises a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide comprising a heterologous amino acid segment having the ability to bind to a binding partner, wherein the VP1 and VP2 polypeptides of the first AAV lack the heterologous amino acid segment, and wherein the second AAV comprises a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide comprising the binding partner, wherein the method comprises contacting the first AAV with the second AAV to form a first AAV-second AAV complex, thereby producing the composition. The first AAV can be an AAV2. The second AAV can be an AAV2. The heterologous amino acid segment can comprise a spytag sequence, a snooptag sequence, a spycatcher sequence, a snoopcather sequence, or the amino acid sequence set forth in SEQ ID NO:1. The VP3 polypeptide of the first AAV can comprise the heterologous amino acid segment at an amino acid position within three amino acid residues of an amino acid residue corresponding to position 453 or position 588 of a reference VP1 polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The VP3 polypeptide of the first AAV can comprise a protease cleavage site. The VP1 and VP2 polypeptides of the second AAV can lack the binding partner. The first and second AAVs of the first AAV-second AAV complex can be covalently attached to each other. The first AAV can be covalently attached to the second AAV via at least one covalent bond between the amino acid segment of the VP3 polypeptide of the first AAV and the binding partner of the VP3 polypeptide of the second AAV.
In another aspect, this document features a method for producing a composition comprising (or consisting essentially of or consisting of) a first AAV attached to a second AAV, wherein the first AAV comprises a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide comprising a first heterologous amino acid segment having the ability to bind to a first binding partner, wherein the VP1 and VP2 polypeptides of the first AAV lack the first heterologous amino acid segment, and wherein the second AAV comprises a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide comprising a second heterologous amino acid segment having the ability to bind to a second binding partner, wherein the method comprises (a) contacting the first AAV with a conjugating polypeptide to form a first AAV-conjugating polypeptide complex, wherein the conjugating polypeptide comprises the first binding partner and the second binding partner, and (b) contacting the first AAV-conjugating polypeptide complex with the second AAV to form a first AAV-conjugating polypeptide-second AAV complex, thereby producing the composition comprising the first AAV attached to the second AAV. The first AAV can be an AAV2. The second AAV can be an AAV2. The first heterologous amino acid segment can comprise a spytag sequence, a snooptag sequence, a spycatcher sequence, a snoopcather sequence, or the amino acid sequence set forth in SEQ ID NO:1. The second heterologous amino acid segment can comprise a spytag sequence, a snooptag sequence, a spycatcher sequence, a snoopcather sequence, or the amino acid sequence set forth in SEQ ID NO:1. The VP3 polypeptide of the first AAV can comprise the first heterologous amino acid segment at an amino acid position within three amino acid residues of an amino acid residue corresponding to position 453 or position 588 of a reference VP1 polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The VP3 polypeptide of the second AAV can comprise the second heterologous amino acid segment at an amino acid position within three amino acid residues of an amino acid residue corresponding to position 453 or position 588 of a reference VP1 polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The VP3 polypeptide of the first AAV or the second AAV can comprise a protease cleavage site. The VP1 and VP2 polypeptides of the second AAV can lack the second heterologous amino acid segment. The first and second AAVs of the first AAV-conjugating polypeptide-second AAV complex can be covalently attached to each other. The first AAV can be covalently attached to the conjugating polypeptide via at least one covalent bond between the first heterologous amino acid segment and the first binding partner of the conjugating polypeptide, and wherein the second AAV can be covalently attached to the conjugating polypeptide via at least one covalent bond between the second heterologous amino acid segment and the second binding partner of the conjugating polypeptide. The contacting step with the first AAV or the second AAV can comprise using a solution comprising a titer of the first AAV or of the second AAV that is greater than 1011.
In another aspect, this document features a method for producing a composition comprising (or consisting essentially of or consisting of) a first AAV attached to a second AAV, wherein the first AAV comprises a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide comprising a first heterologous amino acid segment having the ability to bind to a first binding partner, wherein the VP1 and VP2 polypeptides of the first AAV lack the first heterologous amino acid segment, and wherein the second AAV comprises a VP1 polypeptide, a VP2 polypeptide, and a VP3 polypeptide comprising a second heterologous amino acid segment having the ability to bind to a second binding partner, wherein the method comprises (a) contacting the second AAV with a conjugating polypeptide to form a second AAV-conjugating polypeptide complex, wherein the conjugating polypeptide comprises the first binding partner and the second binding partner, and (b) contacting the second AAV-conjugating polypeptide complex with the first AAV to form a first AAV-conjugating polypeptide-second AAV complex, thereby producing the composition comprising the first AAV attached to the second AAV. The first AAV can be an AAV2. The second AAV can be an AAV2. The first heterologous amino acid segment can comprise a spytag sequence, a snooptag sequence, a spycatcher sequence, a snoopcather sequence, or the amino acid sequence set forth in SEQ ID NO:1. The second heterologous amino acid segment can comprise a spytag sequence, a snooptag sequence, a spycatcher sequence, a snoopcather sequence, or the amino acid sequence set forth in SEQ ID NO:1. The VP3 polypeptide of the first AAV can comprise the first heterologous amino acid segment at an amino acid position within three amino acid residues of an amino acid residue corresponding to position 453 or position 588 of a reference VP1 polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The VP3 polypeptide of the second AAV can comprise the second heterologous amino acid segment at an amino acid position within three amino acid residues of an amino acid residue corresponding to position 453 or position 588 of a reference VP1 polypeptide having the amino acid sequence set forth in SEQ ID NO:2. The VP3 polypeptide of the first AAV or the second AAV can comprise a protease cleavage site. The VP1 and VP2 polypeptides of the second AAV can lack the second heterologous amino acid segment. The first and second AAVs of the first AAV-conjugating polypeptide-second AAV complex can be covalently attached to each other. The first AAV can be covalently attached to the conjugating polypeptide via at least one covalent bond between the first heterologous amino acid segment and the first binding partner of the conjugating polypeptide, and wherein the second AAV can be covalently attached to the conjugating polypeptide via at least one covalent bond between the second heterologous amino acid segment and the second binding partner of the conjugating polypeptide. The contacting step with the first AAV or the second AAV can comprise using a solution comprising a titer of the first AAV or of the second AAV that is greater than 1011.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.
This document provides AAVs containing (a) a VP3 polypeptide containing a heterologous amino acid segment (e.g., a tag sequence that can bind covalently to a binding partner) and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid segment. An appropriate AAV can be engineered to contain (a) a VP3 polypeptide containing a heterologous amino acid segment (e.g., a tag sequence that can bind covalently to a binding partner) and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid segment. For example, AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9 can be engineered to contain (a) a VP3 polypeptide containing a heterologous amino acid segment (e.g., a tag sequence that can bind covalently to a binding partner) and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid segment. In some cases, an AAV2 can be engineered to include (a) a VP3 polypeptide containing a heterologous amino acid segment (e.g., a tag sequence that can bind covalently to a binding partner) and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid segment.
As described herein, an AAV (e.g., AAV2) can be designed such that a heterologous amino acid segment is inserted into a VP3 polypeptide of that AAV without be present within the VP1 and VP2 polypeptides of that AAV. Any appropriate site of a VP3 polypeptide can be used as the site for accommodating insertion of a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner). Examples of such sites include, without limitation, those amino acid positions of a VP3 polypeptide that correspond to amino acid position 262, amino acid position 453, amino acid position 587, amino acid position 588, and amino acid position 589 of a reference VP1 polypeptide with the amino acid sequence set forth in SEQ ID NO:2 (
As described herein, any appropriate heterologous amino acid segment can be inserted into a VP3 polypeptide. For example, a heterologous amino acid segment having the ability to bind to (e.g., to covalently bind to) a binding partner can be inserted into a VP3 polypeptide. Examples of heterologous amino acid segments having the ability to bind to (e.g., to covalently bind to) a binding partner and that can be used as described herein include, without limitation, a spytag sequence that can bind to a spycatcher sequence, a spycatcher sequence that can bind to a spytag sequence, a snooptag sequence that can bind to a snoopcatcher sequence, a snoopcatcher sequence that can bind to a snooptag sequence, the amino acid sequence set forth in SEQ ID NO:1 (or a variant of the amino acid sequence set forth in SEQ ID NO:1) that can bind to a sequence described herein (e.g., SEQ ID NO:56), and a sequence described herein (e.g., SEQ ID NO:56) that can bind to the amino acid sequence set forth in SEQ ID NO:1 (or a variant of the amino acid sequence set forth in SEQ ID NO:1). Additional examples heterologous amino acid segments and binding partner pairs that can be used as described herein are set forth in Table 1.
Any appropriate variant of the amino acid sequence set forth in SEQ ID NO:1 can be used. For example, a variant of the amino acid sequence set forth in SEQ ID NO:1 can be designed to include one, two, three, four, five, six, or seven amino acid additions, deletions, or substitutions and can be used as described herein. In some cases, a variant of the amino acid sequence set forth in SEQ ID NO:1 can be designed to include one, two, or three amino acid additions, deletions, or substitutions. Examples of variants of the amino acid sequence set forth in SEQ ID NO:1 that can be used as described herein include, without limitation, those set forth in Table 2.
A binding partner having the ability to form a covalent bond with an amino acid segment containing the amino acid sequence set forth in SEQ ID NO:1 (or a variant thereof) that can be used as described herein can have any appropriate amino acid sequence. Examples of binding partner sequences having the ability to form a covalent bond with an amino acid segment containing the amino acid sequence set forth in SEQ ID NO:1 (or a variant thereof) that can be used as described herein include, without limitation, those set forth in the following formula:
Other examples of binding partner sequences having the ability to form a covalent bond with an amino acid segment containing the amino acid sequence set forth in SEQ ID NO:1 (or a variant thereof) that can be used as described herein include, without limitation, those set forth in the following formula, designated herein as SEQ ID NO:57:
Other examples of binding partner sequences having the ability to form a covalent bond with an amino acid segment that contains the amino acid sequence set forth in SEQ ID NO:1 (or a variant thereof) that can be used as described herein include, without limitation, those set forth in the following formula:
Still other examples of binding partner sequences having the ability to form a covalent bond with an amino acid segment containing the amino acid sequence set forth in SEQ ID NO:1 (or a variant thereof) that can be used as described herein include, without limitation, those set forth in the following formula, designated herein as SEQ ID NO:59:
In some cases, a binding partner sequence having the ability to form a covalent bond with an amino acid segment containing the amino acid sequence set forth in SEQ ID NO:1 (or a variant thereof) that can be used as described herein can have the amino acid sequence as set forth in any one of the sequences of Table 3 (any one of SEQ ID NOs:60-65). In some cases, a binding partner sequence having the ability to form a covalent bond with an amino acid segment containing the amino acid sequence set forth in SEQ ID NO:1 (or a variant thereof) that can be used as described herein can have the amino acid sequence as set forth any one of SEQ ID NOs:60-65 with 20 or less (e.g., one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acid additions, deletions, or substitutions with the proviso that the underlined amino acid residue remains unchanged. For example, a binding partner sequence having the ability to form a covalent bond with an amino acid segment containing the amino acid sequence set forth in SEQ ID NO:1 (or a variant thereof) that can be used as described herein can have the amino acid sequence as set forth in SEQ ID NO:60 with one, two, three, four, or five amino acid additions, deletions, or substitutions with the proviso that the amino acid located at a position corresponding to position 10 of SEQ ID NO:60 remains a lysine and with the proviso that the amino acid located at a position corresponding to position 56 of SEQ ID NO:60 remains a glutamic acid.
In some cases, an AAV described herein can be designed to include one or more linkers within a VP3 polypeptide. For example, an AAV described herein can be designed to include (a) a linker located N-terminal to the heterologous amino acid segment, (b) a linker located C-terminal to the heterologous amino acid segment, or (c) a linker located N-terminal and a linker located C-terminal to the heterologous amino acid segment. Such linkers can be immediately adjacent to the heterologous amino acid segment or can be within 10, 15, 20, 25, or 30 amino acid residues of the heterologous amino acid segment. For example, an AAV described herein can be designed to include, within a VP3 polypeptide in an N-terminal to C-terminal direction, a VP3 amino acid sequence immediately followed by a first linker immediately followed by the heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) immediately followed by a second linker immediately followed a continuation of the VP3 amino acid sequence. In another example, an AAV described herein can be designed to include, within a VP3 polypeptide in an N-terminal to C-terminal direction, a VP3 amino acid sequence immediately followed by a first linker followed within 10 amino acid residues by the heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) followed within 10 amino acid residues by a second linker immediately followed a continuation of the VP3 amino acid sequence. In another example, an AAV described herein can be designed to include, within a VP3 polypeptide in an N-terminal to C-terminal direction, a VP3 amino acid sequence immediately followed by a first linker immediately followed by a continuation of the VP3 amino acid sequence that is less than 20 amino acid residues in length immediately followed by the heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) immediately followed a continuation of the VP3 amino acid sequence that is less than 20 amino acid residues in length immediately followed by a second linker immediately followed a continuation of the VP3 amino acid sequence.
Any appropriate amino acid or amino acid sequence can be used as a linker to flank (e.g., immediately flank or flank within 10, 15, 20, 25, or 30 amino acid residues) one or both ends of a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) that is inserted into a VP3 polypeptide. In some cases, a single amino acid can be used as a linker. Examples of linkers that can be used to flank (e.g., immediately flank or flank within 10, 15, 20, 25, or 30 amino acid residues) one or both ends of a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) include, without limitation, leucine-alanine (LA), alanine (A), threonine-glycine (TG), glycine-leucine-serine (GLS), glycine-glycine-serine (GGS), and glycine-serine (GS).
When an AAV provided herein is designed to include two or more linkers within a VP3 polypeptide, those linkers can be the same or different. For example, an AAV provided herein can be designed to include a VP3 polypeptide that contains (a) a GLS linker N-terminal to (e.g., immediately N-terminal to or within 10, 15, 20, 25, or 30 amino acid residues to) a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) and (b) a GLS linker C-terminal to (e.g., immediately C-terminal to or within 10, 15, 20, 25, or 30 amino acid residues to) the heterologous amino acid segment. In another example, an AAV provided herein can be designed to include a VP3 polypeptide that contains (a) an LA linker N-terminal to (e.g., immediately N-terminal to or within 10, 15, 20, 25, or 30 amino acid residues to) a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) and (b) an A linker C-terminal to (e.g., immediately C-terminal to or within 10, 15, 20, 25, or 30 amino acid residues to) the heterologous amino acid segment. In another example, an AAV provided herein can be designed to include a VP3 polypeptide that contains (a) an TG linker N-terminal to (e.g., immediately N-terminal to or within 10, 15, 20, 25, or 30 amino acid residues to) a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) and (b) an GLS linker C-terminal to (e.g., immediately C-terminal to or within 10, 15, 20, 25, or 30 amino acid residues to) the heterologous amino acid segment.
In some cases, an AAV described herein can be designed to include one or more (e.g., one, two, three, four, or five) protease cleavage sites within a VP3 polypeptide. For example, an AAV described herein can be designed to include (a) one or more protease cleavage sites located within a VP3 polypeptide N-terminal to a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner), or (b) one or more protease cleavage sites located within a VP3 polypeptide C-terminal to a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner). Typically, the one or more protease cleavage sites are designed to be located within a VP3 polypeptide either N-terminal to the heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) or C-terminal to the heterologous amino acid segment, but not both. For example, when an AAV described herein is designed to include one or more protease cleavage sites located within a VP3 polypeptide N-terminal to a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner), the VP3 polypeptide can lack those protease cleavage sites C-terminal to the heterologous amino acid segment. Likewise, when an AAV described herein is designed to include one or more protease cleavage sites located within a VP3 polypeptide C-terminal to a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner), the VP3 polypeptide can lack those protease cleavage sites N-terminal to the heterologous amino acid segment.
In some cases, one or more protease cleavage sites can be engineered into a VP3 polypeptide described herein such that the site(s) are located immediately adjacent to (either N-terminal to or C-terminal to) a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) or can be within 10, 15, 20, 25, or 30 amino acid residues of (either N-terminal to or C-terminal to) a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner). For example, an AAV described herein can be designed to include, within a VP3 polypeptide in an N-terminal to C-terminal direction, a VP3 amino acid sequence immediately followed by one or more protease cleavage sites immediately followed by a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) immediately followed by a continuation of the VP3 amino acid sequence. In another example, an AAV described herein can be designed to include, within a VP3 polypeptide in an N-terminal to C-terminal direction, a VP3 amino acid sequence immediately followed by a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) immediately followed by one or more protease cleavage sites immediately followed by a continuation of the VP3 amino acid sequence.
Any appropriate protease cleavage site can be used to create an AAV described herein. For example, the following protease cleavage site of a TEV protease can be used as described herein: an ENLYFQ↓G sequence (SEQ ID NO:50). Other examples of protease cleavage sites (with their corresponding protease) include, without limitation, those set forth in Table 4.
In some cases, an AAV described herein can be designed to include one or more (e.g., one, two, three, four, or five) protease cleavage sites in combination with one or more linkers within a VP3 polypeptide. Examples of combinations of protease cleavage sites and linkers that can be used to make an AAV described herein include, without limitation, those set forth in Table 5. Each dash located between two listed components in Table 5 can represent that those two components are either immediately contiguous or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acid residues of each other. In some cases, when locate within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acid residues of each other, the intervening amino acid(s) can be of the VP3 polypeptide.
In some cases, an AAV described herein can be designed to include, within a VP3 polypeptide in an N-terminal to C-terminal direction, a VP3 amino acid sequence immediately followed by a first linker immediately followed by a protease cleavage site immediately followed by a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) immediately followed by a second linker immediately followed a continuation of the VP3 amino acid sequence. In another example, an AAV described herein can be designed to include, within a VP3 polypeptide in an N-terminal to C-terminal direction, a VP3 amino acid sequence immediately followed by a first linker followed within 10 amino acid residues by a protease cleavage site immediately followed by a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) followed within 10 amino acid residues by a second linker immediately followed a continuation of the VP3 amino acid sequence. In another example, an AAV described herein can be designed to include, within a VP3 polypeptide in an N-terminal to C-terminal direction, a VP3 amino acid sequence immediately followed by a first linker immediately followed by a continuation of the VP3 amino acid sequence that is less than 20 amino acid residues in length immediately followed by a protease cleavage site immediately followed by a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) immediately followed a continuation of the VP3 amino acid sequence that is less than 20 amino acid residues in length immediately followed by a second linker immediately followed a continuation of the VP3 amino acid sequence.
An AAV having (a) a VP3 polypeptide that includes a heterologous amino acid segment as described herein and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid segment can have the ability to form a covalent bond with another moiety via that heterologous amino acid segment. For example, an AAV provided herein having a VP3 polypeptide that includes a heterologous amino acid segment as described herein can covalently bind to a polypeptide (e.g., a conjugating polypeptide) or another virus (e.g., another AAV) via that heterologous amino acid segment. To form that covalent bond, the other moiety can be designed to include a binding partner having the ability to form that covalent bond with the heterologous amino acid segment.
As described herein, an AAV having (a) a VP3 polypeptide that includes a heterologous amino acid segment as described herein and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid segment can be covalently bound to another moiety of interest via that heterologous amino acid segment. Any appropriate moiety of interest (e.g., a polypeptide or a virus) can be designed to include a binding partner such that that moiety of interest can be covalently bound to an AAV provided herein via a covalent bond formed between the heterologous amino acid segment of the AAV and the binding partner of the moiety of interest. Examples of moieties of interest that can be designed to include a binding partner described herein and used as described herein include, without limitation, polypeptides such as a conjugating polypeptide, a Rabies G-protein, or a genome editing polypeptide (e.g. a Cas9 polypeptide) and viruses such as AAVs or adenoviruses.
In some cases, a first AAV can be designed to include (a) a VP3 polypeptide that includes a heterologous amino acid segment as described herein and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid segment, and a second AAV can be designed to include a capsid polypeptide (e.g., a VP3 polypeptide) having a binding partner sequence having the ability to covalently bind to the heterologous amino acid segment of the VP3 polypeptide of the first AAV. In such a case, the first AAV can be covalently bound to the second AAV via the heterologous amino acid segment of the VP3 polypeptide of the first AAV and the binding partner sequence of a capsid polypeptide (e.g., a VP1, VP2, and/or VP3 polypeptide) of the second AAV.
For example, with reference to
In some cases, with further reference to
In some cases, with further reference to
In some cases, AAVs (e.g., the “second AAV” of the preceding two paragraphs) having a capsid polypeptide designed to include a binding partner having the ability to covalently bind to the heterologous amino acid segment of another AAV (e.g., the “first AAV”) can be designed such that all three of VP1, VP2, and VP3 contains the introduced binding partner. In other cases, those AAVs (e.g., the “second AAV” of the preceding two paragraphs) can be designed to have (a) a VP3 polypeptide that includes the binding partner sequence and (b) VP1 and VP2 polypeptides that lack that binding partner sequence.
In another example, an AAV can be designed to include (a) a VP3 polypeptide that includes a heterologous amino acid segment as described herein and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid segment, and a polypeptide (e.g., a conjugating polypeptide) can be designed to include a binding partner sequence having the ability to covalently bind to the heterologous amino acid segment of VP3 polypeptide of the AAV. In such a case, the AAV can be covalently bound to the polypeptide (e.g., a conjugating polypeptide) via the heterologous amino acid segment of the VP3 polypeptide of the AAV and the binding partner of the polypeptide (e.g., a conjugating polypeptide). Such a polypeptide (e.g., a conjugating polypeptide) containing a binding partner sequence having the ability to covalently bind to the heterologous amino acid segment that is included within a VP3 polypeptide of an AAV as described herein can be used to covalently attach molecules of interest to that AAV via that polypeptide (e.g., conjugating polypeptide). For example, a conjugating polypeptide can be designed to include an enzyme (i.e., a molecule of interest in this example) and a binding partner sequence having the ability to covalently bind to a heterologous amino acid segment of a VP3 polypeptide of an AAV. In this example, such a conjugating polypeptide can be used to covalently attach that enzyme to the surface of the AAV that includes the VP3 polypeptide having the heterologous amino acid segment capable of binding to the binding partner of the conjugating polypeptide.
In some cases, a conjugating polypeptide can be designed to include two or more binding partners having the ability to covalently bind to a heterologous amino acid segment. In such cases, the two or more binding partners can be identical or different. For example, a conjugating polypeptide can be designed to include a first binding partner having the ability to covalently bind to a first heterologous amino acid segment and a second binding partner having the ability to covalently bind to a second heterologous amino acid segment that is the same or different from the first heterologous amino acid segment. In some cases, a conjugating polypeptide can be designed to include a first binding partner containing a spycather sequence and a second binding partner containing snoopcatcher sequence. In another example, a conjugating polypeptide can be designed to include a first spycather sequence and a second spycatcher sequence. In another example, a conjugating polypeptide can be designed to include a first snoopcather sequence and a second snoopcatcher sequence.
A conjugating polypeptide provided herein and designed to include two or more binding partners each having the ability to covalently bind to a heterologous amino acid segment described herein can be used to covalently link two AAVs together. With reference to
With further reference to
In some cases, a conjugating polypeptide provided herein can be designed to include a first binding partner having the ability to covalently bind to a heterologous amino acid segment containing the amino acid sequence set forth in SEQ ID NO:1 (or a variant thereof) and a second binding partner having the ability to covalently bind to a spytag sequence or a snooptag sequence. In these cases, the conjugating polypeptide can be used to covalently link a first AAV that includes a VP3 polypeptide having a heterologous amino acid segment containing the amino acid sequence set forth in SEQ ID NO:1 (or a variant thereof) to a second AAV that includes a VP3 polypeptide having that spytag sequence or snooptag sequence.
This document also provides compositions containing an AAV described herein. For example, compositions can be produced to contain one or more AAVs having (a) VP3 polypeptides that include a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid segment as described herein. In some cases, such a composition can include the AAVs at a titer that is greater than 1011 (e.g., greater than 1×1011, greater than 2×1011, greater than 3×1011, greater than 4×1011, greater than 5×1011, greater than 6×1011, greater than 7×1011, greater than 8×1011, or greater than 9×1011, greater than 1×1012, greater than 2×1012, greater than 3×1012, greater than 4×1012, greater than 5×1012, greater than 6×1012, greater than 7×1012, greater than 8×1012, or greater than 9×1012, greater than 1×1013, greater than 2×1013, greater than 3×1013, greater than 4×1013, greater than 5×1013, greater than 6×1013, greater than 7×1013, greater than 8×1013, or greater than 9×1013, greater than 1×1014, greater than 2×1014, greater than 3×1014, greater than 4×1014, greater than 5×1014, greater than 6×1014, greater than 7×1014, greater than 8×1014, or greater than 9×1014). In some cases, a composition provided herein can have AAVs with a titer that is greater than (e.g., at least 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold greater than) the titer of an AAV preparation of a comparable AAV that contains VP1, VP2, and VP3 polypeptides having the heterologous amino acid segment. For example, a composition provided herein having an AAV designed to have (a) VP3 polypeptides that include a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid segment can have an AAV titer that is greater than (e.g., at least 5-fold, 10-fold, 25-fold, 50-fold, 75-fold, or 100-fold greater than) an AAV preparation of a comparable AAV that contains VP1, VP2, and VP3 polypeptide having the heterologous amino acid segment.
This document also provides nucleic acid molecules and vector systems for producing an AAV having (a) a VP3 polypeptide that includes a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner) and (b) VP1 and VP2 polypeptides that lack that heterologous amino acid segment. For example, a first isolated nucleic acid molecule provided herein (e.g., an AAV trans vector for VP3) can be designed to encode (a) a VP1 polypeptide, (b) a VP2 polypeptide, and (c) a VP3 polypeptide containing a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner); except that the start codon of the VP1 polypeptide is deleted and except that a stop codon is inserted into the coding frame of the VP2 sequence between the VP2 start codon and the VP3 start codon. In such cases, the start codon of the VP1 sequence can be changed from ATG to any other codon except a stop codon. For example, the start codon of the VP1 sequence can be changed from ATG to ATC, CTT, or TCT. In addition, the stop codon inserted into the coding frame of the VP2 sequence between the VP2 start codon and the VP3 start codon can be any appropriate stop codon (e.g., TAA, TGA, or TAG), and the stop codon can be inserted at any appropriate location between the VP2 start codon and the VP3 start codon. For example, a stop codon can be inserted between 138 and 202 codons before the VP3 start codon. This vector can be used to produce a VP3 polypeptide having a heterologous amino acid segment (e.g., a heterologous amino acid segment having the ability to bind to a binding partner).
A second isolated nucleic acid molecule provided herein (e.g., an AAV trans vector for VP1 and VP2) can be designed to encode (a) a VP1 polypeptide (e.g., a wild-type VP1 polypeptide), (b) a VP2 polypeptide (e.g., a wild-type VP2 polypeptide), and (c) a VP3 polypeptide (e.g., a wild-type VP3 polypeptide); except that the start codon of the VP3 polypeptide is deleted. In this case, the vector can lack the sequence encoding a heterologous amino acid segment having the ability to bind to a binding partner. The start codon of the VP3 sequence can be changed from ATG to any other codon except a stop codon. For example, the start codon of the VP3 sequence can be changed from ATG to ATC, CTT, or TCT. This vector can be used to produce a VP1 polypeptide and a VP2 polypeptide with each not containing a heterologous amino acid segment having the ability to bind to a binding partner.
This document also provides conjugating polypeptides (and nucleic acids encoding conjugating polypeptides) as described herein. For example, conjugating polypeptides provided herein can be designed to contain one or more binding partner sequences (e.g., one binding partner sequence or two binding partner sequences) having the ability to bind covalently to heterologous amino acid segment(s) of an AAV provided herein. In some cases, conjugating polypeptides are provided herein that include a binding partner sequence having the ability to form a covalent bond with an amino acid segment containing the amino acid sequence set forth in SEQ ID NO:1 (or a variant thereof), a spytag sequence, or a snooptag sequence.
In some cases, a conjugating polypeptide provided herein can include two or more binding partner sequences where at least one binding partner includes the ability to bind covalently to the amino acid segment set forth in SEQ ID NO:1 (or a variant thereof). For example, a conjugating polypeptides provided herein can include a first binding partner having the ability to bind covalently to the amino acid segment set forth in SEQ ID NO:1 (or a variant thereof) and a second binding partner having the ability to bind covalently to the amino acid segment set forth in SEQ ID NO:1 (or a variant thereof). In another example, a conjugating polypeptide can include a first binding partner having the ability to bind covalently to the amino acid segment set forth in SEQ ID NO:1 (or a variant thereof) and a second binding partner having the ability to covalently bind to a spytag sequence or a snooptag sequence. In another example, a conjugating polypeptide can include a first binding partner having the ability to bind covalently to a spytag sequence and a second binding partner having the ability to covalently bind to a snooptag sequence. In another example, a conjugating polypeptide can include a first binding partner having the ability to bind covalently to a spytag sequence and a second binding partner having the ability to covalently bind to a spytag sequence. In another example, a conjugating polypeptide can include a first binding partner having the ability to bind covalently to a snooptag sequence and a second binding partner having the ability to covalently bind to a snooptag sequence.
In some cases, a conjugating polypeptides provided herein can include a linker (e.g., any of the exemplary linkers described herein) located between the two binding partners. In some cases, the two or more binding partner sequences can directly abut each other in the conjugating polypeptide.
Also provided in this document are nucleic acid molecules encoding any of the exemplary conjugating polypeptides described herein. For example, isolated nucleic acid molecules provided herein can encode for a conjugating polypeptide that includes a binding partner (e.g., any of the exemplary binding partners described herein) that binds covalently to a heterologous amino acid segment present in a VP3 polypeptide of an AAV. In such cases, a nucleic acid molecule provided herein can encode the amino acid sequence set forth in
In addition, this document provides methods for making a composition that includes two or more different AAVs covalently linked together. For example, compositions can be produced to contain two or more AAVs linked together through one or more covalent bonds (see, e.g.,
In some cases, a covalent linkage can be provided by an interaction between a first AAV having (a) a VP3 polypeptide that includes a heterologous amino acid segment as described herein and (b) VP1 and VP2 polypeptides lacking that heterologous amino acid sequence and a conjugating polypeptide, and an interaction between that conjugating polypeptide and a second AAV having a capsid polypeptide (e.g., a VP1, VP2, and/or VP3 polypeptide) that includes a heterologous amino acid segment (
In some cases, this document provides methods for generating a composition including a first AAV covalently linked to a second AAV. Such methods can include providing (i) a first AAV having (a) a VP3 polypeptide that includes a heterologous amino acid segment as described herein (e.g., a spytag sequence) and (b) VP1 and VP2 polypeptides lacking that heterologous amino acid sequence and (ii) a second AAV including a capsid polypeptide containing a binding partner (e.g., spycatcher sequence). Such a method can then include contacting the first AAV with the second AAV to form a “first AAV-second AAV” complex (see, e.g.,
In some cases, this document provides methods for generating a composition including a first AAV covalently linked to a second AAV via a conjugating polypeptide. Such methods can include providing (i) a first AAV having (a) a VP3 polypeptide that includes a heterologous amino acid segment as described herein (e.g., a spytag sequence) and (b) VP1 and VP2 polypeptides lacking that heterologous amino acid sequence, (ii) a second AAV including a capsid polypeptide (e.g., a VP1, VP2, and/or VP3 polypeptide) containing a heterologous amino acid segment (e.g., a snooptag sequence), and (iii) a conjugating polypeptide having a first binding partner (e.g., a spycatcher sequence) and a second binding partner (e.g., a snoopcatcher sequence). Such a method can then include contacting the first AAV with a conjugating polypeptide to form a first AAV-conjugating polypeptide complex. In such cases, the first AAV-conjugating polypeptide complex can be contacted with the second AAV to form a “first AAV-conjugating polypeptide-second AAV” complex.
In some cases, a composition provided herein can be purified after the formation of a first AAV-conjugating polypeptide complex or after the formation of a second AAV-conjugating polypeptide complex. In such cases, the purification can enrich a solution for an AAV-conjugating polypeptide complex. In some cases, a composition provided herein can undergo a purification step after formation of the “first AAV-conjugating polypeptide-second AAV” complex or after formation of the “second AAV-conjugating polypeptide-first AAV” complex. In such cases, the purification results in a composition enriched for the AAV-conjugating polypeptide-AAV complexes.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
An AAV vector plasmid was mutated so that the start codon of the VP1 polypeptide was deleted and a stop codon was inserted into the coding frame of the VP2 sequence between the VP2 start codon and the VP3 start codon by PCR amplifying the insert regions and annealing with Gibson Assembly (New England Biolabs). Such vectors included the following constructs: VP3-453-FlagTag-SnoopTag (VP1/2 STOP) (
An AAV vector plasmid was mutated so that the start codon of the VP3 polypeptide was changed into either isoleucine, leucine, or serine amino acids by PCR amplifying the insert regions and annealing with Gibson Assembly (New England Biolabs). Such vectors included the following vectors: VP1/2 (VP3 Serine mutation) (
Nucleic acid encoding conjugating polypeptides was cloned into a bacterial expression vector called pET28a by PCR amplifying the insert regions and annealing with Gibson Assembly (New England Biolabs). Such vectors included: FLEX (SPYCATCHER-LONG FLEXIBLE LINKER-SNOOPCATCHER) (
In order to produce AAV vectors incorporating a heterologous amino acid segment having the ability to bind to a binding partner (e.g., a SpyTag, SnoopTag, or SpyTag002), the segment was engineered onto surface exposed regions of AAV capsid by PCR amplifying the insert regions and annealing with Gibson Assembly (New England Biolabs). The heterologous amino acid segments were inserted into position 453 or 588 of VP3 subunit of AAV vectors that does not express VP1 and VP2 polypeptides.
A three vector system was transfected into HEK293 cells by the plasmid co-transfection method described elsewhere (Grieger et al., Nat. Protoc., 1(3):1412-28 (2006)). The first vector was an AAV cis vector carrying a genome encoding a fluorescent protein (or any nucleic acid of interest) flanked by the viral inverted terminal repeats (ITR). The second vector was an AAV trans vector carrying a heterologous amino acid segment (e.g., a SpyTag or SnoopTag) on the VP3 subunit of the capsid. The third vector was another AAV trans vector expressing the VP1 and VP2 subunits. Recombinant AAV was purified by iodixanol gradient ultra-centrifugation followed by a buffer exchange and concentration with Amicon Ultra-15 Centrifugal Filter Units in PBS+0.001% Pluronic F-68. Titers were determined by quantitative PCR relative to a standard curve (Aurnhammer et al., Hum. Gene Ther. Methods, 23(1):18-28 (2012)). The AAV-SpyTag or AAV-Snooptag vectors were mixed with a Spycatcher/SnoopCatcher conjugating polypeptide containing a Flag Tag by incubating them at room temperature for 1 hour, followed by an overnight incubation at 4° C. Western assays were performed by Jess simple western automated immunoassay systems (Bio Techne). A mouse monoclonal antibody against VP1, VP2 and VP3 from Progen and a rabbit monoclonal antibody against Flag tag sequence from Abcam were used to demonstrate linking of the AAV-SpyTag or AAV-SnoopTag vectors to the Spycatcher/SnoopCatcher conjugating polypeptide. See, e.g.,
A Snooptag was fused with a Cas9 polypeptide (
The Cas9-Snooptag fusion polypeptide was tested for its editing ability. To prepare the Cas9 RNP complexes, Cas9-Snooptag fusion polypeptide was incubated with sgRNA for 30 minutes at room temperature. gRNA was designed for targeting the human Rhodopsin gene and synthesized using GeneArt Precision gRNA Synthesis Kit (Thermo Fisher Scientific). To quantify the editing ability at desired genomic loci, T7 endonuclease I assay was performed. The samples that were PCR amplified with primers designed to amplify a ˜1 kb region containing the target site and treated with T7 endonuclease were run on an agarose gel. Results were presented in
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 63/155,624, filed Mar. 2, 2021. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/018543 | 3/2/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63155624 | Mar 2021 | US |
