WO2024129990A2 - Chemically modified aavs - Google Patents

Chemically modified aavs Download PDF

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WO2024129990A2
WO2024129990A2 PCT/US2023/084058 US2023084058W WO2024129990A2 WO 2024129990 A2 WO2024129990 A2 WO 2024129990A2 US 2023084058 W US2023084058 W US 2023084058W WO 2024129990 A2 WO2024129990 A2 WO 2024129990A2
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aav
amino acid
capsid protein
seq
variant
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PCT/US2023/084058
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French (fr)
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WO2024129990A3 (en
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Mark A. Kay
Francesco PUZZO
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The Board Of Trustees Of The Leland Stanford Junior University
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Publication of WO2024129990A2 publication Critical patent/WO2024129990A2/en
Publication of WO2024129990A3 publication Critical patent/WO2024129990A3/en

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  • AAV vectors have been widely employed in many clinical trials making these vectors the preferred delivery system for in vivo gene therapy.
  • AAV capsids through DNA shuffling or peptide display techniques to create vectors with improved AAV transduction in vivo.
  • AAV vectors have been successfully engineered to enhance targeting to different organs including liver, brain, skeletal muscle, and eye amongst others.
  • researchers have been using different approaches to synthetically modify AAV capsids in order to improve properties such as immune escape and receptor-specific cell targeting.
  • compositions and methods that provide cell-specific targeting for AAVs, and such is provided herein.
  • particular insertion of a single unnatural amino acid onto a specific region of the capsid allows specific conjugation of molecules onto the AAV regardless of capsid amino acid composition.
  • the inventors have developed chemically modified AAV vectors (NE-AAVS) through an unnatural amino acid substitution on the capsid surface for post-production vector engineering through biorthogonal copper-free click chemistry.
  • the inventors identified AAV vectors that tolerate the unnatural amino acid substitution on the capsid without disrupting their packaging and transduction efficiency.
  • they then functionalized the NE-AAVS through conjugation with DNA (AS1411 ), or RNA (E3) aptamers or with a targeting moiety (folic acid (FA)).
  • AS1411 DNA
  • E3 aptamers or with a targeting moiety folic acid (FA)
  • E3-, AS1411 -, and FA-AAV showed on average a 3-9 fold increase in transduction compared to their non -conjugated counterparts in different cancer cell lines.
  • the inventors demonstrated ligand-specific transduction. Moreover, in vivo studies confirmed the selective uptake of FA-AAV and AS1411 -AAV without off target transduction in peripheral organs. Overall, the high versatility of the NE-AAVS paves the way to tailoring gene therapy vectors toward specific types of cells both for ex vivo and in vivo applications.
  • FIG. 1 A-1 B Ne-AAV vector production and conjugation.
  • FIG. 1 A Schematic representation of NE-AAV production. Once the unnatural amino acid is incorporated into the AAV capsid the exposed azido group (N 3 ) reacts with the DBCO resulting in the conjugation of R, where the “R” is a generic molecule (peptide, nucleic acid etc.).
  • FIG. 1 B Western blot analysis of NE-AAV upon conjugation with the DBCO-biotin molecule. An anti-AAV antibody, clone B1 , was used to detect the VP1/2/3 capsid proteins of AAV. An anti-streptavidin antibody was used to detect the biotin- conjugated NE-AAV vector.
  • FIG. 2A-2F Development of the FA-AAV vector after folic acid conjugation.
  • FIG. 2A-2C Schematic representation of the plasmids used for (FIG. 2A) NE-AAV, (FIG. 2B) NE-VP2-AAV, and (FIG. 2C) NE-VP3-AAV production.
  • FIG. 2D Chemical formula of the DBCO-PEG (2K)-FA molecule used for the NE-AAV vector conjugation.
  • FIG. 2E Western blot analysis of NE-AAV, NE-VP2-AAV, NE-VP3-AAV upon DBCO- PEG (2K)-FA conjugation. An anti-AAV antibody, clone B1 , was used to detect the VP 1/2/3 capsid proteins of AAV.
  • FIG. 2F Luciferase activity in HeLa cells after transduction with DBCO-FA-conjugated AAV vectors. Mock-conjugated NE-AAV vectors were used as controls. Cells were transduced at 1 ,000 vg/cell.
  • Statistical analysis (FIG.
  • FIG. 3A-3F Assessment of novel NE-AAV vectors upon folic acid conjugation.
  • FIG. 3A Luciferase activity in HeLa cells of the NE-AAV vectors produced in presence (+ azido-lysine) or absence (- azido-lysine) of unnatural amino acid. AAV crude lysates was used for the HeLa transduction evaluation.
  • FIG. 3B-3D Luciferase activity in HeLa and MCF-7 cell lines upon transduction with (FIG. 3B) FA-DJR/A- N589, (FIG. 3C) FA-AAV8-T457, and (FIG. 3D) FA-LK03-N588. The unconjugated NE- AAV vectors were used as control.
  • FIG. 3E Transmission electric microscopy images of AAV-DJR/A, unconjugated NE-DJR/A-N589, and FA-DJR/A-N589 vectors.
  • FIG. 3F Regression plot showing the reverse correlation between the length of the PEG linker and AAV vectors infectivity of FA-AAV8-T457 and FA-DJR/A-N589 vectors.
  • Statistical analysis (FIG. 3B-3D) two-way ANOVA with Sidak’s post hoc the statistical significance was assumed with P value ⁇ 0.05 (*), ⁇ 0.01(**), ⁇ 0.001 (***) and ⁇ 0.0001 (****). Error bars represent standard deviation of the mean.
  • FIG. 4A-4I Evaluation of FA-AAV vector specific uptake in vitro.
  • FIG. 4B-4D Luciferase activity in HeLa cells transduced with (FIG. 4B) FA-DJR/A-N589, (FIG. 4C) FA-AAV8-T457, and (FIG. 4D) FA-DJR/A-A587. DJR/A, AAV8 and the unconjugated Ne-AAV vectors were used as controls. 200
  • FIG. 4E Luciferase activity in MCF-7 cells transduced with FA-DJR/A-N589. DJR/A and the unconjugated Ne-AAV vectors were used as controls. 200
  • FIG. 4F Schematic representation of the uptake assay conducted by pre-incubating the cells with the monoclonal anti-hFOLR1 antibody.
  • FIG. 4G, FIG. 4H Luciferase activity in HeLa cells transduced with (FIG. 4G) FA-DJR/A-N589, and (FIG. 4H) FA-AAV8-T457.
  • DJR/A, AAV8 and the unconjugated Ne-AAV vectors were used as controls. Cells were pre-incubated with an anti-hFOLR1 monoclonal antibody for one hour at 4°C and then transduced with the AAV vectors (+ hFOLR1 -Ab group).
  • FIG. 4I Luciferase activity in MCF-7 cells transduced with FA-DJR/A-N589. DJR/A and the unconjugated Ne-AAV vectors were used as controls. Cells were pre-incubated with an anti-hFOLR1 monoclonal antibody for one hour at 4°C and then transduced with the AAV vectors (+ hFOLR1-Ab group).
  • Statistical analysis (FIG.
  • FIG. 4G-4I two-way ANOVA with Sidak’s post hoc the statistical significance was assumed with P value ⁇ 0.05 (*), ⁇ 0.01 (**), ⁇ 0.001 (***) and ⁇ 0.0001 (****). Error bars represent standard deviation of the mean
  • FIG. 5A-5L Characterization of DNA and RNA aptamers conjugated to Ne-AAV vectors.
  • FIG. 5A Schematic representation of the DBCO-PEG-aptamer molecule.
  • FIG. 5B Western blot analysis of Ne-AAV vector upon conjugation with DBCO-PEG- AS141 1 .
  • An anti-AAV antibody, clone B1 was used to detect the VP 1/2/3 capsid proteins of AAV.
  • An anti-streptavidin antibody was used to detect the AS1411 - conjugated Ne-AAV vector.
  • FIG. 5C Luciferase activity in MCF-7 cells transduced with AS1411 -DJR/A-N589, and the unconjugated Ne-DJR/A-N589 vectors at different MOI.
  • FIG. 5D Schematic representation of the uptake assay conducted by incubating the cells with different concentrations of the AS1411 antidote.
  • FIG. 5E-5G Luciferase activity in (FIG. 5E) MCF-7, (FIG. 5F) A549, and (FIG. 5G) HeLa cells upon transduction with AS1411 -DJR/A-N589. Unconjugated NE-DJR/A-N589 vectors were used as controls.
  • FIG. 5H Luciferase activity in HeLa cells upon transduction with AS1411 - DJR/A-A587. Unconjugated Ne-DJR/A-N589 and CD-DJR/A-A587 vectors were used as controls. Cells were incubated with different concentrations of AS1411 antidote.
  • FIG. 51 Western blot analysis of NE-AAV vector upon conjugation with DBCO-PEG- C36 or DBCO-PEG-E3. An anti-AAV antibody, clone B1 , was used to detect the VP1/2/3 capsid proteins of AAV.
  • FIG. 5J Schematic representation of the uptake assay conducted by incubating the cells with dynasore.
  • FIG. 5K, FIG. 5L Luciferase activity in (FIG. 5K) Huh7, and (FIG. 5L) MCF-7 cells, upon transduction with E3-DJR/A-A587. Unconjugated Ne-DJR/A-A587 and C36-DJR/A-A587 vectors were used as controls. Cells were incubated with 10uM of clathrin inhibitor, Dynasore.
  • FIG. 5L two-way ANOVA with Sidak’s post hoc.
  • FIG. 5H two-way ANOVA with Tukey’s post hoc.
  • FIG. 6A-6E In vivo characterization of FA-AAV and AS1411-AAV vectors.
  • FIG. 6A Schematic representation of the in vivo study.
  • FIG. 6B Dorsal image of mice treated with 5x10 9 vg of NE-AAV, FA-AAV, and AS141 1 -AAV fourteen days after AAV treatment. PBS-injected mouse was used as negative control.
  • FIG. 6C Luciferase signal from tumor of mice treated with 5x10 9 vg of NE-AAV, FA-AAV, and AS1411 - AAV at three, seven, and fourteen days after AAV treatment (FIG.
  • FIG. 6D Ventral image of mice treated with 5x10 9 vg of NE-AAV, FA-AAV, and AS1411 -AAV fourteen days after AAV treatment. PBS-injected mouse was used as negative control.
  • FIG. 6E Luciferase signal from liver of mice treated with 5x10 9 vg of NE-AAV, FA-AAV, and AS141 1-AAV at three, seven, and fourteen days after AAV treatment.
  • FIG. 6C, FIG. 6E two-way ANOVA with Tukey’s post hoc the statistical significance was assumed with P value ⁇ 0.05 (*), ⁇ 0.01 (**), ⁇ 0.001 (***) and ⁇ 0.0001 (****). Error bars represent standard deviation of the mean.
  • FIG. 7A-7G Development of Ne-AAV vectors.
  • FIG. 7A Luciferase activity in HeLa cells transduced with AAVDJ and AAVDJR/A. Cells were treated with different concentration of heparin.
  • FIG. 7B Luciferase activity in HeLa cells transduced with NE-AAVDJR/A-N589, Ne-AAVDJR/A-D555; Ne-AAVDJR/A-A587; Ne-AAVDJR/A- T456.
  • FIG. 7C Western blot analysis of NE-VP2-AAV.
  • FIG. 7D Western blot analysis of NE-VP3-AAV.
  • An anti-AAV antibody, clone B1 was used to detect the VP1/2/3 capsid protein of AAV.
  • FIG. 7E Silver-stained gel analysis of DJR/A, NE- DJR/A-N589, and FA-DJR/A-N589 vectors.
  • FIG. 7F mRNA levels of hFOLRI in HeLa, MCF-7, and A459 cells measured by qPCR.
  • FIG. 8A-8I Characterization of novel NE-AAV vectors.
  • FIG. 8A Titer of the different Ns -AAV vectors expressed as percentage of the wild-type (WT) AAV serotype. The letter and the number correspond respectively to the amino acid and the position on the capsid where the unnatural amino acid is incorporated.
  • FIG. 8B- 8D Luciferase activity in A549 cells upon transduction with (FIG. 8B) FA-DJR/A- N589, (FIG. 8C) FA-AAV8-T457, and (FIG. 8D) FA-LK03-N588. The unconjugated NE- AAV vectors were used as control.
  • FIG. 8A Titer of the different Ns -AAV vectors expressed as percentage of the wild-type (WT) AAV serotype. The letter and the number correspond respectively to the amino acid and the position on the capsid where the unnatural amino acid is incorporated.
  • FIG. 8B- 8D
  • FIG. 8F Luciferase activity in MCF-7 cells after transduction with (FIG. 8E) NE-AAV8-T457 and (FIG. 8F) NE-DJR/A-N589 conjugated with DBCO-FA molecule carrying different lengths of PEG linker. As control the cells were transduced with AAV8, DJR/A and the respectively unconjugated NE-AAV vectors.
  • FIG. 8G Luciferase activity in MCF-7 cells after transduction with Ns-DJR/A-N589 conjugated with the DBCO-PEG (2K)-FA molecule or with DBCO-PEG.
  • FIG. 8H Western blot analysis of FA-DJR/A-N589 conjugated with different concentration of DBCO-FA. An anti-AAV antibody, clone B1 , was used to detect the VP 1/2/3 capsid protein of AAV.
  • FIG. 81 Luciferase activity in MCF-7 cells after transduction with NE-DJR/A-N589 conjugated with 2mM of DBCO- FA molecule.
  • FIG. 9A-9G Assessment of AS1411 -conjugated Ne-AAV vectors.
  • FIG. 9A Transmission electric microscopy images of AAV-DJR/A and AS141 1-DJR/A-N589.
  • FIG. 9B, FIG. 9C Luciferase activity in (FIG. 9B) MCF-7 and (FIG. 9C) HeLa cells upon transduction with AS1411 -DJR/A-A587. Unconjugated Ne-AAV vectors were used as controls. Cells were incubated with 10 ,M of AS1411 antidote.
  • FIG. 9A Transmission electric microscopy images of AAV-DJR/A and AS141 1-DJR/A-N589.
  • FIG. 9B, FIG. 9C Luciferase activity in (FIG. 9B) MCF-7 and (FIG. 9C) HeLa cells upon transduction with AS1411 -DJR/A-A587. Unconjugated Ne-AAV
  • FIG. 9D Western blot analysis of Ne-AAV vector upon conjugation with the DBCO-PEG-CD molecule.
  • An anti-AAV antibody, clone B1 was used to detect the VP1/2/3 capsid protein of AAV.
  • An anti-streptavidin antibody was used to detect the CD-conjugated Ne-AAV vector.
  • FIG. 9E, FIG. 9F Luciferase activity upon transduction with AS1411 - DJR/A-N589 or the unconjugated Ne-DJR/A-N589 in (FIG. 9E) MCF-7 and (FIG. 9F) A549 cells. Cells were incubated with 1 mg/mL of salmon sperm DNA (ssDNA).
  • FIG. 10A-10F Characterization of Ne-AAV vectors for in vivo studies.
  • FIG. 10A Titers of Ne-DJ-CAG-Fluc and Ne-DJ-INS84-Fluc measured by qPCR.
  • FIG. 10B, FIG. 10C Luciferase activity in HeLa cells upon transduction with (B) AS1411 -DJR/A- A587-INS84-Fluc, and
  • FIG. 10C FA-DJR/A-A587-INS84-Fluc. Unconjugated Ne- DJR/A-N589-INS84-Fluc vectors were used as control.
  • FIG. 10D FIG.
  • FIG. 10E In vivo luciferase images of mice treated with 5x10 9 vg of Ne-AAV, FA-AAV, and AS1411 - AAV (FIG. 10D) three days, and (FIG. 10E) seven days after AAV injection. PBS- injected mouse was used as negative control.
  • FIG. 10F Luciferase activity in harvested liver and tumor HeLa cells of mice treated with 5x10 9 vg of Ne-AAV, FA- AAV, and AS1411 -AAV fourteen days after AAV treatment. Luciferase activity was normalized by the protein concentration measured in the tissue sample.
  • Statistical analysis (FIG. 10A-10C) Student t-test. (FIG.
  • AAV is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof, and can be used to refer to a viral vector system for delivery of nucleic acids. The term covers all subtypes and both naturally occurring as well as recombinant and variant forms, except where specified otherwise.
  • the abbreviation “rAAV” refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector”).
  • AAV includes any convenient AAV type, including variant types, e.g., AAV type 1 (AAV1 ), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV 9_hu14, AAV-DJ (also referred to herein as simply “DJ”), AAV- LK03 (also referred to herein as simply “LK03”), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV.
  • AAV type 1 AAV1
  • AAV2 AAV type 2
  • AAV3 AAV type 3
  • AAV4 AAV4
  • AAV type 5 AAV5
  • AAV6 AAV type 6
  • AAV7 AAV type 7
  • Prime AAV refers to AAV capable of infecting primates
  • non-primate AAV refers to AAV capable of infecting non-primate mammals
  • bovine AAV refers to AAV capable of infecting bovine mammals
  • AAV vector can be used to refer to the delivery system as a whole (e.g., a virion or population of virions), and can also be used to refer to a nucleic acid encoding the delivery system - i.e., one that includes a sequence that encodes a capsid polypeptide (i.e., a nucleic acid that includes a nucleotide sequence encoding a capsid polypeptide, also referred to as a AAV capsid protein or AAV capsid polypeptide - the terms “polypeptide” and “protein” are used interchangeably herein), depending on context.
  • a subject AAV vector is a variant (nonwild type) AAV vector
  • a nucleic acid can include a nucleotide sequence encoding a variant capsid polypeptide, also referred to as a variant AAV capsid protein or variant AAV capsid polypeptide.
  • the AAV vectors can also include a heterologous nucleic acid sequence not of AAV origin (e.g., as part of the nucleic acid insert). This heterologous nucleic acid sequence typically comprises a sequence of interest for the genetic transformation of a cell.
  • the heterologous nucleic acid sequence (the “nucleotide sequence of interest”) is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs).
  • ITRs AAV inverted terminal repeat sequences
  • non-variant parent capsid polypeptides include any naturally occurring AAV capsid polypeptides.
  • the non-variant parent capsid polypeptides include AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, bovine AAV and/or avian AAV capsid polypeptides.
  • substantially identical in the context of variant AAV capsid polypeptides and non-variant parent capsid polypeptides refers to sequences with 1 or more amino acid changes. In some embodiments, these changes do not affect the packaging function of the capsid polypeptides. In some embodiments, substantially identical include variant AAV capsid polypeptides about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, or about 90% identical to non-variant parent capsid polypeptides.
  • the variant AAV capsid polypeptides can be substantially identical to non-variant parent capsid polypeptides over a subregion of the variant AAV capsid polypeptide, such as over about 25%, about 50%, about 75%, or about 90% of the total polypeptide sequence length.
  • An "AAV virion” or “AAV virus” or “AAV viral particle” or “AAV vector particle” refers to a viral particle composed of at least one AAV capsid polypeptide (including both variant AAV capsid polypeptides and non-variant parent capsid polypeptides) and an encapsidated polynucleotide AAV transfer vector. If the particle comprises a heterologous nucleic acid (i.e. a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it can be referred to as an "AAV vector particle” or simply an "AAV vector". Thus, production of AAV virion or AAV particle necessarily includes production of AAV vector as such a vector is contained within an AAV virion or AAV particle.
  • Packaging refers to a series of intracellular events resulting in the assembly of AAV virions or AAV particles which encapsidate a nucleic acid sequence and/or other therapeutic molecule.
  • Packaging can refer to encapsidation of nucleic acid sequence and/or other therapeutic molecules into a capsid comprising the variant AAV capsid polypeptides described herein.
  • AAV "rep” and “cap” genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus (AAV). AAV rep (replication) and cap (capsid) are referred to herein as AAV "packaging genes.”
  • a "helper virus” for AAV refers to a virus allowing AAV (e.g. wild-type AAV) to be replicated and packaged by a mammalian cell.
  • a variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia.
  • the adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used as a helper virus.
  • Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC.
  • Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.
  • HSV herpes simplex viruses
  • EBV Epstein-Barr viruses
  • CMV cytomegaloviruses
  • PRV pseudorabies viruses
  • Helper virus function(s) refers to function(s) encoded in a helper virus genome allowing AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, "helper virus function” may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans.
  • an "infectious" virion, virus or viral particle is one comprising a polynucleotide component deliverable into a cell tropic for the viral species. The term does not necessarily imply any replication capacity of the virus.
  • an "infectious" virus or viral particle is one that upon accessing a target cell, can infect a target cell, and can express a heterologous nucleic acid in a target cell.
  • infectivity refers to the ability of a viral particle to access a target cell, enter a target cell, and express a heterologous nucleic acid in a target cell. Infectivity can refer to in vitro infectivity or in vivo infectivity.
  • Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Total viral particles can be expressed as the number of viral genome copies.
  • the ability of a viral particle to express a heterologous nucleic acid in a cell can be referred to as "transduction.”
  • the ability of a viral particle to express a heterologous nucleic acid in a cell can be assayed using a number of techniques, including assessment of a marker gene, such as a green fluorescent protein (GFP) assay (e.g., where the virus comprises a nucleotide sequence encoding GFP), where GFP is produced in a cell infected with the viral particle and is detected and/or measured; or the measurement of a produced protein, for example by an enzyme-linked immunosorbent assay (ELISA) or fluorescence- activated cell sorting (FACS).
  • GFP green fluorescent protein
  • a "replication-competent" virion or virus refers to an infectious phenotypically wild-type virus, and is replicable in an infected cell (i.e. in the presence of a helper virus or helper virus functions).
  • replication competence generally requires the presence of functional AAV packaging genes.
  • AAV vectors, as described herein lack of one or more AAV packaging genes and are replication-incompetent in mammalian cells (especially in human cells).
  • AAV vectors lack any AAV packaging gene sequences, minimizing the possibility of generating replication competent AAV by recombination between AAV packaging genes and an incoming AAV vector.
  • AAV vector preparations as described herein are those containing few if any replication competent AAV (rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV per 10.sup.2 AAV particles, less than about 1 rcAAV per 10.sup.4 AAV particles, less than about 1 rcAAV per 10.sup.8 AAV particles, less than about 1 rcAAV per 10.sup.12 AAV particles, or no rcAAV).
  • rcAAV replication competent AAV
  • polynucleotide and “nucleic acid” are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • Polynucleotides include genomic DNA, cDNA and antisense DNA, and spliced or unspliced mRNA, rRNA, tRNA, IncRNA, RNA antagomirs, and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), aptamers, small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA).
  • RNAi e.g., small or short hairpin (sh)RNA, microRNA (miRNA), aptamers, small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA
  • Polynucleotides also include non-coding RNA, which include for example, but are not limited to, RNAi, miRNAs, IncRNAs, RNA antagomirs, aptamers, and any other non-coding RNAs known to those of skill in the art.
  • Polynucleotides include naturally occurring, synthetic, and intentionally altered or modified polynucleotides as well as analogues and derivatives.
  • polynucleotide also refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof, and is synonymous with nucleic acid sequence.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • polynucleotide refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment as described herein encompassing a polynucleotide encompasses both the double-stranded form and each of two complementary singlestranded forms known or predicted to make up the double-stranded form. Polynucleotides can be single, double, or triplex, linear or circular, and can be of any length. In discussing polynucleotides, a sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.
  • a "small interfering” or “short interfering RNA” or siRNA is a RNA duplex of nucleotides targeted to a gene interest (a "target gene”).
  • An “RNA duplex” refers to the structure formed by the complementary pairing between two regions of a RNA molecule.
  • siRNA is "targeted” to a gene and the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene.
  • the length of the duplex of siRNAs is less than 30 base pairs.
  • the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 base pairs in length.
  • the length of the duplex is 19-25 base pairs in length.
  • the RNA duplex portion of the siRNA can be part of a hairpin structure.
  • the hairpin structure may contain a loop portion positioned between the two sequences forming the duplex.
  • the loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 1 1 , 12 or 13 nucleotides in length.
  • the hairpin structure can also contain 3' or 5' overhang portions. In some embodiments, the overhang is a 3' or a 5' overhang 0, 1 , 2, 3, 4 or 5 nucleotides in length.
  • Recombinant as applied to a polynucleotide means the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures resulting in a construct distinct and/or different from a polynucleotide found in nature.
  • a recombinant virus is a viral particle encapsidating a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.
  • control element or "control sequence” is a nucleotide sequence involved in an interaction of molecules contributing to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature.
  • Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers.
  • a promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription usually downstream (in the 3' direction) from the promoter.
  • “Operatively linked” or “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner.
  • a promoter is operatively linked to a sequence of interest (the sequence of interest can also be said to be operatively linked to the promoter) if the promoter helps initiate transcription of the sequence of interest. There may be intervening residues between the promoter and sequence of interest so long as this functional relationship is maintained.
  • Heterologous means derived from a genotypically distinct entity from the rest of the entity to it is being compared too.
  • a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide.
  • an AAV including a heterologous nucleic acid encoding a heterologous gene product is an AAV including a nucleic acid not normally included in a naturally-occurring, wild-type AAV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally-occurring, wild-type AAV.
  • An AAV including a nucleic acid encoding a variant AAV capsid polypeptide includes a heterologous nucleic acid sequence.
  • heterologous polynucleotide into a host cell, contained within the virion need not be expressed.
  • heterologous is not always used herein in reference to polynucleotides, reference to a polynucleotide even in the absence of the modifier "heterologous” is intended to include heterologous polynucleotides in spite of the omission.
  • genetic alteration and “genetic modification” (and grammatical variants thereof), are used interchangeably herein to refer to a process wherein a genetic element (e.g., a polynucleotide) is introduced into a cell other than by mitosis or meiosis.
  • the element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell.
  • Genetic alteration may be effected, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, calcium phosphate precipitation, or polynucleotide-liposome complexation.
  • Genetic alteration may also be effected, for example, by transduction or infection with a DNA or RNA virus or viral vector.
  • the genetic element is introduced into a chromosome or mini-chromosome in the cell; but any alteration changing the phenotype and/or genotype of the cell and its progeny is included in this term.
  • a cell is said to be “stably” altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during an extended period of time (e.g., extended culture of the cell when the cell is in vitro).
  • a cell can be “heritably” altered (genetically modified) in that a genetic alteration is introduced and can be inherited by progeny of the altered cell.
  • polypeptide polypeptide
  • peptide and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the "polypeptides,” “proteins” and “peptides” encoded by the “polynucleotide sequences,” include full-length native sequences, as with naturally occurring proteins, as well as functional subsequences, modified forms or sequence variants so long as the subsequence, modified form or variant retains some degree of the intended functionality.
  • the terms also encompass a modified amino acid polymer; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, methylation, carboxylation, deamidation, acetylation, or conjugation with a labeling component.
  • Polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivering a gene product to a mammalian subject, and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, retaining the desired biochemical function of the intact protein.
  • an "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components present where the substance or a similar substance naturally occurs or from which it is initially prepared.
  • an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more isolated.
  • An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some embodiments purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure.
  • highly conserved is meant at least about 80% identity, preferably at least 90% identity, and more preferably, over about 97% identity. Identity is readily determined by one of skill in the art by resort to algorithms and computer programs known by those of skill in the art.
  • treatment refers to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. , arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
  • the terms "individual,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).
  • mammalian sport animals e.g., horses
  • mammalian farm animals e.g., sheep, goats, etc.
  • mammalian pets dogs, cats, etc.
  • rodents e.g., mice, rats, etc.
  • pharmaceutically acceptable and “physiologically acceptable” mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery or contact.
  • a “pharmaceutically acceptable” or “physiologically acceptable” composition is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing substantial undesirable biological effects.
  • such a pharmaceutical composition may be used, for example in administering an AAV vector or AAV virion as disclosed herein, or transformed cell to a subject.
  • unit dosage form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, produces a desired effect (e.g., prophylactic or therapeutic effect).
  • unit dosage forms may be within, for example, ampules and vials, including a liquid composition, or a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo.
  • Individual unit dosage forms can be included in multi-dose kits or containers.
  • AAV vectors or AAV virions, and pharmaceutical compositions thereof can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.
  • a "therapeutically effective amount” will fall in a relatively broad range determinable through experimentation and/or clinical trials.
  • a therapeutically effective dose will be on the order of from about 10 6 to about 10 15 of the AAV virions per kilogram bodyweight of the subject.
  • a therapeutically effective dose will be on the order of from about 10 8 to 10 12 AAV virions per kilogram bodyweight of the subject.
  • Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.
  • an "effective amount” or “sufficient amount” refers to an amount providing, in single or multiple doses, alone or in combination, with one or more other compositions (therapeutic agents such as a drug), treatments, protocols, or therapeutic regimens agents (including, for example, vaccine regimens), a detectable response of any duration of time (long or short term), an expected or desired outcome in or a benefit to a subject of any measurable or detectable degree or for any duration of time (e.g., for minutes, hours, days, months, years, or cured).
  • the doses of an "effective amount” or “sufficient amount” for treatment typically are effective to provide a response to one, multiple or all adverse symptoms, consequences or complications of the disease, one or more adverse symptoms, disorders, illnesses, pathologies, or complications, for example, caused by or associated with the disease, to a measurable extent, although decreasing, reducing, inhibiting, suppressing, limiting or controlling progression or worsening of the disease is also a satisfactory outcome.
  • Prophylaxis and grammatical variations thereof mean a method in which contact, administration or in vivo delivery to a subject is prior to disease. Administration or in vivo delivery to a subject can be performed prior to development of an adverse symptom, condition, complication, etc. caused by or associated with the disease.
  • a screen e.g., genetic
  • the subject may not manifest the disease.
  • Such subjects therefore include those screened positive for an insufficient amount or a deficiency in a functional gene product (protein), or producing an aberrant, partially functional or non-functional gene product (protein), leading to disease; and subjects screening positive for an aberrant, or defective (mutant) gene product (protein) leading to disease, even though such subjects do not manifest symptoms of the disease.
  • tropism refers to the ability of an AAV vector or virion to infect one or more specified cell types, but can also encompass how the vector functions to transduce the cell in the one or more specified cell types; i.e., tropism refers to preferential entry of the AAV vector or virion into certain cell or tissue type(s) and/or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types, optionally and preferably followed by expression (e.g., transcription and, optionally, translation) of sequences carried by the AAV vector or virion in the cell, e.g., for a recombinant virus, expression of the heterologous nucleotide sequence(s).
  • transduction refers to the ability of an AAV vector or virion to infect one or more particular cell types; i.e., transduction refers to entry of the AAV vector or virion into the cell and the transfer of genetic material contained within the AAV vector or virion into the cell to obtain expression from the vector genome. In some cases, but not all cases, transduction and tropism may correlate.
  • efficient transduction or "efficient tropism,” or similar terms, can be determined by reference to a suitable control (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 110%, 125%, 150%, 175%, or 200% or more of the transduction or tropism, respectively, of the control). Suitable controls will depend on a variety of factors including the desired tropism profile. Similarly, it can be determined if a capsid and/or virus "does not efficiently transduce” or “does not have efficient tropism” for a target tissue, or similar terms, by reference to a suitable control.
  • variant AAV capsid proteins that include an azide-bearing unnatural amino acid substitution (e.g. in some cases one single unnatural amino acid substitution).
  • the azide-bearing unnatural amino acid substitution is at a position corresponding to: T456, D555, R587/A587, or N589 of the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2; or E330, T457, N499, or N590 of the AAV8 amino acid sequence set forth as SEQ ID NO: 7; or T455, T456, or N588 of the AAV- LK03 amino acid sequence set forth as SEQ ID NO: 13.
  • R587/A587 is used herein because the amino acid at position 587 in the AAV-DJ capsid sequence (see SEQ ID NO: 1 ) is an arginine (R587) while the amino acid at that same position in the AAV-DJR/A capsid sequence is an alanine (A587).
  • R587/A587 is thus used to refer to position 587 when in the context of referencing both SEQ ID NOs: 1 and 2.
  • the azide-bearing unnatural amino acid is at a position inserted between positions corresponding to N590 and T591 of the AAV8 amino acid sequence set forth as SEQ ID NO: 7.
  • the azide- bearing unnatural amino acid substitution is at a position corresponding to: T456, D555, R587/A587, or N589 of the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2.
  • the azide-bearing unnatural amino acid substitution is at a position corresponding to: E330, T457, N499, or N590 of the AAV8 amino acid sequence set forth as SEQ ID NO: 7.
  • the azide-bearing unnatural amino acid substitution is at a position corresponding to: T455, T456, or N588 of the AAV- LK03 amino acid sequence set forth as SEQ ID NO: 13.
  • the variant AAV capsid protein includes an amino acid sequence having: 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 , 2, 7, and 13.
  • the variant AAV capsid protein includes an amino acid sequence having: 90% or more (e.g., 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 , 2, 7, and 13.
  • the variant AAV capsid protein includes an amino acid sequence having: 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16. In some cases, the variant AAV capsid protein includes an amino acid sequence having: 90% or more (e.g., 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16.
  • the variant AAV capsid protein includes the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16 (e.g., the amino acid sequence set forth in any one of SEQ ID NOs: 5-6, 12, or 14).
  • the variant AAV capsid protein includes an amino acid sequence having: (i) 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, wherein said azide-bearing unnatural amino acid substitution is at position N589 or R587/A587; (ii) 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV8 amino acid sequence set forth as SEQ ID NO: 7, wherein said azide-bearing unnatural amino acid substitution is at position T457; or (iii) 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV-DJ amino acid sequence
  • the variant AAV capsid protein includes an amino acid sequence having: 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, wherein said azide-bearing unnatural amino acid substitution is at position N589 or R587/A587.
  • the variant AAV capsid protein includes the azide-bearing unnatural amino acid substitution in an AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid polypeptide.
  • the variant AAV capsid protein includes an amino acid sequence having 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with a wild type AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid protein.
  • the variant AAV capsid protein is a shuffled variant, meaning that the variant AAV capsid protein resulted from the shuffling of multiple parent capsid protein sequences - and thus such a variant AAV capsid protein includes stretches of wild type sequence, but the capsid protein sequence as a whole does not occur in nature.
  • An azide-bearing unnatural amino acid can be any amino acid bearing an azide (e.g., an azido amino acid). Examples include, but are not necessarily limited to: azidolysine, 6-Azido-D-lysine, 6-Azido-L-lysine, azido-alanine, L-azidobutyl-alanine, 3- Azido-D-alanine, 3-Azido-L-alanine, azido-homoalanine, 4-Azido-L-homoalanine, azido-ornithine, azido-phenylalanine, 4-Azido-L-phenylalanine, p-azido-phenylalanine, and 5-azido-L-norvaline.
  • the unnatural amino acid is an azidolysine.
  • the variant AAV capsid protein includes at least one arginine to alanine mutation at a heparan sulfate proteoglycan (HSPG) binding site (e.g., at an amino acid position corresponding to position 587 and/or 590 of SEQ ID NO: 1 ).
  • the variant AAV capsid protein includes at least one amino acid mutation (e.g., 1 , 2, 3, 4, 1-4, 1 -3, 2-4, 2-3, or at least 2 amino acid mutations) that reduces HSPG binding affinity.
  • Such mutations for various AAV types will be known to one of ordinary skill in the art (see, e.g., Cabanes-Creus et al., Mol Ther Methods Clin Dev. 2020 Jun 12; 17).
  • the variant AAV capsid proteins are chemically modified such that the capsid protein is conjugated via the azide-bearing unnatural amino acid to a targeting moiety such as folic acid, a DNA aptamer (e.g., AS1411 ), or an RNA aptamer (e.g., E3).
  • a targeting moiety such as folic acid, a DNA aptamer (e.g., AS1411 ), or an RNA aptamer (e.g., E3).
  • the conjugation is via a linker (e.g., a Polyethylene glycol (PEG) linker).
  • the linker e.g., PEG
  • the linker is less than 5 kDa in length (e.g., 4 kDa or less, 3 kDa or less, or 2 kDa or less).
  • the linker e.g.., PEG
  • the linker is about 2 kDa in length.
  • the targeting moiety can be conjugated to the unnatural amino acid using any convenient approach, e.g., via click chemistry reaction between the azide of the unnatural amino and a click chemistry group (e.g., dibenzocyclooctyne-amine (DBCO)) of the targeting moiety.
  • DBCO dibenzocyclooctyne-amine
  • the targeting moiety may vary and may be selected based, e.g., on the nature of the molecule to be targeted, e.g., cell surface molecule on the target cell, or an extracellular or secreted molecule.
  • a targeting moiety that may be employed include a polypeptide, an antibody, a ligand, an aptamer, and a small molecule.
  • the targeting moiety specifically binds the target molecule, e.g., a cell surface molecule of the target cell, or an extracellular or secreted target molecule.
  • a first molecule “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances, e.g., in a sample.
  • the targeting moiety “specifically binds” the target molecule if it binds to or associates with the target molecule with an affinity or Ka (that is, an association rate constant of a particular binding interaction with units of 1/M) of, for example, greater than or equal to about 104 M-1.
  • affinity may be defined as an equilibrium dissociation constant (KD) of a particular binding interaction with units of M (e.g., 10-2 M to 10-13 M, or less).
  • specific binding means the targeting moiety binds to the target molecule with a KD of less than or equal to about 10-5 M, less than or equal to about 10-6 M, less than or equal to about 10-7 M, less than or equal to about 10-8 M, or less than or equal to about 10-9 M, 10-10 M, 10-11 M, or 10-12 M or less.
  • the binding affinity of the targeting moiety for the target molecule can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 or BIAcore T200 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like.
  • SPR surface plasmon resonance
  • the targeting moiety is an antibody.
  • antibody is meant an antibody or immunoglobulin of any isotype (e.g., IgG (e.g., lgG1 , lgG2, lgG3, or lgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer which in turn is composed of two dimers of a heavy and light chain polypeptide); single chain antibodies (e.g., scFv); fragments of antibodies (e.g., fragments of whole or single chain antibodies) which retain specific binding to the target molecule (e.g., a cell surface molecule of a target cell), including, but not limited to single chain Fv (scFv), Fab, (Fab’)2, (scFv’)2, and diabodies; chimeric antibodies; monoclonal antibodies, human antibodies, humanized antibodies (e.g., human
  • the antibody is selected from an IgG, single chain Fv (scFv), Fab, (Fab)2, (scFv’)2, or a single variable domain located on a heavy chain (VHH).
  • VHH heavy chain
  • the antibody is a VHH (sometimes referred to herein and elsewhere as a “nanobody”).
  • the antibody may be detectably labeled, e.g., with an in vivo imaging agent, a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like.
  • an antibody included as the targeting moiety will vary based on the cell to be targeted.
  • the antibody specifically binds to an antigen on the surface of a target cell.
  • Target cells of interest include, but are not limited to, cells that are relevant to a particular disease or condition, e.g., a mucin-associated condition.
  • the target cell is selected from a cancer cell, an immune cell, and an endothelial cell. As such, in some embodiments, the target cells are cancer cells.
  • cancer cell is meant a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage-independent growth potential, ability to promote tumor growth and/or development in an immunocompromised non-human animal model, and/or any appropriate indicator of cellular transformation.
  • Cancer cell may be used interchangeably herein with “tumor cell”, “malignant cell” or “cancerous cell”, and encompasses cancer cells of a solid tumor, a semi-solid tumor, a primary tumor, a metastatic tumor, and the like. In certain embodiments, the cancer cell is a carcinoma cell.
  • the targeting moiety when the target cell is a cancer cell, specifically binds to a tumor antigen on the surface of the cancer cell.
  • tumor antigens to which the targeting moiety may specifically bind include 5T4, AXL receptor tyrosine kinase (AXL), B-cell maturation antigen (BCMA), c-MET, C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), Cadherin-6, CD19, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, Cripto protein, CS1 , delta-like canonical Notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), EpCAM, ephrin A4 (EFNA4), epidermal growth factor
  • Non-limiting examples of antibodies that specifically bind to tumor antigens which may be employed as a targeting moiety include Adecatumumab, Ascrinvacumab, Cixutumumab, Conatumumab, Daratumumab, Drozitumab, Duligotumab, Durvalumab, Dusigitumab, Enfortumab, Enoticumab, Figitumumab, Ganitumab, Glembatumumab, Intetumumab, Ipilimumab, Iratumumab, Icrucumab, Lexatumumab, Lucatumumab, Mapatumumab, Narnatumab, Necitumumab, Nesvacumab, Ofatumumab, Olaratumab, Panitumumab, Patritumab, Pritumumab, Radretumab, Ramucirumab,
  • variable is meant the antibody specifically binds to the particular antigen (e.g., HER2 for trastuzumab) but has fewer or more amino acids than the parental antibody (e.g., is a fragment (e.g., scFv) of the parental antibody), has one or more amino acid substitutions relative to the parental antibody, or a combination thereof.
  • the targeting moiety is an antibody approved by the United
  • EMA European Medicines Agency
  • targeting moieties are antibodies specific for antigens that include, but are not necessarily limited to: carbonic anhydrase IX, alpha-fetoprotein (AFP), a- actinin-4, A3, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1 , CASP-8/m, CCL19, CCL21 , CD1 , CD1 a, CD2, CD3, CD4, CD5, CD8, CD11 A, CD14, CD15, CD16, CD18, CD19, CD20, CD21 , CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147
  • the mucin-active protease may be stably associated with (e.g., conjugated to, fused to, or the like) any convenient portion of the antibody.
  • the mucin-active protease is stably associated with a light chain of the antibody, e.g., a kappa (K) light chain or fragment thereof or a lambda (A) light chain or fragment thereof.
  • the antibody light chain or fragment thereof includes a light chain variable region (VL).
  • VL light chain variable region
  • Such an antibody light chain or fragment thereof may further include an antibody light chain constant region (CL) or fragment thereof.
  • the antibody light chain or fragment thereof is a full-length antibody light chain - that is, an antibody light chain that includes a VL and a CL.
  • the mucin-active protease is stably associated with a VL (if present) or a CL (if present), e.g., at or near the N-terminus of a VL or at or near the C-terminus of a CL.
  • the mucin-active protease may be stably associated with a heavy chain or fragment thereof of the antibody.
  • the antibody heavy chain or fragment thereof includes a y, a, 5, E, or p antibody heavy chain or fragment thereof.
  • the antibody heavy chain or fragment thereof is an IgG heavy chain or fragment thereof, e.g., a human IgG 1 heavy chain or fragment thereof.
  • the antibody heavy chain or fragment thereof comprises a heavy chain variable region (VH).
  • VH heavy chain variable region
  • Such an antibody heavy chain or fragment thereof may further include a heavy chain constant region or fragment thereof.
  • the antibody heavy chain constant region or fragment thereof may include one or more of a CH1 domain, CH2 domain, and/or CH3 domain.
  • the antibody heavy chain is a full-length antibody heavy chain - that is, an antibody heavy chain that includes a VH, a CH1 domain, a CH2 domain, and a CH3 domain.
  • the mucin-active protease is stably associated with an Fc region of the antibody.
  • the mucin-active protease is stably associated with the antibody at or near the N-terminus of a VH or at or near the C-terminus of a CH3 domain.
  • the targeting moiety is a ligand.
  • a “ligand” is a substance that forms a complex with a biomolecule in nature to serve a biological purpose.
  • the ligand may be a substance selected from a circulating factor, a secreted factor, a cytokine, a growth factor, a hormone, a peptide, a polypeptide, a small molecule, and a nucleic acid, that forms a complex with the target molecule, e.g., a cell surface molecule on the surface of a target cell.
  • the targeting moiety when the targeting moiety is a ligand, the ligand is modified in such a way that complex formation with the target molecule occurs, but the normal biological result of such complex formation does not occur.
  • the ligand is the ligand of a cell surface receptor present on a target cell.
  • Cell surface receptors of interest include, but are not limited to, receptor tyrosine kinases (RTKs), non-receptor tyrosine kinases (non-RTKs), growth factor receptors, etc.
  • the mucin-active protease When the mucin-active protease is stably associated with a ligand as the targeting moiety, the mucin-active protease may be stably associated with any suitable region of the ligand, e.g., a region of attachment that does not interfere or substantially interfere with the ability of the ligand to bind (e.g., specifically bind) the target molecule.
  • the targeting moiety is an aptamer.
  • aptamer is meant a nucleic acid (e.g., an oligonucleotide) that has a specific binding affinity for the target molecule.
  • the aptamer is a DNA aptamer (e.g., AS1411 ).
  • the aptamer is an RNA aptamer (e.g., E3, C36, and the like).
  • the aptamer is a DNA/RNA hybrid (e.g., Apt-dONT-DEN, A10-3-J1 ).
  • RNA aptamers include, but are not limited to, those that target: 4,4’-methylenedianiline (MDA), Acetylcholine receptor (AChR), African trypanosomes, AMPA receptor GluR2Qflip, Beta Secretace (S10), Beta Secretase (THU), CD4, CTLA-4, EGFR (E07), EGFR (J18), , EGFRvll I (E17), Erythrocyte membrane protein 1 (PfEMPI), gp120, HER3, Human keratinocyte growth factor, L-selectin, Neruotensin-1 (NTS-1), NF-KB, Phosphatidylcholine: cholesterol liposomes, PSMA (A10), Raf-1 , RET receptor tyrosine kinase, TCF-1 , Tenascin-C (AptamerTTAI), TGF-
  • RNA aptamers include, but are not limited to: E3, C36, E2, PTCH1 -SMG-E1 , A10, A15, anti- PSMA aptamer, EpDT3, A10, A10-3.2, and SZTI01 (see, e.g., Wang et al., Molecules. 2022 Jun; 27(1 1 ): 3446).
  • DNA aptamers include, but are not limited to: AS1411 , CD, Apt 1 , Ecad01 , DAC, A10, and A9 (see, e.g., Wang et aL, Molecules. 2022 Jun; 27(1 1 ): 3446).
  • Aptamers can exhibit certain desirable properties for targeted delivery, such as ease of selection and synthesis, high binding affinity and specificity, low immunogenicity, and versatile synthetic accessibility.
  • Aptamers that bind to cell surface molecules are known and include, e.g., TTA1 (a tumor targeting aptamer to the extracellular matrix protein tenascin-C).
  • Aptamers that find use in the context of the present disclosure include those described in Zhu et al. (2015) ChemMedChem 10(1 ):39-45; Sun et al. (2014) Mol. Ther. Nucleic Acids 3:e182; Zhang et al. (2011 ) Curr. Med. Chem.
  • the targeting moiety is a small molecule.
  • small molecule is meant a compound having a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less. In certain aspects, the small molecule is not made of repeating molecular units such as are present in a polymer.
  • the target molecule is a cell surface receptor for which the ligand is a small molecule
  • the targeting moiety is the small molecule ligand (or a derivative thereof) of the receptor.
  • Small molecules that find use as targeting moieties are known.
  • folic acid (FA) derivatives have been shown to effectively target certain types of cancer cells by binding to the folate receptor, which is overexpressed, e.g., in many epithelial tumors. See, e.g., Vergote et al. (2015) Ther. Adv. Med. Oncol. 7(4):206-218.
  • the small molecule sigma-2 has proven to be effective in targeting cancer cells. See, e.g., Hashim et al.
  • Sigma-2 is the small molecule ligand for sigma-2 receptors, which are overexpressed in many proliferating tumor cells including pancreatic cancer cells.
  • a small molecule is employed as the targeting moiety, and it has been demonstrated in the context of a small molecule drug conjugate (SMDC) that the small molecule is effective at targeting a drug to a target cell of interest by binding to a cell surface molecule on the target cell.
  • SMDC small molecule drug conjugate
  • nucleic acids encoding a subject variant AAV capsid protein where the nucleotide sequence encoding the variant AAV capsid protein includes a TAG stop codon at the position encoding said unnatural amino acid substitution.
  • This is to allow for the insertion of an azide-bearing unnatural amino acid, e.g., using a cell having the appropriate tRNA to recognize the stop codon and insert the unnatural amino acid (e.g., Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA pyi , see, e.g., Hohl et al., Sci Rep. 2019 Aug 19;9(1):11971 ).
  • PylRS Pyrrolysyl-tRNA synthetase
  • nucleic acids examples include, but are not limited to: plasmids, viral vectors, and the like.
  • the nucleic acid is a viral vector that encodes a variant AAV capsid protein and includes a nucleotide sequence of interest.
  • an rAAV particle includes a variant AAV protein and a payload nucleic acid that includes a nucleotide sequence of interest.
  • nucleic acid payload of interest can be any convenient nucleic acid.
  • the nucleic acid payload of interest (DNA or mRNA) encodes a polypeptide (e.g., a therapeutic protein or a genome-editing enzyme such as a CRISPR/Cas effector protein, a zinc finger nuclease, or a TALEN).
  • the nucleotide sequence encoding the polypeptide is operably linked to the small ubiquitous INS84 promoter (see, e.g., Chai, S. et al. Hum.
  • the nucleic acid payload of interest is a non-coding RNA (e.g., a CRISPR/Cas guide RNA, an shRNA, an siRNA, a miRNA, an aptamer, a ribozyme) or encodes that non-coding RNA.
  • a non-coding RNA e.g., a CRISPR/Cas guide RNA, an shRNA, an siRNA, a miRNA, an aptamer, a ribozyme
  • encodes that non-coding RNA e.g., a CRISPR/Cas guide RNA, an shRNA, an siRNA, a miRNA, an aptamer, a ribozyme
  • a subject nucleic acid in addition to including a sequence that encodes a variant AAV capsid protein, also encodes a nucleic acid insert (also referred to as a heterologous nucleotide sequence or the “nucleotide sequence of interest”).
  • a subject rAAV particle in addition to including a variant AAV capsid protein, also includes (e.g., encapsidates) a nucleic acid payload of interest (which includes a nucleotide sequence of interest).
  • nucleotide sequence of interest can be operably linked to control elements directing the transcription or expression thereof once the sequence is present inside of a cell (e.g., in some cases integrated into the cell’s genome).
  • control elements can comprise control sequences normally associated with the selected gene (e.g., endogenous cellular control elements).
  • heterologous control sequences can be employed.
  • Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes.
  • Examples include, but are not limited to, the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, an endogenous cellular promoter heterologous to the gene of interest, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like.
  • CMV cytomegalovirus
  • CMVIE CMV immediate early promoter region
  • RSV rous sarcoma virus
  • synthetic promoters hybrid promoters, and the like.
  • sequences derived from nonviral genes such as the murine metallothionein gene, can also be used.
  • Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif.).
  • a cell type-specific or a tissue-specific promoter can be operably linked to the nucleotide sequence of interest and allowing for selective or preferential expression in a particular cell type(s) or tissue(s).
  • an inducible promoter can be operably linked to the nucleotide sequence of interest.
  • a nucleic acid payload is packaged with the variant AAV capsid polypeptides of the disclosure.
  • the nucleic acid payload is at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, or 1500 nucleotides (nt) in length.
  • the nucleic acid payload is 50 nucleotides to 4000 nucleotides long (e.g., 50-3000, 50-2000, 50-1500, 50-1200, 50-1000, 50-900, 50-750, 50-500, 100-4000, 100-3000, 100-2000, 100- 1500, 100-1200, 100-1000, 100-900, 100-750, 100-500, 300-4000, 300-3000, 300- 2000, 300-1500, 300-1200, 300-1000, 300-900, 300-750, 300-500, 500-4000, 500- 3000, 500-2000, 500-1500, 500-1200, 500-1000, or 500-900 nt long).
  • the nucleotide sequence of interest is at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides (nt) in length.
  • the nucleotide sequence of interest is 50 nucleotides to 4000 nucleotides long (e.g., 50-3000, 50-2000, 50-1500, 50-1200, 50-1000, 50-900, 50-750, 50-500, 100-4000, 100-3000, 100-2000, 100-1500, 100-1200, 100-1000, 100- 900, 100-750, 100-500, 300-4000, 300-3000, 300-2000, 300-1500, 300-1200, 300- 1000, 300-900, 300-750, 300-500, 500-4000, 500-3000, 500-2000, 500-1500, 500- 1200, 500-1000, or 500-900 nt long).
  • an AAV vector packaged by a variant AAV capsid polypeptide is at least about 2000 nucleotides in total length and up to about 5000 nucleotides in total length. In some embodiments, an AAV vector packaged by the variant AAV capsid polypeptides is about 2000 nucleotides, about 2400 nucleotides, about 2800 nucleotides, about 3000 nucleotides, about 3200 nucleotides, about 3400 nucleotides, about 3600 nucleotides, about 3800 nucleotides, about 4000 nucleotides, about 4200 nucleotides, about 4400 nucleotides, about 4600 nucleotides, about 4700 nucleotides, or about 4800 nucleotides.
  • an AAV vector packaged by the variant AAV capsid polypeptides is between about 2000 nucleotides (2 kb) and about 5000 nucleotides (5 kb). In some embodiments, an AAV vector packaged by the variant AAV capsid polypeptides is between about 2400 nucleotides (2.4 kb) and about 4800 nucleotides (4.8 kb). In some embodiments, an AAV vector packaged by the variant AAV capsid polypeptides is between about 3000 nucleotides (3 kb) and about 5000 nucleotides (5 kb). In some embodiments, an AAV vector packaged by the variant AAV capsid polypeptides is between about 3000 nucleotides (3 kb) and about 4000 nucleotides (4 kb).
  • the AAV vectors or AAV virions disclosed herein can also include conventional control elements operably linked to the nucleic acid insert (also referred to as a heterologous nucleotide sequence or a “nucleotide sequence of interest”) in a manner permitting transcription, translation and/or expression in a cell transfected with the AAV vector or infected with the AAV virion produced according to the present disclosure.
  • nucleic acid insert also referred to as a heterologous nucleotide sequence or a “nucleotide sequence of interest”
  • operably linked sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e. , Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • polyA polyadenylation
  • a great number of expression control sequences, including promoters selected from native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.
  • constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al., Cell, 41 :521 -530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 promoter (Invitrogen).
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only.
  • Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clonetech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art.
  • inducible promoters regulated by exogenously supplied compounds include, the zinc-inducible sheep metalloth ionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al., (1996) Proc. Natl. Acad. Sci. USA, 93:3346-3351), the tetracycline-repressible system (Gossen et al., (1992) Proc. Natl. Acad. Sci.
  • MT zinc-inducible sheep metalloth ionine
  • Dex dexamethasone
  • MMTV mouse mammary tumor virus
  • T7 polymerase promoter system WO 98/10088
  • ecdysone insect promoter No et al., (1996) Proc. Natl. Acad. Sci. USA, 93:
  • inducible promoters useful in this context are those regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.
  • a nucleotide sequence of interest is operably linked to a tissue-specific promoter.
  • a promoter active in muscle should be used. These include the promoters from genes encoding skeletal .beta.-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, as well as synthetic muscle promoters with activities higher than naturally-occurring promoters (see Li et al., Nat. Biotech., 17:241-245 (1999)). Examples of promoters that are tissue-specific are known for liver (albumin, Miyatake et al., (1997) J.
  • NSE neuron-specific enolase
  • AAV vectors or AAV virions carrying one or more therapeutically useful nucleic acid inserts also include selectable markers or reporter genes, e.g., sequences encoding geneticin, hygromycin or puromycin resistance, among others.
  • selectable reporters or marker genes can be used to signal the presence of the plasmids/vectors in bacterial cells, including, for example, examining ampicillin resistance.
  • Other components of the plasmid may include an origin of replication. Selection of these and other promoters and vector elements are conventional and many such sequences are available (see, e.g., Sambrook et al., and references cited therein).
  • a subject nucleotide sequence of interest encodes a non-coding RNA (e.g., a CRISPR/Cas guide RNA, an antisense RNA, a ribozyme, an shRNA, a microRNA, an aptamer).
  • a subject nucleotide sequence of interest encodes a protein (e.g., a therapeutic protein meant to alleviate a disease and/or its symptoms, a genome-editing enzyme such as a CRISPR/Cas effector protein, TALEN, Zinc Finger nuclease, etc. - meant to provide for targeted genome editing, etc.).
  • peptide or polypeptides envisioned as having a therapeutic activity for the multicellular organism in which they are expressed include, but are not limited to: factor VIII, factor IX, p-g lobin, a CRISPR/Cas effector protein (e.g., Cas9, Cpf 1 , and the like), a low- density lipoprotein receptor, adenosine deaminase, purine nucleoside phosphorylase, sphingomyelinase, glucocerebrosidase, cystic fibrosis transmembrane conductance regulator, a1 -antitrypsin, CD-18, PDGF, VEGF, EGF, TGFa, TGB , FGF, TNF, IL-1 , IL-2, IL-6, IL-8, endothelium derived growth factor (EDGF), ornithine transcarba
  • factor VIII factor VIII
  • factor IX p-g lobin
  • a neurotrophin e.g. NGF, BDNF, NT-3, NT-4, CNTF
  • Kifap3, Bcl-xl collapsin response mediator protein 1 , Chkp, calmodulin 2, calcyon, NPT1 , Eef1 a1 , Dhps, Cd151 , Morf412, CTGF, LDH-A, AtH , NPT2, Ehd3, Cox5b, Tubala, y-actin, Rpsa, NPG3, NPG4, NPG5, NPG6, NPG7, NPG8, NPG9, NPG10, dopamine, interleukins, cytokines, small peptides, the genes/proteins listed in Table 1 (see below: BCKDH complex (E1 a, E1 b and E2 subunits); Methylmalonyl-CoA Mutase; Propionyl-CoA Carboxylase (Alpha and Beta subunits); Isovaleryl
  • Nonlimiting examples of targeted nucleases include naturally occurring and recombinant nucleases, e.g. restriction endonucleases, meganucleases homing endonucleases, CRISPR/Cas effector proteins (e.g., CRISPR/Cas endonucleases such as Cas9, Cas12, Cas13, and the like).
  • naturally occurring and recombinant nucleases e.g. restriction endonucleases, meganucleases homing endonucleases, CRISPR/Cas effector proteins (e.g., CRISPR/Cas endonucleases such as Cas9, Cas12, Cas13, and the like).
  • any targeted nuclease(s) that are specific for the integration site of interest and promote the cleavage of an integration site may be encoded by a nucleotide sequence of interest
  • any examples of nucleases are known in the art, including Zinc finger nucleases (ZFNs), Transcription Activator- Like Effector Nucleases (TALENs), CRISPR/Cas effector proteins, meganucleases, homing endonucleases, restriction endonucleases, and the like (e.g., RecBCD endonuclease, T7 endonuclease, T4 endonuclease IV, Bal 31 endonuclease, Endonuclease I (endo I), Endonuclease II (endo VI, exo III), Micrococcal nuclease, Neurospora endonuclease, S1 -nuclease, P1- nuclease, Mung bean nu
  • the disclosure provides variant AAV capsid polypeptides capable of forming capsids capable of packaging a variety of therapeutic molecules, including nucleic acids and polypeptides.
  • the disclosure provides for AAV vectors capable of containing nucleic acid inserts, including for example, transgene inserts or other nucleic acid inserts. This allows for vectors capable of expressing polypeptides.
  • nucleic acids can comprise heterologous nucleic acid, nucleic acid gene products, and polypeptide gene products.
  • the nucleotide sequence of interest encodes a non-coding RNA, encodes a protein coding sequence, is an expression cassette, is a multiexpression cassette, is a sequence for homologous recombination, is a genomic gene targeting cassette, and/or is a therapeutic expression cassette.
  • the expression cassette is a CRISPR/CAS expression system (e.g., including a CRISPR/Cas guide RNA and a CRISPR/Cas effector protein such as Cas9 or Cpf 1 .
  • a nucleic acid insert comprises a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product, e.g., a nucleic acid gene product or a polypeptide gene product.
  • the gene product is an interfering RNA (e.g., shRNA, siRNA, miRNA).
  • the gene product is an aptamer.
  • the gene product can be a self-complementary nucleic acid.
  • the gene product is a polypeptide-coding RNA (e.g., an mRNA).
  • Suitable heterologous gene product includes interfering RNA, antisense RNA, ribozymes, and aptamers.
  • the gene product is an interfering RNA (RNAi)
  • suitable RNAi include RNAi that decrease the level of a target polypeptide in a cell.
  • exemplary polypeptides include neuroprotective polypeptides and/or anti-angiogenic polypeptides (both of which are therapeutic polypeptides).
  • Suitable polypeptides include, but are not limited to, glial derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF-2), neurturin, ciliary neurotrophic factor (CNTF), nerve growth factor (NGF; e.g., nerve growth factor-.
  • BDNF brain derived neurotrophic factor
  • NT-3 neurotrophin-3
  • NT-4 neurotrophin-4
  • NT-6 neurotrophin-6
  • EGF epidermal growth factor
  • PEDF pigment epithelium derived factor
  • Wnt polypeptide soluble Flt-1 , angiostatin, endostatin, VEGF, an anti-VEGF antibody, a soluble VEGFR, Factor VIII (FVIII), Factor IX (FIX), and a member of the hedgehog family (sonic hedgehog, Indian hedgehog, and desert hedgehog, etc.).
  • useful therapeutic products encoded by the heterologous nucleic acid sequence include hormones and growth and differentiation factors including, without limitation, insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), any one of the transforming growth factor alpha superfamily, including TGF.alpha., activins, inhibins, or any of the bone
  • useful heterologous nucleic acid sequence products include proteins that regulate the immune system including, without limitation, cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25 (including IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors alpha and beta., interferons (alpha, beta, and gamma), stem cell factor, flk-2/flt3 ligand.
  • TPO thrombopoietin
  • IL interleukins
  • IL-1 through IL-25 including IL-2, IL-4, IL-12 and IL-18
  • monocyte chemoattractant protein including IL-2, IL-4, IL-12 and IL-18
  • monocyte chemoattractant protein including IL-2, IL-4,
  • immunoglobulins IgG, IgM, IgA, IgD and IgE include, without limitations, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules, as well as engineered immunoglobulins and MHC molecules.
  • Useful gene products also include complement regulatory proteins such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1 , CF2 and CD59.
  • complement regulatory proteins such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1 , CF2 and CD59.
  • useful heterologous nucleic acid sequence products include any one of the receptors for the hormones, growth factors, cytokines, lymphokines, regulatory proteins and immune system proteins.
  • Useful heterologous nucleic acid sequences also include receptors for cholesterol regulation and/or lipid modulation, including the low density lipoprotein (LDL) receptor, high density lipoprotein (HDL) receptor, the very low density lipoprotein (VLDL) receptor, and scavenger receptors.
  • LDL low density lipoprotein
  • HDL high density lipoprotein
  • VLDL very low density lipoprotein
  • scavenger receptors scavenger receptors.
  • the disclosure also encompasses the use of gene products such as members of the steroid hormone receptor superfamily including glucocorticoid receptors and estrogen receptors, Vitamin D receptors and other nuclear receptors.
  • useful gene products include transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1 , AP-2, myb, MyoD and myogenin, ETS-box containing proteins, TFE3, E2F, ATF1 , ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4 C/EBP, SP1 , CCAAT-box binding proteins, interferon regulation factor (IRF-1 ), Wilms tumor protein, ETS-binding protein, STAT, GATA-box binding proteins, e.g., GATA-3, and the forkhead family of winged helix proteins.
  • transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1 , AP-2, myb, MyoD and myogenin, ETS-box containing proteins, TFE3, E2F, ATF1 , ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4 C/EBP,
  • useful heterologous nucleic acid sequence products include, carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, cystathionine beta-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, Flprotein
  • Still other useful gene products include enzymes useful in enzyme replacement therapy, and which are useful in a variety of conditions resulting from deficient activity of enzyme.
  • enzymes containing mannose-6-phosphate may be utilized in therapies for lysosomal storage diseases (e.g., a suitable gene includes that encoding .beta.-glucuronidase (GUSB)).
  • Host cells are necessary for generating infectious AAV vectors as well as for generating AAV virions based on the disclosed AAV vectors. Accordingly, the present disclosure provides host cells for generation and packaging of AAV virions based on the AAV vectors of the present disclosure.
  • a variety of host cells are known in the art and find use in the methods of the present disclosure. Any host cells described herein or known in the art can be employed with the compositions and methods described herein - as long as the cell allows for the inclusion of an azide-bearing unnatural amino acid at a stop codon - as described elsewhere herein (e.g., the cell is modified to express an appropriate amnio-acid bearing tRNA).
  • the present disclosure provides host cells, e.g., comprising a subject rAAV particle (virion) and/or a subject nucleic acid.
  • a subject host cell can be an isolated cell, e.g., a cell in in vitro culture.
  • a subject host cell can be useful for producing a subject AAV vector or AAV virion. Where a subject host cell is used to produce a subject AAV virion, it is referred to as a "packaging cell.”
  • a subject host cell is stably genetically modified with a subject AAV vector.
  • a subject host cell is transiently genetically modified with a subject AAV vector.
  • a subject nucleic acid is introduced stably or transiently into a host cell, using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, baculovirus infection, and the like.
  • a subject nucleic acid will generally further include a selectable marker, e.g., any of several well-known selectable markers such as neomycin resistance, and the like.
  • the host cell for use in generating infectious virions can be selected from any biological organism, including prokaryotic (e.g., bacterial) cells, and eukaryotic cells, including, insect cells, yeast cells and mammalian cells.
  • a subject host cell is generated by introducing a subject nucleic acid (i.e., AAV vector) into any of a variety of cells, e.g., mammalian cells, including, e.g., murine cells, and primate cells (e.g., human cells).
  • AAV vector a subject nucleic acid
  • mammalian cells including, e.g., murine cells
  • primate cells e.g., human cells.
  • Particularly desirable host cells are selected from among any mammalian species.
  • cells include without limitation, cells such as A549, WEHI, 10T1/2, BHK, MDCK, COS 1 , COS 7, BSC 1 , BSC 40, BMT 10, WI38, HeLa, CHO, 293, Vero, NIH 3T3, PC12, Huh-7 Saos, C2C12, RAT1 , Sf9, L cells, HT1080, human embryonic kidney (HEK), human embryonic stem cells, human adult tissue stem cells, pluripotent stem cells, induced pluripotent stem cells, reprogrammed stem cells, organoid stem cells, bone marrow stem cells, HLHepG2, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals including human, monkey, mouse, rat, rabbit, and hamster.
  • HEK human embryonic kidney
  • the selection of the mammalian species providing the cells is not a limitation of this disclosure; nor is the type of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell, etc.
  • the requirement for the cell used is it is capable of infection or transfection by an AAV vector.
  • the host cell is one that has Rep and Cap stably transfected in the cell, including in some embodiments a variant AAV capsid polypeptide as described herein.
  • the host cell expresses a variant AAV capsid polypeptide of the disclosure or part of an AAV vector as described herein, such as a heterologous nucleic acid sequence contained within the AAV vector.
  • the preparation of a host cell according to the disclosure involves techniques such as assembly of selected DNA sequences. This assembly may be accomplished utilizing conventional techniques. Such techniques include cDNA and genomic cloning, which are well known and are described in Sambrook et al., cited above, use of overlapping oligonucleotide sequences of the adenovirus and AAV genomes, combined with polymerase chain reaction, synthetic methods, and any other suitable methods providing the desired nucleotide sequence.
  • introduction of the AAV vector into the host cell may also be accomplished using techniques known to the skilled artisan and as discussed throughout the specification.
  • standard transfection techniques are used, e.g., CaPO4 transfection or electroporation, and/or infection by hybrid adenovirus/AAV vectors into cell lines such as the human embryonic kidney cell line HEK293 (a human kidney cell line containing functional adenovirus E1 genes providing trans-acting E1 proteins).
  • a subject genetically modified host cell includes, in addition to a nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein, as described above, a nucleic acid that comprises a nucleotide sequence encoding one or more AAV Rep proteins.
  • a subject host cell further comprises an AAV vector.
  • An AAV virion can be generated using a subject host cell. Methods of generating an AAV virion are described in, e.g., U.S. Patent Publication No. 2005/0053922 and U.S. Patent Publication No. 2009/0202490.
  • the host cell contains the sequences driving expression of the AAV capsid polypeptide (including variant AAV capsid polypeptides and non-variant parent capsid polypeptides) in the host cell and Rep sequences of the same serotype as the serotype of the AAV Inverted Terminal Repeats (ITRs) found in the nucleic acid insert (also referred to as a heterologous nucleotide sequence or the “nucleotide sequence of interest”), or a crosscomplementing serotype.
  • the AAV Cap and Rep sequences may be independently obtained from an AAV source and may be introduced into the host cell in any manner known to one of skill in the art or as described herein.
  • sequences encoding each of the essential Rep proteins may be supplied by AAV8, or the sequences encoding the Rep proteins may be supplied by different AAV serotypes (e.g., AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and/or AAV9).
  • the host cell stably contains the capsid protein under the control of a suitable promoter (including, for example, the variant AAV capsid polypeptides of the disclosure), such as those described above.
  • the capsid protein is expressed under the control of an inducible promoter.
  • the capsid protein is supplied to the host cell in trans. When delivered to the host cell in trans, the capsid protein may be delivered via a plasmid containing the sequences necessary to direct expression of the selected capsid protein in the host cell.
  • the vector encoding the capsid protein when delivered to the host cell in trans, also carries other sequences required for packaging the AAV, e.g., the Rep sequences.
  • the host cell stably contains the Rep sequences under the control of a suitable promoter, such as those described above.
  • the essential Rep proteins are expressed under the control of an inducible promoter.
  • the Rep proteins are supplied to the host cell in trans.
  • the Rep proteins may be delivered via a plasmid containing the sequences necessary to direct expression of the selected Rep proteins in the host cell.
  • the vector encoding the capsid protein (including, for example, the variant AAV capsid polypeptides of the disclosure) also carries other sequences required for packaging the AAV vector, e.g., the Rep sequences.
  • the Rep and Cap sequences may be transfected into the host cell on a single nucleic acid molecule and exist stably in the cell as an unintegrated episome.
  • the Rep and Cap sequences are stably integrated into the chromosome of the cell.
  • Another embodiment has the Rep and Cap sequences transiently expressed in the host cell.
  • a useful nucleic acid molecule for such transfection comprises, from 5' to 3', a promoter, an optional spacer interposed between the promoter and the start site of the Rep gene sequence, an AAV Rep gene sequence, and an AAV Cap gene sequence.
  • the molecule(s) providing Rep and capsid can exist in the host cell transiently (i.e., through transfection), in some embodiments, one or both of the Rep and capsid proteins and the promoter(s) controlling their expression be stably expressed in the host cell, e.g., as an episome or by integration into the chromosome of the host cell.
  • the methods employed for constructing embodiments of the disclosure are conventional genetic engineering or recombinant engineering techniques such as those described in the references above.
  • the packaging host cell can require helper functions in order to package the AAV vector of the disclosure into an AAV virion.
  • these functions may be supplied by a herpesvirus.
  • the necessary helper functions are each provided from a human or non-human primate adenovirus source, and are available from a variety of sources, including the American Type Culture Collection (ATCC), Manassas, Va. (US).
  • the host cell is provided with and/or contains an E1a gene product, an E1b gene product, an E2a gene product, and/or an E4 ORF6 gene product.
  • the host cell may contain other adenoviral genes such as VAI RNA. In some embodiments, no other adenovirus genes or gene functions are present in the host cell.
  • the disclosure provides a method for generating an AAV virion of the disclosure.
  • a variety of methods for generating AAV virions are known in the art and can be used to generate AAV virions comprising the AAV vectors described herein.
  • the methods involve inserting or transducing an AAV vector of the disclosure into a host cell capable of packaging the AAV vector into an AAV virion. Any convenient method known to one of skill in the art can be employed to generate the AAV virions of the disclosure.
  • An AAV vector comprising a heterologous nucleic acid and used to generate an AAV virion can be constructed using methods that are well known in the art. See, e.g., Koerber et al. (2009) Mol. Then, 17:2088; Koerber et al. (2008) Mol Then, 16: 1703- 1709; as well as U.S. Pat. Nos. 7,439,065, 6,951 ,758, and 6,491 ,907.
  • the heterologous sequence(s) can be directly inserted into an AAV genome with the major AAV open reading frames ("ORFs") excised therefrom.
  • AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions.
  • Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941 ; International Publication Nos. WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988- 3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J.
  • an AAV vector is introduced into a suitable host cell using known techniques, such as by transfection.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.
  • Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al. (1973) Virol.
  • Suitable host cells for producing AAV virions include any species and/or type of cell that can be, or have been, used as recipients of a heterologous AAV DNA molecule, and can support the expression of required AAV production cofactors from helper viruses.
  • Such host cells can include but are not limited to microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a heterologous DNA molecule.
  • the term includes the progeny of the original cell transfected.
  • a "host cell” as used herein generally refers to a cell transfected with an exogenous DNA sequence.
  • HEK293 Cells from the stable human cell line, HEK293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) can be used.
  • the human cell line HEK293 is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral E1 a and E1 b genes (Aiello et al. (1979) Virology 94:460).
  • the HEK293 cell line is readily transfected, and provides a convenient platform in which to produce AAV virions.
  • Methods of producing an AAV virion in insect cells are known in the art, and can be used to produce a subject AAV virion. See, e.g., U.S. Patent Publication No. 2009/0203071 ; U.S. Pat. No. 7,271 ,002; and Chen (2008) Mol. Then 16:924.
  • the AAV virion or AAV vector is packaged into an infectious virion or virus particle, by any of the methods described herein or known in the art.
  • the variant AAV capsid polypeptide allows for similar packaging as compared to a non-variant parent capsid polypeptide.
  • an AAV vector packaged with the variant AAV capsid polypeptides transduce into cells in vivo better than a vector packaged from non-variant parent capsid polypeptides.
  • the AAV vector packaged with the variant AAV capsid polypeptides transduce into cells in vitro better than a vector packaged from non-variant parent capsid polypeptides.
  • the variant AAV capsid polypeptides result in nucleic acid expression higher than a nucleic acid packaged from non-variant parent capsid polypeptides.
  • the AAV vector packaged with said variant AAV capsid polypeptides result in transgene expression better than a transgene packaged from non-variant parent capsid polypeptides.
  • the present disclosure provides pharmaceutical compositions useful in treating subjects according to the methods of the disclosure as described herein. Further, the present disclosure provides dosing regimens for administering the described pharmaceutical compositions.
  • the present disclosure provides pharmaceutical compositions comprising: a) a subject AAV vector or AAV virion, as described herein as well as therapeutic molecules packaged by or within capsids comprising variant polypeptides as described herein; and b) a pharmaceutically acceptable carrier, diluent, excipient, or buffer.
  • the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in a human.
  • excipients include any pharmaceutical agent that can be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol.
  • Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • a subject composition can comprise a liquid comprising a subject variant AAV capsid polypeptide of the disclosure or AAV virion comprising a variant AAV capsid polypeptide in solution, in suspension, or both.
  • liquid compositions include gels.
  • the liquid composition is aqueous.
  • the composition is an in situ gellable aqueous composition, e.g., an in situ gellable aqueous solution.
  • Aqueous compositions have ophthalmically compatible pH and osmolality.
  • compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery.
  • Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents.
  • Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals.
  • Supplementary active compounds e.g., preservatives, antibacterial, antiviral and antifungal agents
  • compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the art.
  • pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.
  • compositions suitable for parenteral administration comprise aqueous and nonaqueous solutions, suspensions or emulsions of the active compound. Preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non- limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable or synthetic oils.
  • penetrants can be included in the pharmaceutical composition.
  • Penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • the active ingredient can be formulated into aerosols, sprays, ointments, salves, gels, or creams as generally known in the art.
  • pharmaceutical compositions typically include ointments, creams, lotions, pastes, gels, sprays, aerosols, or oils.
  • Useful carriers include Vaseline. RTM., lanolin, polyethylene glycols, alcohols, transdermal enhancers, and combinations thereof.
  • Cosolvents and adjuvants may be added to the formulation.
  • cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters.
  • Adjuvants include, for example, surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.
  • compositions and delivery systems appropriate for the AAV vector or AAV virion and methods and uses of are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20.sup.th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18.sup.th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12.sup.th ed., Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 1 1 .sup.th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).
  • Doses can vary and depend upon whether the treatment is prophylactic or therapeutic, the type, onset, progression, severity, frequency, duration, or probability of the disease treatment is directed to, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan.
  • the dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy and the status of the subject. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.
  • Methods and uses of the disclosure as disclosed herein can be practiced within about 1 hour to about 2 hours, about 2 hours to about 4 hours, about 4 hours to about 12 hours, about 12 hours to about 24 hours or about 24 hours to about 72 hours after a subject has been identified as having the disease targeted for treatment, has one or more symptoms of the disease, or has been screened and is identified as positive as set forth herein even though the subject does not have one or more symptoms of the disease.
  • the disclosure as disclosed herein can be practiced within about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 72 hours or more.
  • methods and uses of the disclosure can be practiced about 1 day to about 7 days, about 7 days to about 14 days, about 14 days to about 21 days, about 21 days to about 48 days or more, months or years after a subject has been identified as having the disease targeted for treatment, has one or more symptoms of the disease, or has been screened and is identified as positive as set forth herein.
  • the disclosure as disclosed herein can be practiced within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 14 days, about 21 days, about 36 days, or about 48 days or more.
  • kits with packaging material and one or more components therein typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein.
  • a kit can contain a collection of such components, e.g., a variant AAV capsid polypeptide, an AAV vector, a nucleic acid encoding a variant AAV protein, and/or an AAV virion (in any combination thereof) and optionally a second active ingredient, such as another compound, agent, drug or composition.
  • a kit refers to a physical structure housing one or more components of the kit.
  • Packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.).
  • Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels or inserts can include information identifying the manufacturer, lot numbers, manufacturer location and date, expiration dates. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location and date.
  • Labels or inserts can include information on a disease a kit component may be used for.
  • Labels or inserts can include instructions for the clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimes described herein.
  • Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, complications or reactions, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects or complications could also occur when the subject has, will be or is currently taking one or more other medications that may be incompatible with the composition, or the subject has, will be or is currently undergoing another incompatible treatment protocol or therapeutic regimen and, therefore, instructions could include information regarding such incompatibilities.
  • Labels or inserts include "printed matter,” e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component.
  • Labels or inserts can additionally include a computer readable medium, such as a bar-coded printed label, a disk, optical disk such as CD- or DVD-ROIWRAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media or memory type cards.
  • the present disclosure provides methods for delivering a payload of interest to an individual (e.g., methods of treating a disease in a subject by administering the AAV vectors and/or nucleic acids of the present disclosure), where AAV virus, vectors and/or nucleic acids described herein comprising one or more variant AAV capsid polypeptides of the present disclosure are administered to the individual.
  • the disclosure provides a method of administering a pharmaceutical composition of the disclosure to a subject in need thereof to treat a disease of a subject.
  • the subject is not otherwise in need of administration of a composition of the disclosure.
  • the variant AAV capsid polypeptides package a therapeutic expression cassette comprised of a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product, such as for example a therapeutic protein.
  • the AAV virion or AAV vector comprises a therapeutic expression cassette comprised of a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product, such as for example a therapeutic protein.
  • variant AAV capsid polypeptides of the disclosure are employed as part of vaccine delivery.
  • Vaccine delivery can include delivery of any of the therapeutic proteins as well as nucleic acids described herein.
  • variant AAV capsid polypeptides of the disclosure are employed as part of a vaccine regimen and dosed according to the methods described herein.
  • the variant AAV capsid polypeptides, the AAV virions, or AAV vectors of the disclosure are used in a therapeutic treatment regimen.
  • the variant AAV capsid polypeptides, the AAV virions, or AAV vectors of the disclosure are used for therapeutic polypeptide production.
  • a subject variant AAV capsid polypeptides or AAV vector when introduced into the cells of a subject, provides for high level production of the heterologous gene product packaged by the variant AAV capsid polypeptides or encoded by the AAV vector.
  • a heterologous polypeptide packaged by the variant AAV capsid polypeptides or encoded by the AAV can be produced.
  • subject variant AAV capsid polypeptides, AAV virion, or AAV vector when introduced into a subject, provide for production of the heterologous gene product packaged by the variant AAV capsid polypeptides or encoded by the AAV vector in at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50% at least about 60%, at least about 70%, at least about 80%, or more than 80%, of the target cells.
  • the present disclosure provides a method of treating a disease, the method comprising administering to an individual in need thereof an effective amount of a therapeutic molecule packaged by the variant AAV capsid polypeptides or subject AAV vector as described above.
  • Subject variant AAV capsid polypeptides or subject AAV vectors can be administered systemically, regionally or locally, or by any route, for example, by injection, infusion, orally (e.g., ingestion or inhalation), or topically (e.g., transdermally).
  • Possible delivery and administration methods can include parenteral, intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous, intracavity, intracranial, transdermal (topical), transmucosal and rectal administration.
  • Example administration and delivery routes include intravenous, intraperitoneal, intrarterial, parenteral, subcutaneous, intra-pleural, topical, dermal, intradermal, transdermal, transmucosal, oral (alimentary), mucosal, respiration, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity, iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, and intralymphatic.
  • the delivery route is systemic (e.g., parenteral, intravenous).
  • a therapeutically effective amount of a therapeutic molecule packaged by the variant AAV capsid polypeptides or a subject AAV vectors is an amount that, when administered to an individual in one or more doses, is effective to slow the progression of the disease or disorder in the individual, or is effective to ameliorate symptoms.
  • a therapeutically effective amount of a therapeutic molecule packaged by the variant AAV capsid polypeptides or a subject AAV vectors can be an amount that, when administered to an individual in one or more doses, is effective to slow the progression of the disease by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more than about 80%, compared to the progression of the disease in the absence of treatment with the therapeutic molecule packaged by the variant AAV capsid polypeptides or AAV vectors.
  • a therapeutic or beneficial effect of treatment is therefore any objective or subjective measurable or detectable improvement or benefit provided to a particular subject.
  • a therapeutic or beneficial effect can but need not be complete ablation of all or any particular adverse symptom, disorder, illness, or complication of a disease.
  • a satisfactory clinical endpoint is achieved when there is an incremental improvement or a partial reduction in an adverse symptom, disorder, illness, or complication caused by or associated with a disease, or an inhibition, decrease, reduction, suppression, prevention, limit or control of worsening or progression of one or more adverse symptoms, disorders, illnesses, or complications caused by or associated with the disease, over a short or long duration (hours, days, weeks, months, etc.).
  • Improvement of clinical symptoms can also be monitored by one or more methods known to the art, and used as an indication of therapeutic effectiveness.
  • Clinical symptoms may also be monitored by anatomical or physiological means, such as indirect ophthalmoscopy, fundus photography, fluorescein angiopathy, optical coherence tomography, electroretinography (full-field, multifocal, or other), external eye examination, slit lamp biomicroscopy, applanation tonometry, pachymetry, autorefraction, or other measures of functional vision.
  • a therapeutic molecule including, for example, nucleic acid that includes a nucleotide sequence of interest packaged by the variant AAV capsid polypeptides, a subject AAV vector, or AAV virus, when introduced into a subject, provides for production of a heterologous gene product (e.g., non-coding or coding RNA, a protein) for a period of time from about 2 days to about 6 months, e.g., from about 2 days to about 7 days, from about 1 week to about 4 weeks, from about 1 month to about 2 months, or from about 2 months to about 6 months.
  • a heterologous gene product e.g., non-coding or coding RNA, a protein
  • therapeutic molecules packaged by the variant AAV capsid polypeptides, a subject AAV vector or virus, when introduced into a subject provides for production of the heterologous gene product for a period of time of more than 6 months, e.g., from about 6 months to 20 years or more, or greater than 1 year, e.g., from about 6 months to about 1 year, from about 1 year to about 2 years, from about 2 years to about 5 years, from about 5 years to about 10 years, from about 10 years to about 15 years, from about 15 years to about 20 years, or more than 20 years.
  • Multiple doses of a subject AAV virion can be administered to an individual in need thereof. Where multiple doses are administered over a period of time, an active agent is administered once a month to about once a year, from about once a year to once every 2 years, from about once every 2 years to once every 5 years, or from about once every 5 years to about once every 10 years, over a period of time.
  • a subject AAV virion is administered over a period of from about 3 months to about 2 years, from about 2 years to about 5 years, from about 5 years to about 10 years, from about 10 years to about 20 years, or more than 20 years.
  • the actual frequency of administration, and the actual duration of treatment depends on various factors.
  • the administration regimen is part of a vaccination regimen.
  • the dose to achieve a therapeutic effect e.g., the dose in vector genomes/per kilogram of body weight (vg/kg) will vary based on several factors including, but not limited to: route of administration, the level of heterologous polynucleotide expression required to achieve a therapeutic effect, the specific disease treated, any host immune response to the viral vector, a host immune response to the heterologous polynucleotide or expression product (e.g., RNA or protein), and the stability of the expressed molecule.
  • route of administration e.g., the level of heterologous polynucleotide expression required to achieve a therapeutic effect
  • the specific disease treated e.g., any host immune response to the viral vector, a host immune response to the heterologous polynucleotide or expression product (e.g., RNA or protein), and the stability of the expressed molecule.
  • a host immune response to the heterologous polynucleotide or expression product e.g., RNA or protein
  • doses will range from at least about, or more, for example, 1X10 9 , 1X1 O 10 , 1 X10 11 , 1 X10 12 , 1 X10 13 ,or 1X10 14 , or more, vector genomes per kilogram (vg/kg) of the weight of the subject, to achieve a therapeutic effect.
  • An effective amount or a sufficient amount can, but need not be, provided in a single administration, may require multiple administrations, and, can but need not be, administered alone or in combination with another composition (e.g., agent), treatment, protocol or therapeutic regimen.
  • another composition e.g., agent
  • the amount may be proportionally increased as indicated by the need of the subject, type, status and severity of the disease treated or side effects (if any) of treatment.
  • an effective amount or a sufficient amount need not be effective or sufficient if given in single or multiple doses without a second composition (e.g., another drug or agent), treatment, protocol or therapeutic regimen, since additional doses, amounts or duration above and beyond such doses, or additional compositions (e.g., drugs or agents), treatments, protocols or therapeutic regimens may be included in order to be considered effective or sufficient in a given subject.
  • Amounts considered effective also include amounts that result in a reduction of the use of another treatment, therapeutic regimen or protocol.
  • An effective amount or a sufficient amount need not be effective in each and every subject treated, or a majority of treated subjects in a given group or population.
  • An effective amount or a sufficient amount means effectiveness or sufficiency in a particular subject, not a group or the general population. As is typical for such methods, some subjects will exhibit a greater response, or less or no response to a given treatment method or use. Thus, appropriate amounts will depend upon the condition treated, the therapeutic effect desired, as well as the individual subject (e.g., the bioavailability within the subject, gender, age, etc.).
  • a detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression, or duration of the disease, or complication caused by or associated with the disease, or an improvement in a symptom or an underlying cause or a consequence of the disease, or a reversal of the disease.
  • a successful treatment outcome can lead to a "therapeutic effect,” or “benefit” of decreasing, reducing, inhibiting, suppressing, limiting, controlling or preventing the occurrence, frequency, severity, progression, or duration of a disease, or one or more adverse symptoms or underlying causes or consequences of the disease in a subject.
  • Treatment methods and uses affecting one or more underlying causes of the disease or adverse symptoms are therefore considered to be beneficial.
  • a decrease or reduction in worsening, such as stabilizing the disease, or an adverse symptom thereof, is also a successful treatment outcome.
  • a therapeutic benefit or improvement therefore need not be complete ablation of the disease, or any one, most or all adverse symptoms, complications, consequences or underlying causes associated with the disease.
  • a satisfactory endpoint is achieved when there is an incremental improvement in a subject's disease, or a partial decrease, reduction, inhibition, suppression, limit, control or prevention in the occurrence, frequency, severity, progression, or duration, or inhibition or reversal, of the disease (e.g., stabilizing one or more symptoms or complications), over a short or long duration of time (hours, days, weeks, months, etc.).
  • Effectiveness of a method or use such as a treatment that provides a potential therapeutic benefit or improvement of a disease, can be ascertained by various methods.
  • Disclosed methods and uses can be combined with any compound, agent, drug, treatment or other therapeutic regimen or protocol having a desired therapeutic, beneficial, additive, synergistic or complementary activity or effect.
  • exemplary combination compositions and treatments include second actives, such as, biologies (proteins), agents and drugs.
  • second actives such as, biologies (proteins), agents and drugs.
  • Such biologies (proteins), agents, drugs, treatments and therapies can be administered or performed prior to, substantially contemporaneously with or following any other method or use of the disclosure.
  • the compound, agent, drug, treatment or other therapeutic regimen or protocol can be administered as a combination composition, or administered separately, such as concurrently or in series or sequentially (prior to or following) delivery or administration of an AAV vector or AAV virion as described herein.
  • the disclosure therefore provides combinations where a method or use of the disclosure is in a combination with any compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition, set forth herein or known to one of skill in the art.
  • the compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition can be administered or performed prior to, substantially contemporaneously with or following administration of an AAV vector or AAV virion as described herein, to a subject.
  • Specific non-limiting examples of combination embodiments therefore include the foregoing or other compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition.
  • Methods and uses of the disclosure also include, among other things, methods and uses that result in a reduced need or use of another compound, agent, drug, therapeutic regimen, treatment protocol, process, or remedy.
  • a method or use of the disclosure has a therapeutic benefit if in a given subject a less frequent or reduced dose or elimination of administration of a recombinant clotting factor protein to supplement for the deficient or defective (abnormal or mutant) endogenous clotting factor in the subject.
  • methods and uses of reducing need or use of another treatment or therapy are provided.
  • the disclosure is useful in animals including veterinary medical applications. Suitable subjects therefore include mammals, such as humans, as well as non-human mammals such as non-human primates.
  • the term "subject” refers to an animal, typically a mammal, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and experimental animals (mouse, rat, rabbit, guinea pig).
  • Human subjects include fetal, neonatal, infant, juvenile and adult subjects.
  • Subjects include animal disease models, for example, mouse and other animal models of blood clotting diseases and others known to those of skill in the art.
  • a method or use of the disclosure includes: (a) providing an AAV virion whose capsid comprises a variant AAV capsid polypeptide (e.g., prepared as described herein), wherein the AAV virion comprises a heterologous nucleic acid sequence (e.g., in some cases operably linked to an expression control element conferring transcription of said nucleic acid sequence); and (b) administering an amount of the AAV virion to the mammal such that said heterologous nucleic acid is expressed in the mammal.
  • a heterologous nucleic acid sequence e.g., in some cases operably linked to an expression control element conferring transcription of said nucleic acid sequence
  • a method or use of the disclosure includes: (a) providing a therapeutic molecule packaged by variant AAV capsid polypeptides (e.g., prepared as described herein), wherein the therapeutic molecule comprises a heterologous nucleic acid sequence (e.g., which can in some cases be operably linked to an expression control element conferring transcription of said nucleic acid sequence); and (b) administering an amount of the therapeutic molecule (including, for example, a vaccine) packaged by variant AAV capsid polypeptides to the mammal such that said heterologous nucleic acid is expressed in the mammal.
  • a therapeutic molecule packaged by variant AAV capsid polypeptides e.g., prepared as described herein
  • the therapeutic molecule comprises a heterologous nucleic acid sequence (e.g., which can in some cases be operably linked to an expression control element conferring transcription of said nucleic acid sequence)
  • an amount of the therapeutic molecule including, for example, a vaccine
  • a method or use of the disclosure includes delivering or transferring a heterologous polynucleotide sequence into a mammal or a cell of a mammal, by administering a heterologous polynucleotide packaged by the variant AAV capsid polypeptides, a plurality of heterologous polynucleotides packaged by variant AAV capsid polypeptides, an AAV virion prepared as described herein, or a plurality of AAV virions comprising the heterologous nucleic acid sequence to a mammal or a cell of a mammal, thereby delivering or transferring the heterologous polynucleotide sequence into the mammal or cell of the mammal.
  • the heterologous nucleic acid sequence encodes a protein expressed in the mammal, or where the heterologous nucleic acid sequence encodes an inhibitory sequence or protein that reduces expression of an endogenous protein in the mammal.
  • a method or use of the disclosure includes is a method of delivering a payload of interest to the central nervous system of an individual, and includes administering to the individual a nucleic acid or a recombinant AAV (rAAV) particle as described herein (e.g., where the nucleic acid is a viral vector that encodes a variant AAV capsid protein and includes a nucleotides sequence of interest, where the rAAV particle comprises a variant AAV particle and a payload nucleic acid that includes a nucleotides sequence of interest).
  • rAAV recombinant AAV
  • a method of delivering a payload of interest to a target cell can include contacting the target cell with a subject recombinant AAV particle.
  • the target cell is in vivo and said contacting comprises administering the recombinant AAV particle to an individual.
  • Reagents, compositions, and kits/systems that find use in practicing the subject methods are provided.
  • a triple asterisks denotes a TAG stop codon (DNA nucleotides) inserted onto the AAV capsid sequence in order to replace the amino acid at that position with an unnatural amino acid.
  • the replacement is at the position reported in the name of the sequence (e.g., in “AAV8-T457” a stop TAG codon replaces the Threonine at amino acid position 457 in the AAV8 amino acid sequence - such that an unnatural amino acid (used for conjugation as described elsewhere herein) was instead placed at position 457).
  • a variant adeno-associated virus (AAV) capsid protein comprising: (a) an azide-bearing unnatural amino acid substitution at a position corresponding to:
  • variant AAV capsid protein of 1 comprising an amino acid sequence having:
  • variant AAV capsid protein of 1 comprising an amino acid sequence having 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, wherein said azide-bearing unnatural amino acid substitution is at position N589 or R587/A587.
  • variant AAV capsid protein of 1 comprising an amino acid sequence having 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 , 2, 7, and 13.
  • variant AAV capsid protein of 1 comprising an amino acid sequence having 90% or more (e.g., 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 , 2, 7, and 13.
  • the variant AAV capsid protein of 1 comprising an amino acid sequence having 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16.
  • the variant AAV capsid protein of 1 comprising an amino acid sequence having 90% or more (e.g., 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16. .
  • the variant AAV capsid protein of 1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16. .
  • the variant AAV capsid protein of 1 comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5-6, 12, or 14. 0.
  • the variant AAV capsid protein of 11 wherein the HSPG binding site is at an amino acid position corresponding to position 587 and/or 590 of SEQ ID NO: 1. 3.
  • the variant AAV capsid protein of any one of 1 -12, wherein the variant AAV capsid protein is conjugated to a DNA or RNA aptamer via the azide-bearing unnatural amino acid.
  • the variant AAV capsid protein of 18, wherein the PEG linker length is 4 kDa or less (e.g., 3 kDa or less, 2 kDa or less). 21 .
  • the variant AAV capsid protein of 18, wherein the PEG linker length is about 2 kDa.
  • a chemically modified variant adeno-associated virus (AAV) capsid protein comprising an azide-bearing unnatural amino acid substitution, wherein the capsid protein is conjugated to folic acid, a DNA aptamer, or an RNA aptamer via the azide-bearing unnatural amino acid.
  • AAV adeno-associated virus
  • a nucleic acid comprising a nucleotide sequence encoding the variant AAV capsid protein of any one of 1 -12, wherein said nucleotide sequence comprises a TAG stop codon at the position encoding said unnatural amino acid substitution.
  • a recombinant AAV particle comprising:
  • a polypeptide e.g., a genome-editing enzyme such as a CRISPR/Cas effector protein, a zinc finger nuclease, or a TALEN
  • a non-coding RNA e.g., a CRISPR/Cas guide RNA, an shRNA, an siRNA, a miRNA, an aptamer
  • encodes said non-coding RNA or is an mRNA.
  • a method of delivering a payload of interest to a target cell comprising contacting the target cell with the recombinant AAV particle of any one of 25-26.
  • Ne-AAV a novel class of programmable AAV vectors, Ne-AAV, was created by utilizing single unnatural amino acid insertion.
  • the different mutant capsids of NE-AAV vectors were characterized and the NE-AAV vectors were successfully conjugated by click chemistry.
  • NE- AAVs were successfully programmed to target specific cancer cells via folic acid, or DNA aptamer, or RNA aptamer conjugation.
  • folic acid conjugated AAV vectors and the DNA aptamer AS141 1 conjugated AAV vectors led to highly specific uptake in the intended target cells.
  • Example 1 Production of chemically modified AAV and click chemistry conjugation [00175] The AAV-DJ capsid was first chemically modified. AAV-DJ was previously isolated because of its efficiency in transducing many cells in vitro as well as its high titer production.
  • AAV-DJR/A capsid sequence DJR/A-N589 (see SEQ ID NO: 5), DJR/A-D555 (see SEQ ID NO: 4), DJR/A-A587 (see SEQ ID NO: 6), and DJR/A-T456 (see SEQ ID NO: 3)) as sites to replace the endogenous amino acid with the unnatural amino acid.
  • TAG amber stop codon
  • capsids were used for AAV production using a CAG promoter-firefly luciferase expression cassette for in vitro experiments.
  • the pAcBad ,tR4-MbPyl plasmid which expresses a pyrrolysyl-tRNA and a pyrrolysyl- tRNA synthase, both derived from the Methanosarcina barkeri, was added to the classical triple transfection protocol for AAV production.
  • the unnatural amino acid, azido-lysine was supplemented into the cell media during the vector preparation (Figure 1A).
  • This novel AAV referred to herein as Ne-AAV, carrying a single azido-amino acid insertion on a specific capsid position allowed the conjugation of any molecule containing a dibenzocyclooctyne chemical group (DBCO) to the capsid by simply performing a click chemistry reaction ( Figure 1 A).
  • DBCO dibenzocyclooctyne chemical group
  • Ne-DJR/A mutants were assessed in HeLa cells.
  • the Ne-DJR/A- N589 and Ne-DJR/A-A587 vectors resulted in slightly higher luciferase activity compared to the Ne-DJR/A-D555 and Ne-DJR/A-T456 vectors ( Figure 7B).
  • Ne-DJR/A-N589 vector was conjugated with a DBCO-biotin to detect the biotin-AAV by western blot using a streptavidin-labeled antibody.
  • both the unconjugated and conjugated NE-AAV vectors were detected by the anti-AAV antibody AAVB1 (red signal), while the streptavidin antibody (green signal) recognized only the biotin- conjugated Ne-AAV.
  • the streptavidin signal overlapped with the AAVB1 antibody (yellow signal) demonstrating the specific nature of the biotin conjugation on the AAV capsid proteins VP1/2/3 ( Figure 1 B).
  • Example 2 Design and characterization of Ns-AAVs upon folic acid conjugation [00181] Since the Ne-AAV capsid incorporated the azido-lysine on each of the VP1 , VP2, and VP3 proteins ( Figure 2A), an attempt was also made to specifically target the insertion of the unnatural amino acid onto only the VP2 ( Figure 2B) or VP3 proteins ( Figure 2C).
  • NE-AAVS vectors were produced expressing luciferase and the folic acid-conjugated AAV vectors were tested on cancer cell lines that are reported to express high levels of human folic acid receptor (hFOLRI ) (see htt followed by ps:/ followed by /ww followed by w.p followed by roteinatlas followed by .org/ followed by ENSG00000110195-FOLR1].; Figure 7F).
  • hFOLRI human folic acid receptor
  • the folic acid-conjugated Ne-AAV and Ne-VP3-AAV showed significant higher luciferase activity compared to the unconjugated Ne-AAV control vectors (mock) ( Figure 2F).
  • the FA-conjugated Ne-AAV displayed remarkably enhanced transduction efficiency compared to both the FA-conjugated Ne-VP2-AAV and Ne- VP3-AAV ( Figure 2F).
  • the Ne-VP2-AAV and Ne-VP3-AAV vectors showed significant decreased infectivity even when transduced at higher multiplicity of infection (MOI) ( Figure 7G). Because the Ne-VP2-AAV and Ne-VP3-AAV displayed an overall reduced ability to transduce cells, the Ne-AAV vectors were used in consequent studies (Figure 2A).
  • Example 3 Rational muta enesis screen to identify novel Ne-AAV mutant capsids
  • the unnatural amino acid was introduced on AAV8 and AAVLK03 capsids.
  • new vectors were generated with the azidolysine displayed on the capsid surface.
  • Eight new Ne-AAV variants were produced (five AAV8 and three LK03 mutants, where the capital letter and the number next to the AAV serotype indicate the position and amino acid replaced by the azido-lysine on the vector capsid).
  • all the new Ne-AAV vectors showed a lower titer compared to their wild-type counterpart ( Figure 8A).
  • luciferase-expressing vectors were made using either regular media, where no vector is expected to be produced, or supplemented with the azido-lysine and their infectivity on HeLa cells was assessed. For a quick assessment of transduction, crude AAV lysates were used. Among the new vectors only Ne-AAV8-T457 and Ne-LK03-N588 retained the ability to transduce cells when produced in presence of azido-lysine, while Ne-DJR/A-N589 was used as control ( Figure 3A).
  • Ne-DJR/A-N589 and Ne-AAV8-T457 were conjugated with DBCO-FA containing different lengths of PEG (2kDa, 5kDa and 10kDa).
  • DBCO-FA containing different lengths of PEG (2kDa, 5kDa and 10kDa).
  • both the Ne-DJR/A-N589 and Ne-AAV8-T457 conjugated with the DBCO-FA-2k demonstrated a significant increase in luciferase activity compared to the vectors with longer PEG linkers, DBCO-FA-5k and DBCO-FA-10k and the unconjugated NE-AAV.
  • the FA-AAVs also showed an increased infectivity compared to their wild-type counterpart, DJR/A and AAV8, respectively (Figure 8E, 8F).
  • Figure 8E, 8F an inverse correlation was observed between the length of the PEG linker and FA-AAV infectivity regardless of the NE-AAV capsid utilized, where the increasing length of the linkers significantly reduced the AAV transduction ( Figure 3F).
  • the cells were treated with an antibody directed against the hFOLRI (hFOLRI -Ab) to block its activity and in turn FA-AAV transduction (Figure 4F).
  • hFOLRI -Ab an antibody directed against the hFOLRI
  • Figure 4F the FA- DJR/A-N589 displayed a significant 2-fold decrease in luciferase levels when HeLa cells were pre-incubated with the hFOLRI -Ab compared to cells that were not treated with the antibody.
  • the unconjugated Ne-DJR/A-N589 and the DJR/A showed similar levels of transduction regardless of whether the cells were treated with the hFOLRI -Ab.
  • Example 5 In vitro characterization of aptamer-conjugated NE-AA VS
  • the DBCO-PEG-AS1411 was conjugated to the Ne-AAV vectors.
  • a biotinylated oligonucleotide antidote was designed that would specifically bind to the AS141 1 aptamer.
  • the AS1411-AAV would be detected by western blot using a streptavidin-conjugated antibody. Indeed, western blot analysis showed that only the AS1411 -AAV was detected by the streptavidin antibody (green signal) upon incubation with the biotinylated probe (Figure 5B).
  • the AS1411 -AAV resulted in a significant 9- ,10-, and 5-fold increase in transduction compared to the unconjugated vector in MCF-7, A549 and Hela cells, respectively ( Figure 5E-G). Furthermore, the antidote significantly prevented the transduction of the AS1411 -DJR/A-A587 vector in both HeLa and MCF-7 cells ( Figure 9B, C).
  • the NE-AAV was conjugated with a different aptamer (Zhao et al., Biomaterials 67, 42-51 (2015)), referred to herein as CD-AAV, originally isolated for uptake in AML cells (Figure 9D).
  • CD-AAV a different aptamer
  • Figure 9D Hela cells infected with CD-AAV and in presence of AS141 1 antidote did not show any inhibition of transduction similarly to the unconjugated NE-AAV vector.
  • the AS1411 -AAV showed a dose dependent decrease in luciferase activity (Figure 5H). AS141 1 binding to cells has been shown to be blocked in the presence of salmon sperm DNA (ssDNA).
  • both MCF-7 and A549 cells were treated with 1 mg/mL of ssDNA and transduced with either the AS1411 -DJR/A-N589 or its unconjugated counterpart, NE-DJR/A-N589.
  • the AS1411 -DJR/A-N589 showed a massive ⁇ 20-fold decrease infectivity in cells treated with the ssDNA, while the unconjugated NE-DJR/A-N589 displayed similar transduction levels in cells regardless of the ssDNA ( Figure 9E, F).
  • transduction by AS141 1-AAV is strictly dependent on the AS141 1 aptamer regardless of the NE-AAV capsid used similarly to FA receptor-dependent FA-AAV transduction.
  • the AAV was conjugated with the E3 RNA aptamer (Gray et al., Proc. Natl. Acad. Sci. U. S. A. 115, 4761-4766 (2016)).
  • This aptamer has been reported to specifically target various tumor cells and was successfully conjugated with anti-cancer drugs to treat prostate cancer using a xenograft mouse model.
  • the western blot membrane was probed with a biotinylated E3 oligonucleotide antidote. Similar to the DNA aptamer conjugation, this showed that the E3 aptamer was specifically conjugated to the AAV.
  • Example 6 Highly specific targeting of FA-AA V and AS1411 -AA V vectors in vivo
  • their yield was first improved.
  • researchers had found that the GAG promoter element in the AAV cassette might negatively impact AAV production due to vector genome truncation.
  • the large GAG promoter was thus replaced with a newly characterized small ubiquitous promoter, INS84.
  • the new Ne-AAV expressing firefly luciferase under the small INS84 promoter resulted in significantly higher vector titers compared to the AAV cassette carrying the GAG promoter ( Figure 10A).
  • Ne-DJR/A-A587 (Ne-AAV hereafter) containing the INS84-Fluc cassette was conjugated with either folic acid (FA-AA V), or with the AS1411 aptamer (AS1411- AAV) and was tested in HeLa cells.
  • the FA-AAV and AS141 1-AAVs resulted in significantly higher luciferase activity compared to their unconjugated counterpart but with similar activity compared to the Ne-AAV containing the CAG promoter ( Figure 10B, C).
  • Ne-AAV vectors were subcutaneously transplanted into immunodeficient nude mice and the AAV vectors were subcutaneously injected once the HeLa cells had formed palpable tumors.
  • the luciferase expression of the unconjugated Ne-AAV, FA-AAV and AS1411 -AAV vectors was assessed three, seven and fourteen days after AAV treatment by in vivo imaging (Figure 6A). All three vectors, Ne-AAV, FA-AAV and AS1411 -AAV, were able to infect the tumor cells, and by day fourteen reached maximal expression, albeit expression was detectable at earlier timepoints (Figure 6B; Figure 10D, E).
  • Ne-AAV and FA-AAV displayed a slightly higher luciferase signal in tumor cells compared to AS141 1 -AAV ( Figure 6C).
  • a major difference was the off-target expression in the liver with Ne-AAV exhibiting the most luciferase activity, followed by the FA-AAV, and finally the AS1411 -AAV which did not show any activity in the liver ( Figure 6D, E).
  • luciferase measurement in the harvested tissues after fourteen days from the AAV treatment confirmed that the liver was transduced by the Ne-AAV and FA-AAV vectors but not by the AS1411 -AAV.
  • no significant difference in luciferase activity was detected on the explanted tumor cells among the mice treated with the Ne-AAV, FA-AAV and AS1411 -AAV vectors ( Figure 10F).
  • capsid VP proteins Although the exact number of capsid VP proteins that have been successfully conjugated in the vector preparation is unknow, the data generated in vitro and in vivo clearly support that the ligand-vector conjugation was able to specifically modify the AAV tropism.
  • Tumor cells usually undergo genomic and metabolic dysregulation, which leads to the expression of tumor-specific cellular surface molecules.
  • researchers have been trying to develop ligands to specifically target cancer cells.
  • folic acid and aptamers have been conjugated to drugs, nucleic acids, and lipid nanoparticles. Nevertheless, nanoparticles have been associated with acute cell toxicity depending on their formulation.
  • aptamers possess very poor and limited endosomal escape that may hamper their development as therapeutics.
  • AAV vectors use their intrinsic phospholipase A2 activity which allow their effective release from the endosomal compartment and very efficient nuclear trafficking.
  • the studies described herein show the first successful conjugation of aptamers to AAV capsids in order to efficiently enhance and re-direct their tropism.
  • the transduction specificity of AAV conjugated to a peptide molecule (FA), an RNA aptamer (E3), and a DNA aptamer (AS141 1) were characterized through a series of in vitro uptake assays and the use of different cell lines.
  • the ligand’s characteristics were demonstrated to be transferable, since the NE-AAV serotype did not influence the vector uptake.
  • aptamers to modulate the AAV tropism presents apparent advantages: a) large scale production of nucleic acids is less expensive than protein- or antibodies- based drugs, b) the possible use of a universal vector will further reduce the costs of manufacturing, c) the aptamer selection for a specific type of cells is more simple than screening libraries of antibodies or AAVs and d) chemical synthesis of oligonucleotides facilitates straight forward attachment of moieties that support their subsequent conjugation to the modified AAV capsids described herein.
  • All the plasmids generated in this study have been produced by Gibson assembly using NEBuilder® HiFi DNA Assembly Master Mix (NEB, cat. E2621 S). Single nucleotide mutations were introduced by QuickChange II Site-Directed Mutagenesis Kit (Agilent Technologies, cat. 200523).
  • a pcDNA3.1 plasmid backbone was used for the expression of VP2 or VP3 under the control of a CMV promoter.
  • the pAcBad .tR4-MbPyl plasmid was a gift from Peter Schultz (Addgene plasmid cat. 50832) 27 .
  • the pAAV plasmid with the ubiquitous CAG promoter upstream the firefly luciferase transgene for the in vitro experiments was generated in the Kay lab and available at Addgene (catalog number 83281 ).
  • the CAG promoter was swapped with the minimal ubiquitous INS84 promoter.
  • Transfection was carried out using the following plasmids: pRep/Cap (5
  • the AAV vectors were purified using AAVpro® Purification Kit (Takara, cat. 6666) following manufacturer instructions. For crude lysate AAVs, the cells were harvested and underwent three cycles of freeze/thaw for lysis. They were spun at 5000g at 4°C for 15 minutes to pellet cell debris.
  • Supernatant was collected and treated with 1 pL of benzonase for one hour at 37°C. Cells were spun at 5000g for 10 minutes to pellet debris and the supernatant collected. The supernatant was incubated for one hour at 4°C in ice. Another centrifugation step at 7000g at 4°C for 30 minutes was carried out. Supernatant was collected and stored at -80°C until the day of the AAV transduction.
  • Vector titers were obtained by qPCR on the CFSX384 instrument (Biorad) using Brilliant III Ultra-Fast SYBR QPCR MM (Agilent Technologies, cat. 600882).
  • NE-AAV were conjugated with the DBCO PEG Folic acid or DBCO PEG Biotin (NANOCS, cat. PG2-DBFA, PG2-BNDB).
  • DBCO-PEG chemicals were resuspended in 20% DMSO to a final concentration of 2mM used as stock solution. Before the click reaction, the stock was further diluted to 500mM in 20% of DMSO solution.
  • DBCO- PEG moieties were added to NE-AAV to a final concentration of 10OmM and incubated overnight on a tube rotor at 4°C.
  • the NE-AAV unconjugated control vectors were incubated in the same 20% DMSO solution used for the DBCO-PEG solubilization.
  • DBCO-PEG-Aptamer preparation was carried out as previously reported 71 . Briefly, the DNA aptamers, AS1411 and CD, carrying an ammino (NH2) modified 5’ end were synthesized from Integrated DNA Technology
  • AS141 1 5’-5AmMC6-TTTTTTTTTTTTTTTTTTGGTGGTGGTGGTGTTGGTGGTGGTGG-3’ (SEQ ID NO: 19)
  • E3 and C36 RNA aptamers were synthesized as previously reported 22 . Briefly, the E3 (GGCUUUCGGGCUUUCGGCAACAUCAGCCCCUCAGCC) (SEQ ID NO: 21 ) and C36 (GGCGUAGUGAUUAUGAAUCGUGUGCUAAUACACGCC) (SEQ ID NO: 22) aptamers were synthesized by solid phase synthesis on a MerMade 12 Synthesizer (Biosearch Technologies, as previously described). Briefly, the aptamers were synthesized using 2'-F-modified pyrimidines and 2'OH purines on an inverted dT CPG column.
  • Synthesis reagents were purchased from Glen Research (Sterling, VA) and Chemgenes Corp (Wilmington, MA). All aptamers were synthesized bearing a 5' amine using a C6 phosphoramidite (Glen Research, Sterling, VA).
  • DBCO-PEG4-NHS Ester Click Chemistry Tool, cat. A134-10
  • the resulted DBCO-PEG-Aptamer was finally purified using Cytiva illustraTM MicroSpinTM G-25 Columns (Cytiva life sciences, cat. 27532501) following manufacturer instructions.
  • DBCO-PEG-Aptamer was added to the NE-AAV to a final concentration of 125 pM and incubated overnight on a tube rotor at 4°C.
  • the conjugated AAV was added to 450
  • the cells used for the folic acid experiments were grown in RPMI 1640 Medium (Fisher, 27016021 ) supplemented with 10% fetal bovine serum (FBS), 100 lU/ml pen icillin/streptomycin .
  • the rest of the experiments were carried out using DMEM Medium (Thermo Fisher, cat. 15017CV) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 lll/ml penicillin/streptomycin. All the cells were maintained in a humidified incubator at 37°C with 5% CO2.
  • Total mRNA was extracted from HeLa, MCF-7 and A549 using RNeasy mini kit (QIAGEN) following manufacturer instructions.
  • mRNA was converted to cDNA using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher, cat. K1671 ).
  • Cells were incubated with AAV vectors for 48 hours using a multiplicity of infection (MOI) of 5000 unless specified otherwise in the figure legend.
  • MOI multiplicity of infection
  • Cell lysis and luciferase assay were performed using the Promega Luciferase 1000 Assay System (Promega, cat. E4550) following manufacturer instructions.
  • For the uptake experiments 1.5 ug per well of anti-hFOLR1 antibody (Thermo Fisher, cat. MA5-23917), 200
  • AAV samples were loaded on a 4-15% gradient polyacrylamide gel (Fisher, cat. 34028). Protein transfer was performed using the iBIot system (Thermo Fisher, cat. IB23002). The membrane was blocked with Odyssey buffer (Fisher, cat. NC0730870) and incubated with the anti-AAV antibody (Gene Tex, cat. GTX44495) or a streptavidin-conjugated antibody (Fisher, cat. NC9386176) both diluted 1 :1000 in odyssey buffer. The membrane was washed and incubated with a secondary antibody (Fisher, cat. 92532210) diluted 1 :100000 and visualized by Odyssey imaging system (Li-Cor Biosciences).
  • the membrane After the blocking step, was incubated with 1 gM of AS1411 -(5’- CCACCACCACCACAACCACCACCACCACC-Biotin-3’) (SEQ ID NO: 28), CD-(5’- CCCCCCATGGCACCATCCTG-Biotin-3’) (SEQ ID NO: 29),E3-(5’- GATGTTGCCGAAAGCCCGAA-Biotin-3’) (SEQ ID NO: 30) probes overnight at room temperature, and subsequently incubated with a streptavidin-conjugated antibody for two hours at room temperature (Fisher, cat. NC9386176).
  • AAV vectors were placed on a 300-mesh carbon/formvar coated Cu grids and allow to settle 3 minutes. Samples were washed two times with MilliQ-l-kO and stained 1 minute with 1% Uranyl Acetate in mQ-H 2 O. AAV samples were allowed to dry. Images acquisition was performed on the JEOL-JEM1400 microscope at 120kV.
  • luciferase imaging was performed using the Lago optimal imaging system (Spectral Instruments Imaging). Luciferase imagines were analyzed using Aura Software (Spectral Instruments Imaging). Explanted liver and HeLa tumor were freshly homogenized in PBS using a Bullet Blender (Next Advance). The luciferase assay was performed using the Promega Luciferase 1000 Assay System (Promega, cat. E4550) following manufacturer instructions. Protein concentration in tissue samples was measured with Pierce BCA protein assay kit (Thermo Fisher, cat. 23227) following manufacturer instructions.

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Abstract

Provided are variant AAV capsid proteins including an azide-bearing unnatural amino acid substitution. In some embodiments, the azide-bearing unnatural amino acid substitution is at a position corresponding to: T456, D555, R587/A587, or N589 of the AAV-DJ or DJR/A amino acid sequence; or E330, T457, N499, or N590 of the AAV8 amino acid sequence; or T455, T456, or N588 of the AAV- LK03 amino acid sequence. In some embodiments, the azide-bearing unnatural amino acid is at a position inserted between positions corresponding to N590 and T591 of the AAV8 sequence. In some embodiments, a variant AAV capsid protein is chemically modified such that it is conjugated via the azide-bearing unnatural amino acid to a targeting moiety such as folic acid, a DNA aptamer (e.g., AS1411), or an RNA aptamer (e.g., E3). Also provided are nucleic acids encoding a subject variant AAV capsid protein, and recombinant AAV particles.

Description

CHEMICALLY MODIFIED AAVS
CROSS-REFERENCE
[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63/432,467 filed December 14, 2022, which application is incorporated herein by reference in its entirety.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH
[0002] This invention was made with Government support under contract AI1 16698 awarded by the National Institutes of Health. The Government has certain rights in the invention.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS AN XML FILE
[0003] A Sequence Listing is provided herewith as a Sequence Listing XML, “STAN- 1881 WO Seq List. xml” created on December 14, 2023 and having a size of 45,519 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.
I. INTRODUCTION
[0004] In the last two decades recombinant adeno-associated viral (AAV) vectors have been widely employed in many clinical trials making these vectors the preferred delivery system for in vivo gene therapy. Several groups have developed AAV capsids through DNA shuffling or peptide display techniques to create vectors with improved AAV transduction in vivo. Thus, AAV vectors have been successfully engineered to enhance targeting to different organs including liver, brain, skeletal muscle, and eye amongst others. More recently, researchers have been using different approaches to synthetically modify AAV capsids in order to improve properties such as immune escape and receptor-specific cell targeting.
[0005] In recent years, progress in synthetic biology has led to expansion of the genetic code by taking advantage of “unnatural” amino acids from prokaryotic microorganisms. Consequently, researchers have been able to utilize them in mammalian systems and create hybrid proteins bearing unnatural amino acids in their structure. This was achieved by engineering pairs of orthogonal prokaryotic tRNA/tRNA synthases which were genetically expressed in mammalian cells. Hence, the unnatural amino acid can be recognized by the mammalian translational machinery and inserted into a nascent polypeptide. These unnatural amino acids have been mostly employed for studying protein functions or creating de novo protein properties for therapeutic purposes. Recently, the use of the unnatural amino acids was applied to AAV engineering - particularly for the placement of an azido group (N3) on the capsid surface, which allows click chemistry conjugation of molecules on AAV2. Other chemical modification of AAV vectors have also been described. Nevertheless, these approaches are strictly dependent on the amino acid composition of the capsid surface.
[0006] Thus, there is a need for compositions and methods that provide cell-specific targeting for AAVs, and such is provided herein. For example, particular insertion of a single unnatural amino acid onto a specific region of the capsid allows specific conjugation of molecules onto the AAV regardless of capsid amino acid composition.
II. SUMMARY
[0007] The inventors have developed chemically modified AAV vectors (NE-AAVS) through an unnatural amino acid substitution on the capsid surface for post-production vector engineering through biorthogonal copper-free click chemistry. The inventors identified AAV vectors that tolerate the unnatural amino acid substitution on the capsid without disrupting their packaging and transduction efficiency. As illustrative examples, they then functionalized the NE-AAVS through conjugation with DNA (AS1411 ), or RNA (E3) aptamers or with a targeting moiety (folic acid (FA)). Surprisingly, E3-, AS1411 -, and FA-AAV showed on average a 3-9 fold increase in transduction compared to their non -conjugated counterparts in different cancer cell lines. Using specific competitors, the inventors demonstrated ligand-specific transduction. Moreover, in vivo studies confirmed the selective uptake of FA-AAV and AS1411 -AAV without off target transduction in peripheral organs. Overall, the high versatility of the NE-AAVS paves the way to tailoring gene therapy vectors toward specific types of cells both for ex vivo and in vivo applications.
III. BRIEF DESCRIPTION OF THE DRAWINGS
[0008] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [0009] FIG. 1 A-1 B. Ne-AAV vector production and conjugation. (FIG. 1 A) Schematic representation of NE-AAV production. Once the unnatural amino acid is incorporated into the AAV capsid the exposed azido group (N3) reacts with the DBCO resulting in the conjugation of R, where the “R” is a generic molecule (peptide, nucleic acid etc.). (FIG. 1 B) Western blot analysis of NE-AAV upon conjugation with the DBCO-biotin molecule. An anti-AAV antibody, clone B1 , was used to detect the VP1/2/3 capsid proteins of AAV. An anti-streptavidin antibody was used to detect the biotin- conjugated NE-AAV vector.
[0010] FIG. 2A-2F. Development of the FA-AAV vector after folic acid conjugation.
(FIG. 2A-2C) Schematic representation of the plasmids used for (FIG. 2A) NE-AAV, (FIG. 2B) NE-VP2-AAV, and (FIG. 2C) NE-VP3-AAV production. (FIG. 2D) Chemical formula of the DBCO-PEG (2K)-FA molecule used for the NE-AAV vector conjugation. (FIG. 2E) Western blot analysis of NE-AAV, NE-VP2-AAV, NE-VP3-AAV upon DBCO- PEG (2K)-FA conjugation. An anti-AAV antibody, clone B1 , was used to detect the VP 1/2/3 capsid proteins of AAV. Asterisks indicate the VP proteins where the folic acid has been conjugated. (FIG. 2F) Luciferase activity in HeLa cells after transduction with DBCO-FA-conjugated AAV vectors. Mock-conjugated NE-AAV vectors were used as controls. Cells were transduced at 1 ,000 vg/cell. Statistical analysis: (FIG. 2F) two-way ANOVA with Tukey’s post hoc the statistical significance was assumed with P value <0.05 (*), <0.01 , <0.001 (***) and <0.0001 (*‘“); with Sidak’s post hoc the statistical significance was assumed with P value <0.05 (#), <0.01 (##), <0.001 (###) and <0.0001 (####). Error bars represent standard deviation of the mean.
[0011] FIG. 3A-3F. Assessment of novel NE-AAV vectors upon folic acid conjugation.
(FIG. 3A) Luciferase activity in HeLa cells of the NE-AAV vectors produced in presence (+ azido-lysine) or absence (- azido-lysine) of unnatural amino acid. AAV crude lysates was used for the HeLa transduction evaluation. (FIG. 3B-3D) Luciferase activity in HeLa and MCF-7 cell lines upon transduction with (FIG. 3B) FA-DJR/A- N589, (FIG. 3C) FA-AAV8-T457, and (FIG. 3D) FA-LK03-N588. The unconjugated NE- AAV vectors were used as control. (FIG. 3E) Transmission electric microscopy images of AAV-DJR/A, unconjugated NE-DJR/A-N589, and FA-DJR/A-N589 vectors. (FIG. 3F) Regression plot showing the reverse correlation between the length of the PEG linker and AAV vectors infectivity of FA-AAV8-T457 and FA-DJR/A-N589 vectors. Statistical analysis: (FIG. 3B-3D) two-way ANOVA with Sidak’s post hoc the statistical significance was assumed with P value <0.05 (*), <0.01(**), <0.001 (***) and <0.0001 (****). Error bars represent standard deviation of the mean.
[0012] FIG. 4A-4I. Evaluation of FA-AAV vector specific uptake in vitro. (FIG. 4A)
Schematic representation of the uptake assay conducted by adding 200 |iM of folic acid into the cell media during AAV transduction. (FIG. 4B-4D) Luciferase activity in HeLa cells transduced with (FIG. 4B) FA-DJR/A-N589, (FIG. 4C) FA-AAV8-T457, and (FIG. 4D) FA-DJR/A-A587. DJR/A, AAV8 and the unconjugated Ne-AAV vectors were used as controls. 200 |iM of folic acid was added into the cell media at the time of AAV transduction (+ free folic acid group). (FIG. 4E) Luciferase activity in MCF-7 cells transduced with FA-DJR/A-N589. DJR/A and the unconjugated Ne-AAV vectors were used as controls. 200 |iM of folic acid was added into the cell media at the time of AAV transduction (+ free folic acid group). (FIG. 4F) Schematic representation of the uptake assay conducted by pre-incubating the cells with the monoclonal anti-hFOLR1 antibody. (FIG. 4G, FIG. 4H) Luciferase activity in HeLa cells transduced with (FIG. 4G) FA-DJR/A-N589, and (FIG. 4H) FA-AAV8-T457. DJR/A, AAV8 and the unconjugated Ne-AAV vectors were used as controls. Cells were pre-incubated with an anti-hFOLR1 monoclonal antibody for one hour at 4°C and then transduced with the AAV vectors (+ hFOLR1 -Ab group). (FIG. 4I) Luciferase activity in MCF-7 cells transduced with FA-DJR/A-N589. DJR/A and the unconjugated Ne-AAV vectors were used as controls. Cells were pre-incubated with an anti-hFOLR1 monoclonal antibody for one hour at 4°C and then transduced with the AAV vectors (+ hFOLR1-Ab group). Statistical analysis: (FIG. 4B-4E, FIG. 4G-4I) two-way ANOVA with Sidak’s post hoc the statistical significance was assumed with P value <0.05 (*), <0.01 (**), <0.001 (***) and <0.0001 (****). Error bars represent standard deviation of the mean
[0013] FIG. 5A-5L. Characterization of DNA and RNA aptamers conjugated to Ne-AAV vectors. (FIG. 5A) Schematic representation of the DBCO-PEG-aptamer molecule. (FIG. 5B) Western blot analysis of Ne-AAV vector upon conjugation with DBCO-PEG- AS141 1 . An anti-AAV antibody, clone B1 , was used to detect the VP 1/2/3 capsid proteins of AAV. An anti-streptavidin antibody was used to detect the AS1411 - conjugated Ne-AAV vector. (FIG. 5C) Luciferase activity in MCF-7 cells transduced with AS1411 -DJR/A-N589, and the unconjugated Ne-DJR/A-N589 vectors at different MOI. (FIG. 5D) Schematic representation of the uptake assay conducted by incubating the cells with different concentrations of the AS1411 antidote. (FIG. 5E-5G) Luciferase activity in (FIG. 5E) MCF-7, (FIG. 5F) A549, and (FIG. 5G) HeLa cells upon transduction with AS1411 -DJR/A-N589. Unconjugated NE-DJR/A-N589 vectors were used as controls. Cells were incubated with different concentrations of AS141 1 antidote. (FIG. 5H) Luciferase activity in HeLa cells upon transduction with AS1411 - DJR/A-A587. Unconjugated Ne-DJR/A-N589 and CD-DJR/A-A587 vectors were used as controls. Cells were incubated with different concentrations of AS1411 antidote. (FIG. 51) Western blot analysis of NE-AAV vector upon conjugation with DBCO-PEG- C36 or DBCO-PEG-E3. An anti-AAV antibody, clone B1 , was used to detect the VP1/2/3 capsid proteins of AAV. An anti-streptavidin antibody was used to detect the specifically E3-conjugated Ne-AAV vector. (FIG. 5J) Schematic representation of the uptake assay conducted by incubating the cells with dynasore. (FIG. 5K, FIG. 5L) Luciferase activity in (FIG. 5K) Huh7, and (FIG. 5L) MCF-7 cells, upon transduction with E3-DJR/A-A587. Unconjugated Ne-DJR/A-A587 and C36-DJR/A-A587 vectors were used as controls. Cells were incubated with 10uM of clathrin inhibitor, Dynasore.Statistical analysis: (FIG. 5E-5G, FIG. 5K, FIG. 5L) two-way ANOVA with Sidak’s post hoc. O^iM of AS1411 -DJR/A-N589=3; 1 j M of AS141 1 -DJR/A-N589=3; 10p,M of AS141 1-DJR/A-N589=3; OgM of Ne-DJR/A-N589=3; 1gM of NE-DJR/A- N589=3; 1 OpiM of NE-DJR/A-N589=3. (FIG. 5H) two-way ANOVA with Tukey’s post hoc. The statistical significance was assumed with P value <0.05 (*), <0.01 (“), <0.001 (***), and <0.0001 (M“), and with P value <0.05 (#), <0.01 (##), <0.001 (###) and <0.0001 (####). Error bars represent standard deviation of the mean.
[0014] FIG. 6A-6E. In vivo characterization of FA-AAV and AS1411-AAV vectors. (FIG. 6A) Schematic representation of the in vivo study. (FIG. 6B) Dorsal image of mice treated with 5x109 vg of NE-AAV, FA-AAV, and AS141 1 -AAV fourteen days after AAV treatment. PBS-injected mouse was used as negative control. (FIG. 6C) Luciferase signal from tumor of mice treated with 5x109 vg of NE-AAV, FA-AAV, and AS1411 - AAV at three, seven, and fourteen days after AAV treatment (FIG. 6D) Ventral image of mice treated with 5x109 vg of NE-AAV, FA-AAV, and AS1411 -AAV fourteen days after AAV treatment. PBS-injected mouse was used as negative control. (FIG. 6E) Luciferase signal from liver of mice treated with 5x109 vg of NE-AAV, FA-AAV, and AS141 1-AAV at three, seven, and fourteen days after AAV treatment. Statistical analysis: (FIG. 6C, FIG. 6E) two-way ANOVA with Tukey’s post hoc the statistical significance was assumed with P value <0.05 (*), <0.01 (**), <0.001 (***) and <0.0001 (****). Error bars represent standard deviation of the mean. [0015] FIG. 7A-7G. Development of Ne-AAV vectors. (FIG. 7A) Luciferase activity in HeLa cells transduced with AAVDJ and AAVDJR/A. Cells were treated with different concentration of heparin. (FIG. 7B) Luciferase activity in HeLa cells transduced with NE-AAVDJR/A-N589, Ne-AAVDJR/A-D555; Ne-AAVDJR/A-A587; Ne-AAVDJR/A- T456. (FIG. 7C) Western blot analysis of NE-VP2-AAV. An anti-AAV antibody, clone B1 , was used to detect the VP1/2/3 capsid protein of AAV. (FIG. 7D) Western blot analysis of NE-VP3-AAV. An anti-AAV antibody, clone B1 , was used to detect the VP1/2/3 capsid protein of AAV. (FIG. 7E) Silver-stained gel analysis of DJR/A, NE- DJR/A-N589, and FA-DJR/A-N589 vectors. (FIG. 7F) mRNA levels of hFOLRI in HeLa, MCF-7, and A459 cells measured by qPCR. (FIG. 7G) Luciferase activity in MCF-7 cells transduced with DBCO-FA-VP2-AAV and DBCO-FA-VP3-AAV vectors at different MOI. As control the mock-unconjugated NE-VP2-AAV and NE-VP3-AAV (grey bars) were used. Statistical analysis: (FIG. 7A) two-way ANOVA with Tukey’s post hoc; (FIG. 7B) one-way ANOVA with Tukey’s post hoc; (FIG. 7G) two-way ANOVA with Sidak’s post hoc. The statistical significance was assumed with P value <0.05 (*), <0.01 (**), <0.001 (***) and <0.0001 (****). Error bars represent standard deviation of the mean.
[0016] FIG. 8A-8I. Characterization of novel NE-AAV vectors. (FIG. 8A) Titer of the different Ns -AAV vectors expressed as percentage of the wild-type (WT) AAV serotype. The letter and the number correspond respectively to the amino acid and the position on the capsid where the unnatural amino acid is incorporated. (FIG. 8B- 8D) Luciferase activity in A549 cells upon transduction with (FIG. 8B) FA-DJR/A- N589, (FIG. 8C) FA-AAV8-T457, and (FIG. 8D) FA-LK03-N588. The unconjugated NE- AAV vectors were used as control. (FIG. 8E, FIG. 8F) Luciferase activity in MCF-7 cells after transduction with (FIG. 8E) NE-AAV8-T457 and (FIG. 8F) NE-DJR/A-N589 conjugated with DBCO-FA molecule carrying different lengths of PEG linker. As control the cells were transduced with AAV8, DJR/A and the respectively unconjugated NE-AAV vectors. (FIG. 8G) Luciferase activity in MCF-7 cells after transduction with Ns-DJR/A-N589 conjugated with the DBCO-PEG (2K)-FA molecule or with DBCO-PEG. As control the cells were transduced with DJR/A and the unconjugated NE-AAV vector. (FIG. 8H) Western blot analysis of FA-DJR/A-N589 conjugated with different concentration of DBCO-FA. An anti-AAV antibody, clone B1 , was used to detect the VP 1/2/3 capsid protein of AAV. (FIG. 81) Luciferase activity in MCF-7 cells after transduction with NE-DJR/A-N589 conjugated with 2mM of DBCO- FA molecule. As control the cells were transduced with AAV8, DJR/A and the respectively unconjugated Ne-AAV vectors.
[0017] FIG. 9A-9G. Assessment of AS1411 -conjugated Ne-AAV vectors. (FIG. 9A) Transmission electric microscopy images of AAV-DJR/A and AS141 1-DJR/A-N589. (FIG. 9B, FIG. 9C) Luciferase activity in (FIG. 9B) MCF-7 and (FIG. 9C) HeLa cells upon transduction with AS1411 -DJR/A-A587. Unconjugated Ne-AAV vectors were used as controls. Cells were incubated with 10 ,M of AS1411 antidote. (FIG. 9D) Western blot analysis of Ne-AAV vector upon conjugation with the DBCO-PEG-CD molecule. An anti-AAV antibody, clone B1 , was used to detect the VP1/2/3 capsid protein of AAV. An anti-streptavidin antibody was used to detect the CD-conjugated Ne-AAV vector. (FIG. 9E, FIG. 9F) Luciferase activity upon transduction with AS1411 - DJR/A-N589 or the unconjugated Ne-DJR/A-N589 in (FIG. 9E) MCF-7 and (FIG. 9F) A549 cells. Cells were incubated with 1 mg/mL of salmon sperm DNA (ssDNA). (G) Luciferase activity in liver cancer cell lines, Huh7 and Hepa1 -6 upon transduction with E3-DJR/A-A587. Unconjugated Ne-DJR/A-A587 and C36-DJR/A-A587 vectors were used as controls. Statistical analysis: (FIG. 9B, FIG. 9C, FIG. 9E, FIG. 9F) two-way ANOVA with Sidak’s post hoc. (FIG. 9G) two-way ANOVA with Tukey’s post hoc. The statistical significance was assumed with P value <0.05 (*), <0.01(**), <0.001 (***) and <0.0001 (****). Error bars represent standard deviation of the mean.
[0018] FIG. 10A-10F. Characterization of Ne-AAV vectors for in vivo studies. (FIG. 10A) Titers of Ne-DJ-CAG-Fluc and Ne-DJ-INS84-Fluc measured by qPCR. (FIG. 10B, FIG. 10C) Luciferase activity in HeLa cells upon transduction with (B) AS1411 -DJR/A- A587-INS84-Fluc, and (FIG. 10C) FA-DJR/A-A587-INS84-Fluc. Unconjugated Ne- DJR/A-N589-INS84-Fluc vectors were used as control. (FIG. 10D, FIG. 10E) In vivo luciferase images of mice treated with 5x109 vg of Ne-AAV, FA-AAV, and AS1411 - AAV (FIG. 10D) three days, and (FIG. 10E) seven days after AAV injection. PBS- injected mouse was used as negative control. (FIG. 10F) Luciferase activity in harvested liver and tumor HeLa cells of mice treated with 5x109 vg of Ne-AAV, FA- AAV, and AS1411 -AAV fourteen days after AAV treatment. Luciferase activity was normalized by the protein concentration measured in the tissue sample. Statistical analysis: (FIG. 10A-10C) Student t-test. (FIG. 10F) one-way ANOVA with Tukey’s post hoc. The statistical significance was assumed with P value <0.05 (*), <0.01(**), <0.001 (***) and <0.0001 (****). Error bars represent standard deviation of the mean. IV. DEFINITIONS
[0019] "AAV" is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof, and can be used to refer to a viral vector system for delivery of nucleic acids. The term covers all subtypes and both naturally occurring as well as recombinant and variant forms, except where specified otherwise. The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector").
[0020] The term "AAV" includes any convenient AAV type, including variant types, e.g., AAV type 1 (AAV1 ), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV 9_hu14, AAV-DJ (also referred to herein as simply “DJ”), AAV- LK03 (also referred to herein as simply “LK03”), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV capable of infecting primates, "non-primate AAV" refers to AAV capable of infecting non-primate mammals, "bovine AAV" refers to AAV capable of infecting bovine mammals, etc.
[0021] As would be clear to one of ordinary skill in the art, it is to be understood that the term “AAV vector” can be used to refer to the delivery system as a whole (e.g., a virion or population of virions), and can also be used to refer to a nucleic acid encoding the delivery system - i.e., one that includes a sequence that encodes a capsid polypeptide (i.e., a nucleic acid that includes a nucleotide sequence encoding a capsid polypeptide, also referred to as a AAV capsid protein or AAV capsid polypeptide - the terms “polypeptide” and “protein” are used interchangeably herein), depending on context. In some embodiments, a subject AAV vector is a variant (nonwild type) AAV vector (e.g., a nucleic acid can include a nucleotide sequence encoding a variant capsid polypeptide, also referred to as a variant AAV capsid protein or variant AAV capsid polypeptide). The AAV vectors (including variant AAV vectors) can also include a heterologous nucleic acid sequence not of AAV origin (e.g., as part of the nucleic acid insert). This heterologous nucleic acid sequence typically comprises a sequence of interest for the genetic transformation of a cell. In some cases, the heterologous nucleic acid sequence (the “nucleotide sequence of interest”) is flanked by at least one, and generally by two AAV inverted terminal repeat sequences (ITRs). [0022] The phrase "non-variant parent capsid polypeptides" (or “wild type capsid protein”) includes any naturally occurring AAV capsid polypeptides. In some embodiments, the non-variant parent capsid polypeptides include AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, bovine AAV and/or avian AAV capsid polypeptides.
[0023] The term "substantially identical" in the context of variant AAV capsid polypeptides and non-variant parent capsid polypeptides refers to sequences with 1 or more amino acid changes. In some embodiments, these changes do not affect the packaging function of the capsid polypeptides. In some embodiments, substantially identical include variant AAV capsid polypeptides about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, or about 90% identical to non-variant parent capsid polypeptides. In some embodiments, the variant AAV capsid polypeptides can be substantially identical to non-variant parent capsid polypeptides over a subregion of the variant AAV capsid polypeptide, such as over about 25%, about 50%, about 75%, or about 90% of the total polypeptide sequence length.
[0024] An "AAV virion" or "AAV virus" or "AAV viral particle" or "AAV vector particle" refers to a viral particle composed of at least one AAV capsid polypeptide (including both variant AAV capsid polypeptides and non-variant parent capsid polypeptides) and an encapsidated polynucleotide AAV transfer vector. If the particle comprises a heterologous nucleic acid (i.e. a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it can be referred to as an "AAV vector particle" or simply an "AAV vector". Thus, production of AAV virion or AAV particle necessarily includes production of AAV vector as such a vector is contained within an AAV virion or AAV particle.
[0025] "Packaging" refers to a series of intracellular events resulting in the assembly of AAV virions or AAV particles which encapsidate a nucleic acid sequence and/or other therapeutic molecule. Packaging can refer to encapsidation of nucleic acid sequence and/or other therapeutic molecules into a capsid comprising the variant AAV capsid polypeptides described herein.
[0026] The phrase "therapeutic molecule" as used herein can include nucleic acids (including, for example, vectors), polypeptides (including, for example, antibodies), and vaccines, as well as any other therapeutic molecule that could be packaged by the variant AAV capsid polypeptides of the invention. [0027] AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and encapsidation proteins of adeno-associated virus (AAV). AAV rep (replication) and cap (capsid) are referred to herein as AAV "packaging genes."
[0028] A "helper virus" for AAV refers to a virus allowing AAV (e.g. wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used as a helper virus. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.
[0029] "Helper virus function(s)" refers to function(s) encoded in a helper virus genome allowing AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, "helper virus function" may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans.
[0030] An "infectious" virion, virus or viral particle is one comprising a polynucleotide component deliverable into a cell tropic for the viral species. The term does not necessarily imply any replication capacity of the virus. As used herein, an "infectious" virus or viral particle is one that upon accessing a target cell, can infect a target cell, and can express a heterologous nucleic acid in a target cell. Thus, "infectivity" refers to the ability of a viral particle to access a target cell, enter a target cell, and express a heterologous nucleic acid in a target cell. Infectivity can refer to in vitro infectivity or in vivo infectivity. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity can be expressed as the ratio of infectious viral particles to total viral particles. Total viral particles can be expressed as the number of viral genome copies. The ability of a viral particle to express a heterologous nucleic acid in a cell can be referred to as "transduction." The ability of a viral particle to express a heterologous nucleic acid in a cell can be assayed using a number of techniques, including assessment of a marker gene, such as a green fluorescent protein (GFP) assay (e.g., where the virus comprises a nucleotide sequence encoding GFP), where GFP is produced in a cell infected with the viral particle and is detected and/or measured; or the measurement of a produced protein, for example by an enzyme-linked immunosorbent assay (ELISA) or fluorescence- activated cell sorting (FACS).
[0031] A "replication-competent" virion or virus (e.g. a replication-competent AAV) refers to an infectious phenotypically wild-type virus, and is replicable in an infected cell (i.e. in the presence of a helper virus or helper virus functions). In the case of AAV, replication competence generally requires the presence of functional AAV packaging genes. In some embodiments, AAV vectors, as described herein, lack of one or more AAV packaging genes and are replication-incompetent in mammalian cells (especially in human cells). In some embodiments, AAV vectors lack any AAV packaging gene sequences, minimizing the possibility of generating replication competent AAV by recombination between AAV packaging genes and an incoming AAV vector. In many embodiments, AAV vector preparations as described herein are those containing few if any replication competent AAV (rcAAV, also referred to as RCA) (e.g., less than about 1 rcAAV per 10.sup.2 AAV particles, less than about 1 rcAAV per 10.sup.4 AAV particles, less than about 1 rcAAV per 10.sup.8 AAV particles, less than about 1 rcAAV per 10.sup.12 AAV particles, or no rcAAV).
[0032] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Polynucleotides include genomic DNA, cDNA and antisense DNA, and spliced or unspliced mRNA, rRNA, tRNA, IncRNA, RNA antagomirs, and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh)RNA, microRNA (miRNA), aptamers, small or short interfering (si)RNA, trans-splicing RNA, or antisense RNA). Polynucleotides also include non-coding RNA, which include for example, but are not limited to, RNAi, miRNAs, IncRNAs, RNA antagomirs, aptamers, and any other non-coding RNAs known to those of skill in the art. Polynucleotides include naturally occurring, synthetic, and intentionally altered or modified polynucleotides as well as analogues and derivatives. The term "polynucleotide" also refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof, and is synonymous with nucleic acid sequence. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment as described herein encompassing a polynucleotide encompasses both the double-stranded form and each of two complementary singlestranded forms known or predicted to make up the double-stranded form. Polynucleotides can be single, double, or triplex, linear or circular, and can be of any length. In discussing polynucleotides, a sequence or structure of a particular polynucleotide may be described herein according to the convention of providing the sequence in the 5' to 3' direction.
[0033] A "small interfering" or "short interfering RNA" or siRNA is a RNA duplex of nucleotides targeted to a gene interest (a "target gene"). An "RNA duplex" refers to the structure formed by the complementary pairing between two regions of a RNA molecule. siRNA is "targeted" to a gene and the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene. In some embodiments, the length of the duplex of siRNAs is less than 30 base pairs. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 base pairs in length. In some embodiments, the length of the duplex is 19-25 base pairs in length. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion positioned between the two sequences forming the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 1 1 , 12 or 13 nucleotides in length. The hairpin structure can also contain 3' or 5' overhang portions. In some embodiments, the overhang is a 3' or a 5' overhang 0, 1 , 2, 3, 4 or 5 nucleotides in length.
[0034] "Recombinant," as applied to a polynucleotide means the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures resulting in a construct distinct and/or different from a polynucleotide found in nature. A recombinant virus is a viral particle encapsidating a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.
[0035] A "control element" or "control sequence" is a nucleotide sequence involved in an interaction of molecules contributing to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription usually downstream (in the 3' direction) from the promoter.
[0036] "Operatively linked" or "operably linked" refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a sequence of interest (the sequence of interest can also be said to be operatively linked to the promoter) if the promoter helps initiate transcription of the sequence of interest. There may be intervening residues between the promoter and sequence of interest so long as this functional relationship is maintained.
[0037] "Heterologous" means derived from a genotypically distinct entity from the rest of the entity to it is being compared too. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. A promoter removed from its native coding sequence and operatively linked to a coding sequence it is not naturally found linked to a heterologous promoter. For example, an AAV including a heterologous nucleic acid encoding a heterologous gene product is an AAV including a nucleic acid not normally included in a naturally-occurring, wild-type AAV, and the encoded heterologous gene product is a gene product not normally encoded by a naturally-occurring, wild-type AAV. An AAV including a nucleic acid encoding a variant AAV capsid polypeptide includes a heterologous nucleic acid sequence. Once transferred/delivered into a host cell, a heterologous polynucleotide, contained within the virion, can be expressed (e.g., transcribed, and translated if appropriate). Alternatively, a transferred/delivered heterologous polynucleotide into a host cell, contained within the virion, need not be expressed. Although the term "heterologous" is not always used herein in reference to polynucleotides, reference to a polynucleotide even in the absence of the modifier "heterologous" is intended to include heterologous polynucleotides in spite of the omission.
[0038] The terms "genetic alteration" and "genetic modification" (and grammatical variants thereof), are used interchangeably herein to refer to a process wherein a genetic element (e.g., a polynucleotide) is introduced into a cell other than by mitosis or meiosis. The element may be heterologous to the cell, or it may be an additional copy or improved version of an element already present in the cell. Genetic alteration may be effected, for example, by transfecting a cell with a recombinant plasmid or other polynucleotide through any process known in the art, such as electroporation, calcium phosphate precipitation, or polynucleotide-liposome complexation. Genetic alteration may also be effected, for example, by transduction or infection with a DNA or RNA virus or viral vector. Generally, the genetic element is introduced into a chromosome or mini-chromosome in the cell; but any alteration changing the phenotype and/or genotype of the cell and its progeny is included in this term.
[0039] A cell is said to be "stably" altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during an extended period of time (e.g., extended culture of the cell when the cell is in vitro). Such a cell can be "heritably" altered (genetically modified) in that a genetic alteration is introduced and can be inherited by progeny of the altered cell.
[0040] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The "polypeptides," "proteins" and "peptides" encoded by the "polynucleotide sequences," include full-length native sequences, as with naturally occurring proteins, as well as functional subsequences, modified forms or sequence variants so long as the subsequence, modified form or variant retains some degree of the intended functionality. The terms also encompass a modified amino acid polymer; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, methylation, carboxylation, deamidation, acetylation, or conjugation with a labeling component. Polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivering a gene product to a mammalian subject, and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, retaining the desired biochemical function of the intact protein.
[0041] An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components present where the substance or a similar substance naturally occurs or from which it is initially prepared. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more isolated. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some embodiments purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure. [0042] By the term "highly conserved" is meant at least about 80% identity, preferably at least 90% identity, and more preferably, over about 97% identity. Identity is readily determined by one of skill in the art by resort to algorithms and computer programs known by those of skill in the art.
[0043] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e. , arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.
[0044] The terms "individual," "subject," and "patient" are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).
[0045] The terms "pharmaceutically acceptable" and "physiologically acceptable" mean a biologically acceptable formulation, gaseous, liquid or solid, or mixture thereof, suitable for one or more routes of administration, in vivo delivery or contact. A "pharmaceutically acceptable" or "physiologically acceptable" composition is a material that is not biologically or otherwise undesirable, e.g., the material may be administered to a subject without causing substantial undesirable biological effects. Thus, such a pharmaceutical composition may be used, for example in administering an AAV vector or AAV virion as disclosed herein, or transformed cell to a subject.
[0046] The phrase a "unit dosage form" as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle or filling agent) which, when administered in one or more doses, produces a desired effect (e.g., prophylactic or therapeutic effect). In some embodiments, unit dosage forms may be within, for example, ampules and vials, including a liquid composition, or a composition in a freeze-dried or lyophilized state; a sterile liquid carrier, for example, can be added prior to administration or delivery in vivo. Individual unit dosage forms can be included in multi-dose kits or containers. AAV vectors or AAV virions, and pharmaceutical compositions thereof can be packaged in single or multiple unit dosage form for ease of administration and uniformity of dosage.
[0047] A "therapeutically effective amount" will fall in a relatively broad range determinable through experimentation and/or clinical trials. For example, for in vivo injection, e.g., injection directly into the tissue of a subject (for example, muscle tissue), a therapeutically effective dose will be on the order of from about 106 to about 1015 of the AAV virions per kilogram bodyweight of the subject. In some embodiments, a therapeutically effective dose will be on the order of from about 108 to 1012 AAV virions per kilogram bodyweight of the subject. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves.
[0048] An "effective amount" or "sufficient amount" refers to an amount providing, in single or multiple doses, alone or in combination, with one or more other compositions (therapeutic agents such as a drug), treatments, protocols, or therapeutic regimens agents (including, for example, vaccine regimens), a detectable response of any duration of time (long or short term), an expected or desired outcome in or a benefit to a subject of any measurable or detectable degree or for any duration of time (e.g., for minutes, hours, days, months, years, or cured).
[0049] The doses of an "effective amount" or "sufficient amount" for treatment (e.g., to ameliorate or to provide a therapeutic benefit or improvement) typically are effective to provide a response to one, multiple or all adverse symptoms, consequences or complications of the disease, one or more adverse symptoms, disorders, illnesses, pathologies, or complications, for example, caused by or associated with the disease, to a measurable extent, although decreasing, reducing, inhibiting, suppressing, limiting or controlling progression or worsening of the disease is also a satisfactory outcome.
[0050] "Prophylaxis" and grammatical variations thereof mean a method in which contact, administration or in vivo delivery to a subject is prior to disease. Administration or in vivo delivery to a subject can be performed prior to development of an adverse symptom, condition, complication, etc. caused by or associated with the disease. For example, a screen (e.g., genetic) can be used to identify such subjects as candidates for the described methods and uses, but the subject may not manifest the disease. Such subjects therefore include those screened positive for an insufficient amount or a deficiency in a functional gene product (protein), or producing an aberrant, partially functional or non-functional gene product (protein), leading to disease; and subjects screening positive for an aberrant, or defective (mutant) gene product (protein) leading to disease, even though such subjects do not manifest symptoms of the disease.
[0051] The phrases "tropism" and "transduction" are interrelated, but there are differences. The term "tropism" as used herein refers to the ability of an AAV vector or virion to infect one or more specified cell types, but can also encompass how the vector functions to transduce the cell in the one or more specified cell types; i.e., tropism refers to preferential entry of the AAV vector or virion into certain cell or tissue type(s) and/or preferential interaction with the cell surface that facilitates entry into certain cell or tissue types, optionally and preferably followed by expression (e.g., transcription and, optionally, translation) of sequences carried by the AAV vector or virion in the cell, e.g., for a recombinant virus, expression of the heterologous nucleotide sequence(s). As used herein, the term "transduction" refers to the ability of an AAV vector or virion to infect one or more particular cell types; i.e., transduction refers to entry of the AAV vector or virion into the cell and the transfer of genetic material contained within the AAV vector or virion into the cell to obtain expression from the vector genome. In some cases, but not all cases, transduction and tropism may correlate.
[0052] Unless indicated otherwise, "efficient transduction" or "efficient tropism," or similar terms, can be determined by reference to a suitable control (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 110%, 125%, 150%, 175%, or 200% or more of the transduction or tropism, respectively, of the control). Suitable controls will depend on a variety of factors including the desired tropism profile. Similarly, it can be determined if a capsid and/or virus "does not efficiently transduce" or "does not have efficient tropism" for a target tissue, or similar terms, by reference to a suitable control.
V. DETAILED DESCRIPTION
[0053] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. [0054] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[0055] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.
[0056] Unless defined otherwise, 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 belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.
[0057] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
[0058] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. As such, the articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the polypeptide” includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
[0059] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. For example, 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 subcombinations 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.
[0060] While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §1 12.
Variant AA V capsid proteins
[0061] Provided are variant AAV capsid proteins that include an azide-bearing unnatural amino acid substitution (e.g. in some cases one single unnatural amino acid substitution). In some embodiments, the azide-bearing unnatural amino acid substitution is at a position corresponding to: T456, D555, R587/A587, or N589 of the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2; or E330, T457, N499, or N590 of the AAV8 amino acid sequence set forth as SEQ ID NO: 7; or T455, T456, or N588 of the AAV- LK03 amino acid sequence set forth as SEQ ID NO: 13. As would be understood by one of ordinary skill in the art, the term “R587/A587” is used herein because the amino acid at position 587 in the AAV-DJ capsid sequence (see SEQ ID NO: 1 ) is an arginine (R587) while the amino acid at that same position in the AAV-DJR/A capsid sequence is an alanine (A587). The term “R587/A587” is thus used to refer to position 587 when in the context of referencing both SEQ ID NOs: 1 and 2. In some embodiments, the azide-bearing unnatural amino acid is at a position inserted between positions corresponding to N590 and T591 of the AAV8 amino acid sequence set forth as SEQ ID NO: 7. In some embodiments, the azide- bearing unnatural amino acid substitution is at a position corresponding to: T456, D555, R587/A587, or N589 of the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the azide-bearing unnatural amino acid substitution is at a position corresponding to: E330, T457, N499, or N590 of the AAV8 amino acid sequence set forth as SEQ ID NO: 7. In some embodiments, the azide-bearing unnatural amino acid substitution is at a position corresponding to: T455, T456, or N588 of the AAV- LK03 amino acid sequence set forth as SEQ ID NO: 13.
[0062] In some cases, the variant AAV capsid protein includes an amino acid sequence having: 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 , 2, 7, and 13. In some cases, the variant AAV capsid protein includes an amino acid sequence having: 90% or more (e.g., 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 , 2, 7, and 13. In some cases, the variant AAV capsid protein includes an amino acid sequence having: 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16. In some cases, the variant AAV capsid protein includes an amino acid sequence having: 90% or more (e.g., 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16. In some cases, the variant AAV capsid protein includes the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16 (e.g., the amino acid sequence set forth in any one of SEQ ID NOs: 5-6, 12, or 14).
[0063] In some cases, the variant AAV capsid protein includes an amino acid sequence having: (i) 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, wherein said azide-bearing unnatural amino acid substitution is at position N589 or R587/A587; (ii) 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV8 amino acid sequence set forth as SEQ ID NO: 7, wherein said azide-bearing unnatural amino acid substitution is at position T457; or (iii) 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV- LK03 amino acid sequence set forth as SEQ ID NO: 13, wherein said azide-bearing unnatural amino acid substitution is at position N588. In some cases, the variant AAV capsid protein includes an amino acid sequence having: 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, wherein said azide-bearing unnatural amino acid substitution is at position N589 or R587/A587.
[0064] In some embodiments, the variant AAV capsid protein includes the azide-bearing unnatural amino acid substitution in an AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid polypeptide. In some cases, the variant AAV capsid protein includes an amino acid sequence having 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with a wild type AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid protein.
[0065] In some cases, the variant AAV capsid protein is a shuffled variant, meaning that the variant AAV capsid protein resulted from the shuffling of multiple parent capsid protein sequences - and thus such a variant AAV capsid protein includes stretches of wild type sequence, but the capsid protein sequence as a whole does not occur in nature.
Azide-bearing unnatural amino acid
[0066] An azide-bearing unnatural amino acid can be any amino acid bearing an azide (e.g., an azido amino acid). Examples include, but are not necessarily limited to: azidolysine, 6-Azido-D-lysine, 6-Azido-L-lysine, azido-alanine, L-azidobutyl-alanine, 3- Azido-D-alanine, 3-Azido-L-alanine, azido-homoalanine, 4-Azido-L-homoalanine, azido-ornithine, azido-phenylalanine, 4-Azido-L-phenylalanine, p-azido-phenylalanine, and 5-azido-L-norvaline. In some embodiments, the unnatural amino acid is an azidolysine.
[0067] In some cases, the variant AAV capsid protein includes at least one arginine to alanine mutation at a heparan sulfate proteoglycan (HSPG) binding site (e.g., at an amino acid position corresponding to position 587 and/or 590 of SEQ ID NO: 1 ). In some cases, the variant AAV capsid protein includes at least one amino acid mutation (e.g., 1 , 2, 3, 4, 1-4, 1 -3, 2-4, 2-3, or at least 2 amino acid mutations) that reduces HSPG binding affinity. Such mutations for various AAV types will be known to one of ordinary skill in the art (see, e.g., Cabanes-Creus et al., Mol Ther Methods Clin Dev. 2020 Jun 12; 17).
Targeting Moiety
[0068] In some embodiments, the variant AAV capsid proteins are chemically modified such that the capsid protein is conjugated via the azide-bearing unnatural amino acid to a targeting moiety such as folic acid, a DNA aptamer (e.g., AS1411 ), or an RNA aptamer (e.g., E3). In some cases the conjugation is via a linker (e.g., a Polyethylene glycol (PEG) linker). In some cases, the linker (e.g.., PEG) is less than 5 kDa in length (e.g., 4 kDa or less, 3 kDa or less, or 2 kDa or less). In some cases, the linker (e.g.., PEG) is about 2 kDa in length. The targeting moiety can be conjugated to the unnatural amino acid using any convenient approach, e.g., via click chemistry reaction between the azide of the unnatural amino and a click chemistry group (e.g., dibenzocyclooctyne-amine (DBCO)) of the targeting moiety.
[0069] The targeting moiety may vary and may be selected based, e.g., on the nature of the molecule to be targeted, e.g., cell surface molecule on the target cell, or an extracellular or secreted molecule. Non-limiting examples of a targeting moiety that may be employed include a polypeptide, an antibody, a ligand, an aptamer, and a small molecule.
[0070] In certain embodiments, the targeting moiety specifically binds the target molecule, e.g., a cell surface molecule of the target cell, or an extracellular or secreted target molecule. As used herein, a first molecule “specifically binds” or “preferentially binds” to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration than it binds to other substances, e.g., in a sample. In certain embodiments, the targeting moiety “specifically binds” the target molecule if it binds to or associates with the target molecule with an affinity or Ka (that is, an association rate constant of a particular binding interaction with units of 1/M) of, for example, greater than or equal to about 104 M-1. Alternatively, affinity may be defined as an equilibrium dissociation constant (KD) of a particular binding interaction with units of M (e.g., 10-2 M to 10-13 M, or less). In certain aspects, specific binding means the targeting moiety binds to the target molecule with a KD of less than or equal to about 10-5 M, less than or equal to about 10-6 M, less than or equal to about 10-7 M, less than or equal to about 10-8 M, or less than or equal to about 10-9 M, 10-10 M, 10-11 M, or 10-12 M or less. The binding affinity of the targeting moiety for the target molecule can be readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 or BIAcore T200 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the like.
[0071] According to some embodiments, the targeting moiety is an antibody. By “antibody” is meant an antibody or immunoglobulin of any isotype (e.g., IgG (e.g., lgG1 , lgG2, lgG3, or lgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer which in turn is composed of two dimers of a heavy and light chain polypeptide); single chain antibodies (e.g., scFv); fragments of antibodies (e.g., fragments of whole or single chain antibodies) which retain specific binding to the target molecule (e.g., a cell surface molecule of a target cell), including, but not limited to single chain Fv (scFv), Fab, (Fab’)2, (scFv’)2, and diabodies; chimeric antibodies; monoclonal antibodies, human antibodies, humanized antibodies (e.g., humanized whole antibodies, humanized half antibodies, or humanized antibody fragments, e.g., humanized scFv); and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. In certain embodiments, the antibody is selected from an IgG, single chain Fv (scFv), Fab, (Fab)2, (scFv’)2, or a single variable domain located on a heavy chain (VHH). According to some embodiments, the antibody is a VHH (sometimes referred to herein and elsewhere as a “nanobody”). The antibody may be detectably labeled, e.g., with an in vivo imaging agent, a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like.
[0072] An antibody included as the targeting moiety will vary based on the cell to be targeted. In some embodiments, the antibody specifically binds to an antigen on the surface of a target cell. Target cells of interest include, but are not limited to, cells that are relevant to a particular disease or condition, e.g., a mucin-associated condition. According to some embodiments, the target cell is selected from a cancer cell, an immune cell, and an endothelial cell. As such, in some embodiments, the target cells are cancer cells. By “cancer cell” is meant a cell exhibiting a neoplastic cellular phenotype, which may be characterized by one or more of, for example, abnormal cell growth, abnormal cellular proliferation, loss of density dependent growth inhibition, anchorage-independent growth potential, ability to promote tumor growth and/or development in an immunocompromised non-human animal model, and/or any appropriate indicator of cellular transformation. “Cancer cell” may be used interchangeably herein with “tumor cell”, “malignant cell” or “cancerous cell”, and encompasses cancer cells of a solid tumor, a semi-solid tumor, a primary tumor, a metastatic tumor, and the like. In certain embodiments, the cancer cell is a carcinoma cell.
[0073] According to some embodiments, when the target cell is a cancer cell, the targeting moiety specifically binds to a tumor antigen on the surface of the cancer cell. Nonlimiting examples of tumor antigens to which the targeting moiety may specifically bind include 5T4, AXL receptor tyrosine kinase (AXL), B-cell maturation antigen (BCMA), c-MET, C4.4a, carbonic anhydrase 6 (CA6), carbonic anhydrase 9 (CA9), Cadherin-6, CD19, CD22, CD25, CD27L, CD30, CD33, CD37, CD44v6, CD56, CD70, CD74, CD79b, CD123, CD138, carcinoembryonic antigen (CEA), cKit, Cripto protein, CS1 , delta-like canonical Notch ligand 3 (DLL3), endothelin receptor type B (EDNRB), EpCAM, ephrin A4 (EFNA4), epidermal growth factor receptor (EGFR), EGFRvlll, ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3), EPH receptor A2 (EPHA2), fibroblast growth factor receptor 2 (FGFR2), fibroblast growth factor receptor 3 (FGFR3), FMS-like tyrosine kinase 3 (FLT3), folate receptor 1 (FOLR1 ), GLUT3, glycoprotein non-metastatic B (GPNMB), guanylate cyclase 2 C (GUCY2C), HCAM, human epidermal growth factor receptor 2 (HER2), human epidermal growth factor receptor 3 (HER3), Integrin alpha, lysosomal-associated membrane protein 1 (LAMP-1 ), Lewis Y, LIV-1 , leucine rich repeat containing 15 (LRRC15), mesothelin (MSLN), sodium-dependent phosphate transport protein 2B (NaPi2b), Nectin-4, NMB, NOTCH3, p-cadherin (p-CAD), prostate-specific membrane antigen (PSMA), protein tyrosine kinase 7 (PTK7), solute carrier family 44 member 4 (SLC44A4), SLIT like family member 6 (SLITRK6), STEAP family member 1 (STEAP1 ), tissue factor (TF), T cell immunoglobulin and mucin protein-1 (TIM-1 ), trophoblast cell-surface antigen (TROP-2), and VEGF-A. [0074] Non-limiting examples of antibodies that specifically bind to tumor antigens which may be employed as a targeting moiety include Adecatumumab, Ascrinvacumab, Cixutumumab, Conatumumab, Daratumumab, Drozitumab, Duligotumab, Durvalumab, Dusigitumab, Enfortumab, Enoticumab, Figitumumab, Ganitumab, Glembatumumab, Intetumumab, Ipilimumab, Iratumumab, Icrucumab, Lexatumumab, Lucatumumab, Mapatumumab, Narnatumab, Necitumumab, Nesvacumab, Ofatumumab, Olaratumab, Panitumumab, Patritumab, Pritumumab, Radretumab, Ramucirumab, Rilotumumab, Robatumumab, Seribantumab, Tarextumab, Teprotumumab, Tovetumab, Vantictumab, Vesencumab, Votumumab, Zalutumumab, Flanvotumab, Altumomab, Anatumomab, Arcitumomab, Bectumomab, Blinatumomab, Detumomab, Ibritumomab, Minretumomab, Mitumomab, Moxetumomab, Naptumomab, Nofetumomab, Pemtumomab, Pintumomab, Racotumomab, Satumomab, Solitomab, Taplitumomab, Tenatumomab, Tositumomab, Tremelimumab, Abagovomab, Igovomab, Oregovomab, Capromab, Edrecolomab, Nacolomab, Amatuximab, Bavituximab, Brentuximab, Cetuximab, Derlotuximab, Dinutuximab, Ensituximab, Futuximab, Girentuximab, Indatuximab, Isatuximab, Margetuximab, Rituximab, Siltuximab, Ublituximab, Ecromeximab, Abituzumab, Alemtuzumab, Bevacizumab, Bivatuzumab, Brontictuzumab, Cantuzumab, Cantuzumab, Citatuzumab, Clivatuzumab, Dacetuzumab, Demcizumab, Dalotuzumab, Denintuzumab, Elotuzumab, Emactuzumab, Emibetuzumab, Enoblituzumab, Etaracizumab, Farletuzumab, Ficlatuzumab, Gemtuzumab, Imgatuzumab, Inotuzumab, Labetuzumab, Lifastuzumab, Lintuzumab, Lorvotuzumab, Lumretuzumab, Matuzumab, Milatuzumab, Nimotuzumab, Obinutuzumab, Ocaratuzumab, Otlertuzumab, Onartuzumab, Oportuzumab, Parsatuzumab, Pertuzumab, Pinatuzumab, Polatuzumab, Sibrotuzumab, Simtuzumab, Tacatuzumab, Tigatuzumab, Trastuzumab, Tucotuzumab, Vandortuzumab, Vanucizumab, Veltuzumab, Vorsetuzumab, Sofituzumab, Catumaxomab, Ertumaxomab, Depatuxizumab, Ontuxizumab, Blontuvetmab, Tamtuvetmab, or a tumor antigenbinding variant thereof. As used herein, “variant” is meant the antibody specifically binds to the particular antigen (e.g., HER2 for trastuzumab) but has fewer or more amino acids than the parental antibody (e.g., is a fragment (e.g., scFv) of the parental antibody), has one or more amino acid substitutions relative to the parental antibody, or a combination thereof.
[0075] In certain embodiments, the targeting moiety is an antibody approved by the United
States Food and Drug Administration and/or the European Medicines Agency (EMA) for use as a therapeutic antibody (e.g., for targeting certain disease-associated cells in a patient, etc.), or a fragment thereof (e.g., a single-chain version of such an antibody, such as an scFv version of the antibody) that retains the ability to specifically bind the target antigen.
[0076] Examples of targeting moieties are antibodies specific for antigens that include, but are not necessarily limited to: carbonic anhydrase IX, alpha-fetoprotein (AFP), a- actinin-4, A3, ART-4, B7, Ba 733, BAGE, BrE3-antigen, CA125, CAMEL, CAP-1 , CASP-8/m, CCL19, CCL21 , CD1 , CD1 a, CD2, CD3, CD4, CD5, CD8, CD11 A, CD14, CD15, CD16, CD18, CD19, CD20, CD21 , CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD70L, CD74, CD79a, CD80, CD83, CD95, CD126, CD132, CD133, CD138, CD147, CD154, CDC27, CDK-4/m, CDKN2A, CTLA-4, CXCR4, CXCR7, CXCL12, HIF-1 a, colon-specific antigen-p (CSAp), CEACAM5, CEACAM6, c-Met, DAM, epidermal growth factor receptor (EGFR), EGFRvlll, EGP-1 (TROP-2), EGP-2, ELF2-M, Ep-CAM, fibroblast growth factor (FGF), Flt-1 , Flt-3, folate receptor, G250 antigen, GAGE, gp100, GRO- , HLA-DR, HM1.24, human chorionic gonadotropin (HCG) and its subunits, HER2/neu, histone H2B, histone H3, histone H4, HMGB-1 , hypoxia inducible factor (HIF-1 ), HSP70-2M, HST-2, insulin-like growth factor-1 receptor (IGF-1 R), IFN-y IFN-a, IFN-p, IFN-A, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-23, IL-25, insulin-like growth factor-1 (IGF-1), KC4-antigen, KS-1 -antigen, KS1 -4, Le-Y, LDR/FUT, macrophage migration inhibitory factor (MIF), MAGE, MAGE-3, MART-1 , MART-2, NY-ESO-1 , TRAG-3, mCRP, MCP-1 , M IP- 1 A, M IP- 1 B, MIF, MUC1 , MUC2, MUC3, MUC4, MUC5ac, MUC13, MUC16, MUM-1/2, MUM-3, NCA66, NCA95, NCA90, PAM4 antigen, PD-1 , PD-L1 , PD-1 receptor, placental growth factor, p53, PLAGL2, prostatic acid phosphatase, PSA, PRAME, PSMA, P1 GF, ILGF, ILGF-1 R, IL-6, IL-25, RS5, RANTES, T101 , SAGE, 5100, survivin, survivin-2B, TAC, TAG-72, tenascin, TRAIL receptors, TNF-o, Tn antigen, tumor necrosis antigens, VEGFR, ED-B fibronectin, WT-1 , 17-1 A-antigen, complement factors C3, 03a, C3b, 05a, and C5
[0077] When an antibody is employed as the targeting moiety, the mucin-active protease may be stably associated with (e.g., conjugated to, fused to, or the like) any convenient portion of the antibody. In certain embodiments, the mucin-active protease is stably associated with a light chain of the antibody, e.g., a kappa (K) light chain or fragment thereof or a lambda (A) light chain or fragment thereof. According to some embodiments, the antibody light chain or fragment thereof includes a light chain variable region (VL). Such an antibody light chain or fragment thereof may further include an antibody light chain constant region (CL) or fragment thereof. In certain embodiments, the antibody light chain or fragment thereof is a full-length antibody light chain - that is, an antibody light chain that includes a VL and a CL. In certain embodiments, the mucin-active protease is stably associated with a VL (if present) or a CL (if present), e.g., at or near the N-terminus of a VL or at or near the C-terminus of a CL.
[0078] When an antibody is employed as the targeting moiety, the mucin-active protease may be stably associated with a heavy chain or fragment thereof of the antibody. In certain embodiments, the antibody heavy chain or fragment thereof includes a y, a, 5, E, or p antibody heavy chain or fragment thereof. According to some embodiments, the antibody heavy chain or fragment thereof is an IgG heavy chain or fragment thereof, e.g., a human IgG 1 heavy chain or fragment thereof. In certain embodiments, the antibody heavy chain or fragment thereof comprises a heavy chain variable region (VH). Such an antibody heavy chain or fragment thereof may further include a heavy chain constant region or fragment thereof. For example, when the antibody includes a heavy chain constant region or fragment thereof, the antibody heavy chain constant region or fragment thereof may include one or more of a CH1 domain, CH2 domain, and/or CH3 domain. According to some embodiments, the antibody heavy chain is a full-length antibody heavy chain - that is, an antibody heavy chain that includes a VH, a CH1 domain, a CH2 domain, and a CH3 domain. In certain embodiments, the mucin-active protease is stably associated with an Fc region of the antibody. According to some embodiments, the mucin-active protease is stably associated with the antibody at or near the N-terminus of a VH or at or near the C-terminus of a CH3 domain.
[0079] According to certain embodiments, the targeting moiety is a ligand. As used herein, a “ligand” is a substance that forms a complex with a biomolecule in nature to serve a biological purpose. The ligand may be a substance selected from a circulating factor, a secreted factor, a cytokine, a growth factor, a hormone, a peptide, a polypeptide, a small molecule, and a nucleic acid, that forms a complex with the target molecule, e.g., a cell surface molecule on the surface of a target cell. In certain aspects, when the targeting moiety is a ligand, the ligand is modified in such a way that complex formation with the target molecule occurs, but the normal biological result of such complex formation does not occur. In certain embodiments, the ligand is the ligand of a cell surface receptor present on a target cell. Cell surface receptors of interest include, but are not limited to, receptor tyrosine kinases (RTKs), non-receptor tyrosine kinases (non-RTKs), growth factor receptors, etc. When the mucin-active protease is stably associated with a ligand as the targeting moiety, the mucin-active protease may be stably associated with any suitable region of the ligand, e.g., a region of attachment that does not interfere or substantially interfere with the ability of the ligand to bind (e.g., specifically bind) the target molecule.
[0080] In certain embodiments, the targeting moiety is an aptamer. By “aptamer” is meant a nucleic acid (e.g., an oligonucleotide) that has a specific binding affinity for the target molecule. In some cases, the aptamer is a DNA aptamer (e.g., AS1411 ). In some cases, the aptamer is an RNA aptamer (e.g., E3, C36, and the like). In some cases, the aptamer is a DNA/RNA hybrid (e.g., Apt-dONT-DEN, A10-3-J1 ). Examples of RNA aptamers include, but are not limited to, those that target: 4,4’-methylenedianiline (MDA), Acetylcholine receptor (AChR), African trypanosomes, AMPA receptor GluR2Qflip, Beta Secretace (S10), Beta Secretase (THU), CD4, CTLA-4, EGFR (E07), EGFR (J18), , EGFRvll I (E17), Erythrocyte membrane protein 1 (PfEMPI), gp120, HER3, Human keratinocyte growth factor, L-selectin, Neruotensin-1 (NTS-1), NF-KB, Phosphatidylcholine: cholesterol liposomes, PSMA (A10), Raf-1 , RET receptor tyrosine kinase, TCF-1 , Tenascin-C (AptamerTTAI), TGF-|3 type III receptor, Tumor necrosis factor superfamily member 4-1 BB, Tumor necrosis factor superfamily member 0X40, VEGF, Wilms tumor protein (WT1 ), and av[33 integrin (see, e.g., Germer et al., Int J Biochem Mol Biol. 2013; 4(1): 27-40). Examples of RNA aptamers include, but are not limited to: E3, C36, E2, PTCH1 -SMG-E1 , A10, A15, anti- PSMA aptamer, EpDT3, A10, A10-3.2, and SZTI01 (see, e.g., Wang et al., Molecules. 2022 Jun; 27(1 1 ): 3446). Examples of DNA aptamers include, but are not limited to: AS1411 , CD, Apt 1 , Ecad01 , DAC, A10, and A9 (see, e.g., Wang et aL, Molecules. 2022 Jun; 27(1 1 ): 3446). Aptamers can exhibit certain desirable properties for targeted delivery, such as ease of selection and synthesis, high binding affinity and specificity, low immunogenicity, and versatile synthetic accessibility. Aptamers that bind to cell surface molecules are known and include, e.g., TTA1 (a tumor targeting aptamer to the extracellular matrix protein tenascin-C). Aptamers that find use in the context of the present disclosure include those described in Zhu et al. (2015) ChemMedChem 10(1 ):39-45; Sun et al. (2014) Mol. Ther. Nucleic Acids 3:e182; Zhang et al. (2011 ) Curr. Med. Chem. 18(27):4185-4194; and Germer et al., Int J Biochem Mol Biol. 2013; 4(1): 27-40. [0081] In some embodiments, the targeting moiety is a small molecule. By “small molecule” is meant a compound having a molecular weight of 1000 atomic mass units (amu) or less. In some embodiments, the small molecule is 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less. In certain aspects, the small molecule is not made of repeating molecular units such as are present in a polymer. In certain aspects, the target molecule is a cell surface receptor for which the ligand is a small molecule, and the targeting moiety is the small molecule ligand (or a derivative thereof) of the receptor. Small molecules that find use as targeting moieties are known. As just one example, folic acid (FA) derivatives have been shown to effectively target certain types of cancer cells by binding to the folate receptor, which is overexpressed, e.g., in many epithelial tumors. See, e.g., Vergote et al. (2015) Ther. Adv. Med. Oncol. 7(4):206-218. In another example, the small molecule sigma-2 has proven to be effective in targeting cancer cells. See, e.g., Hashim et al. (2014) Molecular Oncology 8(5) :956-967. Sigma-2 is the small molecule ligand for sigma-2 receptors, which are overexpressed in many proliferating tumor cells including pancreatic cancer cells. In certain embodiments, a small molecule is employed as the targeting moiety, and it has been demonstrated in the context of a small molecule drug conjugate (SMDC) that the small molecule is effective at targeting a drug to a target cell of interest by binding to a cell surface molecule on the target cell.
Nucleic acids
[0082] Also provided are nucleic acids encoding a subject variant AAV capsid protein, where the nucleotide sequence encoding the variant AAV capsid protein includes a TAG stop codon at the position encoding said unnatural amino acid substitution. This is to allow for the insertion of an azide-bearing unnatural amino acid, e.g., using a cell having the appropriate tRNA to recognize the stop codon and insert the unnatural amino acid (e.g., Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNApyi , see, e.g., Hohl et al., Sci Rep. 2019 Aug 19;9(1):11971 ). Examples of such nucleic acids include, but are not limited to: plasmids, viral vectors, and the like. In some cases, the nucleic acid is a viral vector that encodes a variant AAV capsid protein and includes a nucleotide sequence of interest. In some cases, an rAAV particle includes a variant AAV protein and a payload nucleic acid that includes a nucleotide sequence of interest.
[0083] Also provided are recombinant AAV particles that include a subject variant AAV capsid protein and a nucleic acid payload of interest. [0084] The nucleic acid payload of interest can be any convenient nucleic acid. For example, in some cases the nucleic acid payload of interest (DNA or mRNA) encodes a polypeptide (e.g., a therapeutic protein or a genome-editing enzyme such as a CRISPR/Cas effector protein, a zinc finger nuclease, or a TALEN). In some cases, the nucleotide sequence encoding the polypeptide is operably linked to the small ubiquitous INS84 promoter (see, e.g., Chai, S. et al. Hum. Gene Then, 2022). In some cases, the nucleic acid payload of interest is a non-coding RNA (e.g., a CRISPR/Cas guide RNA, an shRNA, an siRNA, a miRNA, an aptamer, a ribozyme) or encodes that non-coding RNA.
[0085] In some cases a subject nucleic acid, in addition to including a sequence that encodes a variant AAV capsid protein, also encodes a nucleic acid insert (also referred to as a heterologous nucleotide sequence or the “nucleotide sequence of interest”). Likewise, in some cases a subject rAAV particle, in addition to including a variant AAV capsid protein, also includes (e.g., encapsidates) a nucleic acid payload of interest (which includes a nucleotide sequence of interest). The “nucleotide sequence of interest can be operably linked to control elements directing the transcription or expression thereof once the sequence is present inside of a cell (e.g., in some cases integrated into the cell’s genome). Such control elements can comprise control sequences normally associated with the selected gene (e.g., endogenous cellular control elements). Alternatively, heterologous control sequences can be employed. Useful heterologous control sequences generally include those derived from sequences encoding mammalian or viral genes. Examples include, but are not limited to, the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, an endogenous cellular promoter heterologous to the gene of interest, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like. In addition, sequences derived from nonviral genes, such as the murine metallothionein gene, can also be used. Such promoter sequences are commercially available from, e.g., Stratagene (San Diego, Calif.).
[0086] In some embodiments, a cell type-specific or a tissue-specific promoter can be operably linked to the nucleotide sequence of interest and allowing for selective or preferential expression in a particular cell type(s) or tissue(s). Thus, in some embodiments, an inducible promoter can be operably linked to the nucleotide sequence of interest. [0087] In some embodiments, a nucleic acid payload is packaged with the variant AAV capsid polypeptides of the disclosure. In some embodiments, the nucleic acid payload is at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1 100, 1200, 1300, 1400, or 1500 nucleotides (nt) in length. In some embodiments, the nucleic acid payload is 50 nucleotides to 4000 nucleotides long (e.g., 50-3000, 50-2000, 50-1500, 50-1200, 50-1000, 50-900, 50-750, 50-500, 100-4000, 100-3000, 100-2000, 100- 1500, 100-1200, 100-1000, 100-900, 100-750, 100-500, 300-4000, 300-3000, 300- 2000, 300-1500, 300-1200, 300-1000, 300-900, 300-750, 300-500, 500-4000, 500- 3000, 500-2000, 500-1500, 500-1200, 500-1000, or 500-900 nt long). In some embodiments, the nucleotide sequence of interest is at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides (nt) in length. In some embodiments, the nucleotide sequence of interest is 50 nucleotides to 4000 nucleotides long (e.g., 50-3000, 50-2000, 50-1500, 50-1200, 50-1000, 50-900, 50-750, 50-500, 100-4000, 100-3000, 100-2000, 100-1500, 100-1200, 100-1000, 100- 900, 100-750, 100-500, 300-4000, 300-3000, 300-2000, 300-1500, 300-1200, 300- 1000, 300-900, 300-750, 300-500, 500-4000, 500-3000, 500-2000, 500-1500, 500- 1200, 500-1000, or 500-900 nt long).
[0088] In some embodiments, an AAV vector packaged by a variant AAV capsid polypeptide is at least about 2000 nucleotides in total length and up to about 5000 nucleotides in total length. In some embodiments, an AAV vector packaged by the variant AAV capsid polypeptides is about 2000 nucleotides, about 2400 nucleotides, about 2800 nucleotides, about 3000 nucleotides, about 3200 nucleotides, about 3400 nucleotides, about 3600 nucleotides, about 3800 nucleotides, about 4000 nucleotides, about 4200 nucleotides, about 4400 nucleotides, about 4600 nucleotides, about 4700 nucleotides, or about 4800 nucleotides. In some embodiments, an AAV vector packaged by the variant AAV capsid polypeptides is between about 2000 nucleotides (2 kb) and about 5000 nucleotides (5 kb). In some embodiments, an AAV vector packaged by the variant AAV capsid polypeptides is between about 2400 nucleotides (2.4 kb) and about 4800 nucleotides (4.8 kb). In some embodiments, an AAV vector packaged by the variant AAV capsid polypeptides is between about 3000 nucleotides (3 kb) and about 5000 nucleotides (5 kb). In some embodiments, an AAV vector packaged by the variant AAV capsid polypeptides is between about 3000 nucleotides (3 kb) and about 4000 nucleotides (4 kb).
[0089] The AAV vectors or AAV virions disclosed herein can also include conventional control elements operably linked to the nucleic acid insert (also referred to as a heterologous nucleotide sequence or a “nucleotide sequence of interest”) in a manner permitting transcription, translation and/or expression in a cell transfected with the AAV vector or infected with the AAV virion produced according to the present disclosure. As used herein, "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
[0090] Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e. , Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of expression control sequences, including promoters selected from native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.
[0091] Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al., Cell, 41 :521 -530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the beta-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 promoter (Invitrogen). Inducible promoters allow regulation of gene expression and can be regulated by exogenously supplied compounds, environmental factors such as temperature, or the presence of a specific physiological state, e.g., acute phase, a particular differentiation state of the cell, or in replicating cells only. Inducible promoters and inducible systems are available from a variety of commercial sources, including, without limitation, Invitrogen, Clonetech and Ariad. Many other systems have been described and can be readily selected by one of skill in the art. Examples of inducible promoters regulated by exogenously supplied compounds, include, the zinc-inducible sheep metalloth ionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system (WO 98/10088); the ecdysone insect promoter (No et al., (1996) Proc. Natl. Acad. Sci. USA, 93:3346-3351), the tetracycline-repressible system (Gossen et al., (1992) Proc. Natl. Acad. Sci. USA, 89:5547-5551 ), the tetracycline-inducible system (Gossen et al., (1995) Science, 268:1766-1769, see also Harvey et al., (1998) Curr. Opin. Chem. Biol., 2:512-518), the RU486-inducible system (Wang et al., (1997) Nat. Biotech., 15:239-243 and Wang et al., (1997) Gene Ther., 4:432-441 ) and the rapamycin-inducible system (Magari et al., (1997) J. Clin. Invest., 100:2865-2872). Other types of inducible promoters useful in this context are those regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or in replicating cells only.
[0092] In some cases a nucleotide sequence of interest is operably linked to a tissue-specific promoter. For instance, if expression in skeletal muscle is desired, a promoter active in muscle should be used. These include the promoters from genes encoding skeletal .beta.-actin, myosin light chain 2A, dystrophin, muscle creatine kinase, as well as synthetic muscle promoters with activities higher than naturally-occurring promoters (see Li et al., Nat. Biotech., 17:241-245 (1999)). Examples of promoters that are tissue-specific are known for liver (albumin, Miyatake et al., (1997) J. Virol., 71 :5124- 32; hepatitis B virus core promoter, Sandig et al., (1996) Gene Ther., 3:1002-9; alphafetoprotein (AFP), Arbuthnot et al., (1996) Hum. Gene Then, 7:1503-14), bone osteocalcin (Stein et al., (1997) Mol. Biol. Rep., 24:185-96); bone sialoprotein (Chen et al., (1996) J. Bone Miner. Res., 11 :654-64), lymphocytes (CD2, Hansal et al., (1998) J. Immunol., 161 :1063-8; immunoglobulin heavy chain; T cell receptor chain), neuronal such as neuron-specific enolase (NSE) promoter (Andersen et al., (1993) Cell. Mol. Neurobiol., 13:503-15), neurofilament light-chain gene (Piccioli et al., (1991 ) Proc. Natl. Acad. Sci. USA, 88:5611 -5), and the neuron-specific vgf gene (Piccioli et al., (1995) Neuron, 15:373-84), among others.
[0093] In various embodiments, AAV vectors or AAV virions carrying one or more therapeutically useful nucleic acid inserts (also referred to as a heterologous nucleotide sequences or “nucleotide sequences of interest”) also include selectable markers or reporter genes, e.g., sequences encoding geneticin, hygromycin or puromycin resistance, among others. Selectable reporters or marker genes can be used to signal the presence of the plasmids/vectors in bacterial cells, including, for example, examining ampicillin resistance. Other components of the plasmid may include an origin of replication. Selection of these and other promoters and vector elements are conventional and many such sequences are available (see, e.g., Sambrook et al., and references cited therein).
[0094] In some cases a subject nucleotide sequence of interest encodes a non-coding RNA (e.g., a CRISPR/Cas guide RNA, an antisense RNA, a ribozyme, an shRNA, a microRNA, an aptamer). In some cases a subject nucleotide sequence of interest encodes a protein (e.g., a therapeutic protein meant to alleviate a disease and/or its symptoms, a genome-editing enzyme such as a CRISPR/Cas effector protein, TALEN, Zinc Finger nuclease, etc. - meant to provide for targeted genome editing, etc.). Examples of peptide or polypeptides envisioned as having a therapeutic activity for the multicellular organism in which they are expressed (e.g., via a nucleic acid encoding the peptide or polypeptide) include, but are not limited to: factor VIII, factor IX, p-g lobin, a CRISPR/Cas effector protein (e.g., Cas9, Cpf 1 , and the like), a low- density lipoprotein receptor, adenosine deaminase, purine nucleoside phosphorylase, sphingomyelinase, glucocerebrosidase, cystic fibrosis transmembrane conductance regulator, a1 -antitrypsin, CD-18, PDGF, VEGF, EGF, TGFa, TGB , FGF, TNF, IL-1 , IL-2, IL-6, IL-8, endothelium derived growth factor (EDGF), ornithine transcarbamylase, argininosuccinate synthetase, phenylalanine hydroxylase, branched-chain a-ketoacid dehydrogenase, fumarylacetoacetate hydrolase, glucose 6-phosphatase, a-L-fucosidase, p-glucuronidase, a-L-iduronidase, galactose 1 - phosphate uridyltransferase; a neuroprotective factor, e.g. a neurotrophin (e.g. NGF, BDNF, NT-3, NT-4, CNTF), Kifap3, Bcl-xl, collapsin response mediator protein 1 , Chkp, calmodulin 2, calcyon, NPT1 , Eef1 a1 , Dhps, Cd151 , Morf412, CTGF, LDH-A, AtH , NPT2, Ehd3, Cox5b, Tubala, y-actin, Rpsa, NPG3, NPG4, NPG5, NPG6, NPG7, NPG8, NPG9, NPG10, dopamine, interleukins, cytokines, small peptides, the genes/proteins listed in Table 1 (see below: BCKDH complex (E1 a, E1 b and E2 subunits); Methylmalonyl-CoA Mutase; Propionyl-CoA Carboxylase (Alpha and Beta subunits); Isovaleryl CoA dehydrogenase; HADHA; HADHB; LCHAD; ACADM; ACADVL; G6PC (GSD1a); G6PT1 (GSD1 b); SLC17A3; SLC37A4 (GSD1c); Acid alpha-glucosidase; OCTN2; CPT1 ; CACT; CPT2; CPS1 ; ARG1 ; ASL; OTC; UGT1 A1 ; FAH; COL7A1 ; COL17A1 ; MMP1 ; KRT5; LAMA3; LAMB3; LAMC2; ITGB4; and/or ATP7B), and the like. The above list of proteins refers to mammalian proteins, and in many embodiments human proteins, where the nucleotide and amino acid sequences of the above proteins are generally known to those of skill in the art.
[0095] Nonlimiting examples of targeted nucleases (genome-editing enzymes) include naturally occurring and recombinant nucleases, e.g. restriction endonucleases, meganucleases homing endonucleases, CRISPR/Cas effector proteins (e.g., CRISPR/Cas endonucleases such as Cas9, Cas12, Cas13, and the like). Any targeted nuclease(s) that are specific for the integration site of interest and promote the cleavage of an integration site may be encoded by a nucleotide sequence of interest, any examples of nucleases are known in the art, including Zinc finger nucleases (ZFNs), Transcription Activator- Like Effector Nucleases (TALENs), CRISPR/Cas effector proteins, meganucleases, homing endonucleases, restriction endonucleases, and the like (e.g., RecBCD endonuclease, T7 endonuclease, T4 endonuclease IV, Bal 31 endonuclease, Endonuclease I (endo I), Endonuclease II (endo VI, exo III), Micrococcal nuclease, Neurospora endonuclease, S1 -nuclease, P1- nuclease, Mung bean nuclease I, Ustilago nuclease, Dnase I, AP endonuclease, EndoR, etc.).
[0096] In various embodiments, the disclosure provides variant AAV capsid polypeptides capable of forming capsids capable of packaging a variety of therapeutic molecules, including nucleic acids and polypeptides. In various embodiments, the disclosure provides for AAV vectors capable of containing nucleic acid inserts, including for example, transgene inserts or other nucleic acid inserts. This allows for vectors capable of expressing polypeptides. Such nucleic acids can comprise heterologous nucleic acid, nucleic acid gene products, and polypeptide gene products.
[0097] In some embodiments, the nucleotide sequence of interest encodes a non-coding RNA, encodes a protein coding sequence, is an expression cassette, is a multiexpression cassette, is a sequence for homologous recombination, is a genomic gene targeting cassette, and/or is a therapeutic expression cassette. In some embodiments, the expression cassette is a CRISPR/CAS expression system (e.g., including a CRISPR/Cas guide RNA and a CRISPR/Cas effector protein such as Cas9 or Cpf 1 . In some embodiments, a nucleic acid insert comprises a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product, e.g., a nucleic acid gene product or a polypeptide gene product. As noted above, in some embodiments, the gene product is an interfering RNA (e.g., shRNA, siRNA, miRNA). In some embodiments, the gene product is an aptamer. The gene product can be a self-complementary nucleic acid. In some embodiments, the gene product is a polypeptide-coding RNA (e.g., an mRNA).
[0098] Suitable heterologous gene product includes interfering RNA, antisense RNA, ribozymes, and aptamers. Where the gene product is an interfering RNA (RNAi), suitable RNAi include RNAi that decrease the level of a target polypeptide in a cell.
[0099] In some embodiments, exemplary polypeptides include neuroprotective polypeptides and/or anti-angiogenic polypeptides (both of which are therapeutic polypeptides). Suitable polypeptides include, but are not limited to, glial derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF-2), neurturin, ciliary neurotrophic factor (CNTF), nerve growth factor (NGF; e.g., nerve growth factor-. beta.), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-6 (NT-6), epidermal growth factor (EGF), pigment epithelium derived factor (PEDF), a Wnt polypeptide, soluble Flt-1 , angiostatin, endostatin, VEGF, an anti-VEGF antibody, a soluble VEGFR, Factor VIII (FVIII), Factor IX (FIX), and a member of the hedgehog family (sonic hedgehog, Indian hedgehog, and desert hedgehog, etc.).
[00100] In some embodiments, useful therapeutic products encoded by the heterologous nucleic acid sequence include hormones and growth and differentiation factors including, without limitation, insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), any one of the transforming growth factor alpha superfamily, including TGF.alpha., activins, inhibins, or any of the bone morphogenic proteins (BMP) BMPs 1 -15, any one of the heregulin/neuregulin/ARIA/neu differentiation factor (NDF) family of growth factors, nerve growth factor (NGF), brain- derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT-4/5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), neurturin, agrin, any one of the family of semaphorins/collapsins, netrin-1 and netrin- 2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.
[00101] In some embodiments, useful heterologous nucleic acid sequence products include proteins that regulate the immune system including, without limitation, cytokines and lymphokines such as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25 (including IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors alpha and beta., interferons (alpha, beta, and gamma), stem cell factor, flk-2/flt3 ligand. Gene products produced by the immune system are also useful in the present disclosure. These include, without limitations, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules, as well as engineered immunoglobulins and MHC molecules. Useful gene products also include complement regulatory proteins such as complement regulatory proteins, membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1 , CF2 and CD59.
[00102] In some embodiments, useful heterologous nucleic acid sequence products include any one of the receptors for the hormones, growth factors, cytokines, lymphokines, regulatory proteins and immune system proteins. Useful heterologous nucleic acid sequences also include receptors for cholesterol regulation and/or lipid modulation, including the low density lipoprotein (LDL) receptor, high density lipoprotein (HDL) receptor, the very low density lipoprotein (VLDL) receptor, and scavenger receptors. The disclosure also encompasses the use of gene products such as members of the steroid hormone receptor superfamily including glucocorticoid receptors and estrogen receptors, Vitamin D receptors and other nuclear receptors. In addition, useful gene products include transcription factors such as jun, fos, max, mad, serum response factor (SRF), AP-1 , AP-2, myb, MyoD and myogenin, ETS-box containing proteins, TFE3, E2F, ATF1 , ATF2, ATF3, ATF4, ZF5, NFAT, CREB, HNF-4 C/EBP, SP1 , CCAAT-box binding proteins, interferon regulation factor (IRF-1 ), Wilms tumor protein, ETS-binding protein, STAT, GATA-box binding proteins, e.g., GATA-3, and the forkhead family of winged helix proteins.
[00103] In some embodiments, useful heterologous nucleic acid sequence products include, carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetoacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, cystathionine beta-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, Flprotein, T-protein, a cystic fibrosis transmembrane regulator (CFTR) sequence, and a dystrophin cDNA sequence. Still other useful gene products include enzymes useful in enzyme replacement therapy, and which are useful in a variety of conditions resulting from deficient activity of enzyme. For example, enzymes containing mannose-6-phosphate may be utilized in therapies for lysosomal storage diseases (e.g., a suitable gene includes that encoding .beta.-glucuronidase (GUSB)).
Host Cells and Packaging
[00104] Host cells are necessary for generating infectious AAV vectors as well as for generating AAV virions based on the disclosed AAV vectors. Accordingly, the present disclosure provides host cells for generation and packaging of AAV virions based on the AAV vectors of the present disclosure. A variety of host cells are known in the art and find use in the methods of the present disclosure. Any host cells described herein or known in the art can be employed with the compositions and methods described herein - as long as the cell allows for the inclusion of an azide-bearing unnatural amino acid at a stop codon - as described elsewhere herein (e.g., the cell is modified to express an appropriate amnio-acid bearing tRNA).
[00105] The present disclosure provides host cells, e.g., comprising a subject rAAV particle (virion) and/or a subject nucleic acid. A subject host cell can be an isolated cell, e.g., a cell in in vitro culture. A subject host cell can be useful for producing a subject AAV vector or AAV virion. Where a subject host cell is used to produce a subject AAV virion, it is referred to as a "packaging cell." In some embodiments, a subject host cell is stably genetically modified with a subject AAV vector. In other embodiments, a subject host cell is transiently genetically modified with a subject AAV vector.
[00106] In some embodiments, a subject nucleic acid is introduced stably or transiently into a host cell, using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, baculovirus infection, and the like. For stable transformation, a subject nucleic acid will generally further include a selectable marker, e.g., any of several well-known selectable markers such as neomycin resistance, and the like.
[00107] In some embodiments, the host cell for use in generating infectious virions can be selected from any biological organism, including prokaryotic (e.g., bacterial) cells, and eukaryotic cells, including, insect cells, yeast cells and mammalian cells. A subject host cell is generated by introducing a subject nucleic acid (i.e., AAV vector) into any of a variety of cells, e.g., mammalian cells, including, e.g., murine cells, and primate cells (e.g., human cells). Particularly desirable host cells are selected from among any mammalian species. In some embodiments, cells include without limitation, cells such as A549, WEHI, 10T1/2, BHK, MDCK, COS 1 , COS 7, BSC 1 , BSC 40, BMT 10, WI38, HeLa, CHO, 293, Vero, NIH 3T3, PC12, Huh-7 Saos, C2C12, RAT1 , Sf9, L cells, HT1080, human embryonic kidney (HEK), human embryonic stem cells, human adult tissue stem cells, pluripotent stem cells, induced pluripotent stem cells, reprogrammed stem cells, organoid stem cells, bone marrow stem cells, HLHepG2, HepG2 and primary fibroblast, hepatocyte and myoblast cells derived from mammals including human, monkey, mouse, rat, rabbit, and hamster. The selection of the mammalian species providing the cells is not a limitation of this disclosure; nor is the type of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell, etc. The requirement for the cell used is it is capable of infection or transfection by an AAV vector. In some embodiments, the host cell is one that has Rep and Cap stably transfected in the cell, including in some embodiments a variant AAV capsid polypeptide as described herein. In some embodiments, the host cell expresses a variant AAV capsid polypeptide of the disclosure or part of an AAV vector as described herein, such as a heterologous nucleic acid sequence contained within the AAV vector.
[00108] In some embodiments, the preparation of a host cell according to the disclosure involves techniques such as assembly of selected DNA sequences. This assembly may be accomplished utilizing conventional techniques. Such techniques include cDNA and genomic cloning, which are well known and are described in Sambrook et al., cited above, use of overlapping oligonucleotide sequences of the adenovirus and AAV genomes, combined with polymerase chain reaction, synthetic methods, and any other suitable methods providing the desired nucleotide sequence.
[00109] In some embodiments, introduction of the AAV vector into the host cell may also be accomplished using techniques known to the skilled artisan and as discussed throughout the specification. In a preferred embodiment, standard transfection techniques are used, e.g., CaPO4 transfection or electroporation, and/or infection by hybrid adenovirus/AAV vectors into cell lines such as the human embryonic kidney cell line HEK293 (a human kidney cell line containing functional adenovirus E1 genes providing trans-acting E1 proteins).
[00110] In some embodiments, a subject genetically modified host cell includes, in addition to a nucleic acid comprising a nucleotide sequence encoding a variant AAV capsid protein, as described above, a nucleic acid that comprises a nucleotide sequence encoding one or more AAV Rep proteins. In other embodiments, a subject host cell further comprises an AAV vector. An AAV virion can be generated using a subject host cell. Methods of generating an AAV virion are described in, e.g., U.S. Patent Publication No. 2005/0053922 and U.S. Patent Publication No. 2009/0202490.
[00111] In addition to an AAV vector, in some cases the host cell contains the sequences driving expression of the AAV capsid polypeptide (including variant AAV capsid polypeptides and non-variant parent capsid polypeptides) in the host cell and Rep sequences of the same serotype as the serotype of the AAV Inverted Terminal Repeats (ITRs) found in the nucleic acid insert (also referred to as a heterologous nucleotide sequence or the “nucleotide sequence of interest”), or a crosscomplementing serotype. The AAV Cap and Rep sequences may be independently obtained from an AAV source and may be introduced into the host cell in any manner known to one of skill in the art or as described herein. Additionally, when pseudotyping an AAV vector in an AAV8 capsid for example, the sequences encoding each of the essential Rep proteins may be supplied by AAV8, or the sequences encoding the Rep proteins may be supplied by different AAV serotypes (e.g., AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, and/or AAV9).
[00112] In some embodiments, the host cell stably contains the capsid protein under the control of a suitable promoter (including, for example, the variant AAV capsid polypeptides of the disclosure), such as those described above. In some embodiments, the capsid protein is expressed under the control of an inducible promoter. In some embodiments, the capsid protein is supplied to the host cell in trans. When delivered to the host cell in trans, the capsid protein may be delivered via a plasmid containing the sequences necessary to direct expression of the selected capsid protein in the host cell. In some embodiments, when delivered to the host cell in trans, the vector encoding the capsid protein (including, for example, the variant AAV capsid polypeptides of the disclosure) also carries other sequences required for packaging the AAV, e.g., the Rep sequences.
[00113] In some embodiments, the host cell stably contains the Rep sequences under the control of a suitable promoter, such as those described above. In some embodiments, the essential Rep proteins are expressed under the control of an inducible promoter. In another embodiment, the Rep proteins are supplied to the host cell in trans. When delivered to the host cell in trans, the Rep proteins may be delivered via a plasmid containing the sequences necessary to direct expression of the selected Rep proteins in the host cell. In some embodiments, when delivered to the host cell in trans, the vector encoding the capsid protein (including, for example, the variant AAV capsid polypeptides of the disclosure) also carries other sequences required for packaging the AAV vector, e.g., the Rep sequences.
[00114] In some embodiments, the Rep and Cap sequences may be transfected into the host cell on a single nucleic acid molecule and exist stably in the cell as an unintegrated episome. In another embodiment, the Rep and Cap sequences are stably integrated into the chromosome of the cell. Another embodiment has the Rep and Cap sequences transiently expressed in the host cell. For example, a useful nucleic acid molecule for such transfection comprises, from 5' to 3', a promoter, an optional spacer interposed between the promoter and the start site of the Rep gene sequence, an AAV Rep gene sequence, and an AAV Cap gene sequence. [00115] Although the molecule(s) providing Rep and capsid can exist in the host cell transiently (i.e., through transfection), in some embodiments, one or both of the Rep and capsid proteins and the promoter(s) controlling their expression be stably expressed in the host cell, e.g., as an episome or by integration into the chromosome of the host cell. The methods employed for constructing embodiments of the disclosure are conventional genetic engineering or recombinant engineering techniques such as those described in the references above.
[00116] In some embodiments, the packaging host cell can require helper functions in order to package the AAV vector of the disclosure into an AAV virion. In some embodiments, these functions may be supplied by a herpesvirus. In some embodiments, the necessary helper functions are each provided from a human or non-human primate adenovirus source, and are available from a variety of sources, including the American Type Culture Collection (ATCC), Manassas, Va. (US). In some embodiments, the host cell is provided with and/or contains an E1a gene product, an E1b gene product, an E2a gene product, and/or an E4 ORF6 gene product. In some embodiments, the host cell may contain other adenoviral genes such as VAI RNA. In some embodiments, no other adenovirus genes or gene functions are present in the host cell.
Methods for Generating an AA V Virion
[00117] In various embodiments, the disclosure provides a method for generating an AAV virion of the disclosure. A variety of methods for generating AAV virions are known in the art and can be used to generate AAV virions comprising the AAV vectors described herein. Generally, the methods involve inserting or transducing an AAV vector of the disclosure into a host cell capable of packaging the AAV vector into an AAV virion. Any convenient method known to one of skill in the art can be employed to generate the AAV virions of the disclosure.
[00118] An AAV vector comprising a heterologous nucleic acid and used to generate an AAV virion can be constructed using methods that are well known in the art. See, e.g., Koerber et al. (2009) Mol. Then, 17:2088; Koerber et al. (2008) Mol Then, 16: 1703- 1709; as well as U.S. Pat. Nos. 7,439,065, 6,951 ,758, and 6,491 ,907. For example, the heterologous sequence(s) can be directly inserted into an AAV genome with the major AAV open reading frames ("ORFs") excised therefrom. Other portions of the AAV genome can also be deleted, so long as a sufficient portion of the ITRs remain to allow for replication and packaging functions. Such constructs can be designed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941 ; International Publication Nos. WO 92/01070 (published Jan. 23, 1992) and WO 93/03769 (published Mar. 4, 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988- 3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Curr. Topics Microbiol. Immunol. 158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801 ; Shelling and Smith (1994) Gene Therapy 1 :165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875.
[00119] In order to produce AAV virions, an AAV vector is introduced into a suitable host cell using known techniques, such as by transfection. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Particularly suitable transfection methods include calcium phosphate co-precipitation (Graham et al. (1973) Virol. 52:456-467), direct micro-injection into cultured cells (Capecchi, M. R. (1980) Cell 22:479-488), electroporation (Shigekawa et al. (1988) BioTechniques 6:742-751 ), liposome-mediated gene transfer (Mannino et al. (1988) BioTechniques 6:682-690), lipid-mediated transduction (Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84:7413- 7417), and nucleic acid delivery using high-velocity microprojectiles (Klein et al. (1987) Nature 327:70-73).
[00120] Suitable host cells for producing AAV virions include any species and/or type of cell that can be, or have been, used as recipients of a heterologous AAV DNA molecule, and can support the expression of required AAV production cofactors from helper viruses. Such host cells can include but are not limited to microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of a heterologous DNA molecule. The term includes the progeny of the original cell transfected. Thus, a "host cell" as used herein generally refers to a cell transfected with an exogenous DNA sequence. Cells from the stable human cell line, HEK293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) can be used. The human cell line HEK293 is a human embryonic kidney cell line that has been transformed with adenovirus type-5 DNA fragments (Graham et al. (1977) J. Gen. Virol. 36:59), and expresses the adenoviral E1 a and E1 b genes (Aiello et al. (1979) Virology 94:460). The HEK293 cell line is readily transfected, and provides a convenient platform in which to produce AAV virions.
[00121] Methods of producing an AAV virion in insect cells are known in the art, and can be used to produce a subject AAV virion. See, e.g., U.S. Patent Publication No. 2009/0203071 ; U.S. Pat. No. 7,271 ,002; and Chen (2008) Mol. Then 16:924.
[00122] In some embodiments, the AAV virion or AAV vector is packaged into an infectious virion or virus particle, by any of the methods described herein or known in the art.
[00123] In some embodiments, the variant AAV capsid polypeptide allows for similar packaging as compared to a non-variant parent capsid polypeptide. In some embodiments, an AAV vector packaged with the variant AAV capsid polypeptides transduce into cells in vivo better than a vector packaged from non-variant parent capsid polypeptides. In some embodiments, the AAV vector packaged with the variant AAV capsid polypeptides transduce into cells in vitro better than a vector packaged from non-variant parent capsid polypeptides. In some embodiments, the variant AAV capsid polypeptides result in nucleic acid expression higher than a nucleic acid packaged from non-variant parent capsid polypeptides. In some embodiments, the AAV vector packaged with said variant AAV capsid polypeptides result in transgene expression better than a transgene packaged from non-variant parent capsid polypeptides.
Pharmaceutical Compositions & Dosing
[00124] The present disclosure provides pharmaceutical compositions useful in treating subjects according to the methods of the disclosure as described herein. Further, the present disclosure provides dosing regimens for administering the described pharmaceutical compositions. The present disclosure provides pharmaceutical compositions comprising: a) a subject AAV vector or AAV virion, as described herein as well as therapeutic molecules packaged by or within capsids comprising variant polypeptides as described herein; and b) a pharmaceutically acceptable carrier, diluent, excipient, or buffer. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in a human.
[00125] Such excipients, carriers, diluents, and buffers include any pharmaceutical agent that can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol and ethanol. Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro, (2000) Remington: The Science and Practice of Pharmacy, 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Ansel et al., eds., 7.sup.th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds., 3.sup.rd ed. Amer. Pharmaceutical Assoc.
[00126] A subject composition can comprise a liquid comprising a subject variant AAV capsid polypeptide of the disclosure or AAV virion comprising a variant AAV capsid polypeptide in solution, in suspension, or both. As used herein, liquid compositions include gels. In some cases, the liquid composition is aqueous. In some embodiments, the composition is an in situ gellable aqueous composition, e.g., an in situ gellable aqueous solution. Aqueous compositions have ophthalmically compatible pH and osmolality.
[00127] Such compositions include solvents (aqueous or non-aqueous), solutions (aqueous or non-aqueous), emulsions (e.g., oil-in-water or water-in-oil), suspensions, syrups, elixirs, dispersion and suspension media, coatings, isotonic and absorption promoting or delaying agents, compatible with pharmaceutical administration or in vivo contact or delivery. Aqueous and non-aqueous solvents, solutions and suspensions may include suspending agents and thickening agents. Such pharmaceutically acceptable carriers include tablets (coated or uncoated), capsules (hard or soft), microbeads, powder, granules and crystals. Supplementary active compounds (e.g., preservatives, antibacterial, antiviral and antifungal agents) can also be incorporated into the compositions.
[00128] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration or delivery, as set forth herein or known to one of skill in the art. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.
[00129] Compositions suitable for parenteral administration comprise aqueous and nonaqueous solutions, suspensions or emulsions of the active compound. Preparations are typically sterile and can be isotonic with the blood of the intended recipient. Non- limiting illustrative examples include water, saline, dextrose, fructose, ethanol, animal, vegetable or synthetic oils.
[00130] For transmucosal or transdermal administration (e.g., topical contact), penetrants can be included in the pharmaceutical composition. Penetrants are known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. For transdermal administration, the active ingredient can be formulated into aerosols, sprays, ointments, salves, gels, or creams as generally known in the art. For contact with skin, pharmaceutical compositions typically include ointments, creams, lotions, pastes, gels, sprays, aerosols, or oils. Useful carriers include Vaseline. RTM., lanolin, polyethylene glycols, alcohols, transdermal enhancers, and combinations thereof.
[00131] Cosolvents and adjuvants may be added to the formulation. Non-limiting examples of cosolvents contain hydroxyl groups or other polar groups, for example, alcohols, such as isopropyl alcohol; glycols, such as propylene glycol, polyethyleneglycol, polypropylene glycol, glycol ether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Adjuvants include, for example, surfactants such as, soya lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.
[00132] Pharmaceutical compositions and delivery systems appropriate for the AAV vector or AAV virion and methods and uses of are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003) 20.sup.th ed., Mack Publishing Co., Easton, Pa.; Remington's Pharmaceutical Sciences (1990) 18.sup.th ed., Mack Publishing Co., Easton, Pa.; The Merck Index (1996) 12.sup.th ed., Merck Publishing Group, Whitehouse, N.J.; Pharmaceutical Principles of Solid Dosage Forms (1993), Technonic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001) 1 1 .sup.th ed., Lippincott Williams & Wilkins, Baltimore, Md.; and Poznansky et al., Drug Delivery Systems (1980), R. L. Juliano, ed., Oxford, N.Y., pp. 253-315).
[00133] Doses can vary and depend upon whether the treatment is prophylactic or therapeutic, the type, onset, progression, severity, frequency, duration, or probability of the disease treatment is directed to, the clinical endpoint desired, previous or simultaneous treatments, the general health, age, gender, race or immunological competency of the subject and other factors that will be appreciated by the skilled artisan. The dose amount, number, frequency or duration may be proportionally increased or reduced, as indicated by any adverse side effects, complications or other risk factors of the treatment or therapy and the status of the subject. The skilled artisan will appreciate the factors that may influence the dosage and timing required to provide an amount sufficient for providing a therapeutic or prophylactic benefit.
[00134] Methods and uses of the disclosure as disclosed herein can be practiced within about 1 hour to about 2 hours, about 2 hours to about 4 hours, about 4 hours to about 12 hours, about 12 hours to about 24 hours or about 24 hours to about 72 hours after a subject has been identified as having the disease targeted for treatment, has one or more symptoms of the disease, or has been screened and is identified as positive as set forth herein even though the subject does not have one or more symptoms of the disease. In some embodiments, the disclosure as disclosed herein can be practiced within about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, or about 72 hours or more. Of course, methods and uses of the disclosure can be practiced about 1 day to about 7 days, about 7 days to about 14 days, about 14 days to about 21 days, about 21 days to about 48 days or more, months or years after a subject has been identified as having the disease targeted for treatment, has one or more symptoms of the disease, or has been screened and is identified as positive as set forth herein. In some embodiments, the disclosure as disclosed herein can be practiced within about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 14 days, about 21 days, about 36 days, or about 48 days or more.
[00135] In some embodiments, the present disclosure provides kits with packaging material and one or more components therein. A kit typically includes a label or packaging insert including a description of the components or instructions for use in vitro, in vivo, or ex vivo, of the components therein. A kit can contain a collection of such components, e.g., a variant AAV capsid polypeptide, an AAV vector, a nucleic acid encoding a variant AAV protein, and/or an AAV virion (in any combination thereof) and optionally a second active ingredient, such as another compound, agent, drug or composition.
[00136] A kit refers to a physical structure housing one or more components of the kit. Packaging material can maintain the components sterilely, and can be made of material commonly used for such purposes (e.g., paper, corrugated fiber, glass, plastic, foil, ampules, vials, tubes, etc.). [00137] Labels or inserts can include identifying information of one or more components therein, dose amounts, clinical pharmacology of the active ingredient(s) including mechanism of action, pharmacokinetics and pharmacodynamics. Labels or inserts can include information identifying the manufacturer, lot numbers, manufacturer location and date, expiration dates. Labels or inserts can include information identifying manufacturer information, lot numbers, manufacturer location and date. Labels or inserts can include information on a disease a kit component may be used for. Labels or inserts can include instructions for the clinician or subject for using one or more of the kit components in a method, use, or treatment protocol or therapeutic regimen. Instructions can include dosage amounts, frequency or duration, and instructions for practicing any of the methods, uses, treatment protocols or prophylactic or therapeutic regimes described herein.
[00138] Labels or inserts can include information on any benefit that a component may provide, such as a prophylactic or therapeutic benefit. Labels or inserts can include information on potential adverse side effects, complications or reactions, such as warnings to the subject or clinician regarding situations where it would not be appropriate to use a particular composition. Adverse side effects or complications could also occur when the subject has, will be or is currently taking one or more other medications that may be incompatible with the composition, or the subject has, will be or is currently undergoing another incompatible treatment protocol or therapeutic regimen and, therefore, instructions could include information regarding such incompatibilities.
[00139] Labels or inserts include "printed matter," e.g., paper or cardboard, or separate or affixed to a component, a kit or packing material (e.g., a box), or attached to an ampule, tube or vial containing a kit component. Labels or inserts can additionally include a computer readable medium, such as a bar-coded printed label, a disk, optical disk such as CD- or DVD-ROIWRAM, DVD, MP3, magnetic tape, or an electrical storage media such as RAM and ROM or hybrids of these such as magnetic/optical storage media, FLASH media or memory type cards.
Method of Treating a Disease
[00140] The present disclosure provides methods for delivering a payload of interest to an individual (e.g., methods of treating a disease in a subject by administering the AAV vectors and/or nucleic acids of the present disclosure), where AAV virus, vectors and/or nucleic acids described herein comprising one or more variant AAV capsid polypeptides of the present disclosure are administered to the individual. In an example embodiment, the disclosure provides a method of administering a pharmaceutical composition of the disclosure to a subject in need thereof to treat a disease of a subject. In various embodiments, the subject is not otherwise in need of administration of a composition of the disclosure.
[00141] In some embodiments, the variant AAV capsid polypeptides package a therapeutic expression cassette comprised of a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product, such as for example a therapeutic protein. In some embodiments, the AAV virion or AAV vector comprises a therapeutic expression cassette comprised of a heterologous nucleic acid comprising a nucleotide sequence encoding a heterologous gene product, such as for example a therapeutic protein.
[00142] In some embodiments, the variant AAV capsid polypeptides of the disclosure are employed as part of vaccine delivery. Vaccine delivery can include delivery of any of the therapeutic proteins as well as nucleic acids described herein. In some embodiments, variant AAV capsid polypeptides of the disclosure are employed as part of a vaccine regimen and dosed according to the methods described herein.
[00143] In some embodiments, the variant AAV capsid polypeptides, the AAV virions, or AAV vectors of the disclosure are used in a therapeutic treatment regimen.
[00144] In some embodiments, the variant AAV capsid polypeptides, the AAV virions, or AAV vectors of the disclosure are used for therapeutic polypeptide production.
[00145] In some cases, a subject variant AAV capsid polypeptides or AAV vector, when introduced into the cells of a subject, provides for high level production of the heterologous gene product packaged by the variant AAV capsid polypeptides or encoded by the AAV vector. For example, a heterologous polypeptide packaged by the variant AAV capsid polypeptides or encoded by the AAV can be produced.
[00146] In some cases, subject variant AAV capsid polypeptides, AAV virion, or AAV vector, when introduced into a subject, provide for production of the heterologous gene product packaged by the variant AAV capsid polypeptides or encoded by the AAV vector in at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 50% at least about 60%, at least about 70%, at least about 80%, or more than 80%, of the target cells. [00147] In some embodiments, the present disclosure provides a method of treating a disease, the method comprising administering to an individual in need thereof an effective amount of a therapeutic molecule packaged by the variant AAV capsid polypeptides or subject AAV vector as described above.
[00148] Subject variant AAV capsid polypeptides or subject AAV vectors can be administered systemically, regionally or locally, or by any route, for example, by injection, infusion, orally (e.g., ingestion or inhalation), or topically (e.g., transdermally). Possible delivery and administration methods can include parenteral, intravenous, intramuscular, intraperitoneal, intradermal, subcutaneous, intracavity, intracranial, transdermal (topical), transmucosal and rectal administration. Example administration and delivery routes include intravenous, intraperitoneal, intrarterial, parenteral, subcutaneous, intra-pleural, topical, dermal, intradermal, transdermal, transmucosal, oral (alimentary), mucosal, respiration, intranasal, intubation, intrapulmonary, intrapulmonary instillation, buccal, sublingual, intravascular, intrathecal, intracavity, iontophoretic, intraocular, ophthalmic, optical, intraglandular, intraorgan, and intralymphatic. In some cases, the delivery route is systemic (e.g., parenteral, intravenous).
[00149] In some cases, a therapeutically effective amount of a therapeutic molecule packaged by the variant AAV capsid polypeptides or a subject AAV vectors is an amount that, when administered to an individual in one or more doses, is effective to slow the progression of the disease or disorder in the individual, or is effective to ameliorate symptoms. For example, a therapeutically effective amount of a therapeutic molecule packaged by the variant AAV capsid polypeptides or a subject AAV vectors can be an amount that, when administered to an individual in one or more doses, is effective to slow the progression of the disease by at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or more than about 80%, compared to the progression of the disease in the absence of treatment with the therapeutic molecule packaged by the variant AAV capsid polypeptides or AAV vectors.
[00150] A therapeutic or beneficial effect of treatment is therefore any objective or subjective measurable or detectable improvement or benefit provided to a particular subject. A therapeutic or beneficial effect can but need not be complete ablation of all or any particular adverse symptom, disorder, illness, or complication of a disease. Thus, a satisfactory clinical endpoint is achieved when there is an incremental improvement or a partial reduction in an adverse symptom, disorder, illness, or complication caused by or associated with a disease, or an inhibition, decrease, reduction, suppression, prevention, limit or control of worsening or progression of one or more adverse symptoms, disorders, illnesses, or complications caused by or associated with the disease, over a short or long duration (hours, days, weeks, months, etc.).
[00151] Improvement of clinical symptoms can also be monitored by one or more methods known to the art, and used as an indication of therapeutic effectiveness. Clinical symptoms may also be monitored by anatomical or physiological means, such as indirect ophthalmoscopy, fundus photography, fluorescein angiopathy, optical coherence tomography, electroretinography (full-field, multifocal, or other), external eye examination, slit lamp biomicroscopy, applanation tonometry, pachymetry, autorefraction, or other measures of functional vision. In some embodiments, a therapeutic molecule (including, for example, nucleic acid that includes a nucleotide sequence of interest) packaged by the variant AAV capsid polypeptides, a subject AAV vector, or AAV virus, when introduced into a subject, provides for production of a heterologous gene product (e.g., non-coding or coding RNA, a protein) for a period of time from about 2 days to about 6 months, e.g., from about 2 days to about 7 days, from about 1 week to about 4 weeks, from about 1 month to about 2 months, or from about 2 months to about 6 months. In some embodiments, therapeutic molecules packaged by the variant AAV capsid polypeptides, a subject AAV vector or virus, when introduced into a subject provides for production of the heterologous gene product for a period of time of more than 6 months, e.g., from about 6 months to 20 years or more, or greater than 1 year, e.g., from about 6 months to about 1 year, from about 1 year to about 2 years, from about 2 years to about 5 years, from about 5 years to about 10 years, from about 10 years to about 15 years, from about 15 years to about 20 years, or more than 20 years.
[00152] Multiple doses of a subject AAV virion can be administered to an individual in need thereof. Where multiple doses are administered over a period of time, an active agent is administered once a month to about once a year, from about once a year to once every 2 years, from about once every 2 years to once every 5 years, or from about once every 5 years to about once every 10 years, over a period of time. For example, a subject AAV virion is administered over a period of from about 3 months to about 2 years, from about 2 years to about 5 years, from about 5 years to about 10 years, from about 10 years to about 20 years, or more than 20 years. The actual frequency of administration, and the actual duration of treatment, depends on various factors. In some embodiments, the administration regimen is part of a vaccination regimen.
[00153] The dose to achieve a therapeutic effect, e.g., the dose in vector genomes/per kilogram of body weight (vg/kg), will vary based on several factors including, but not limited to: route of administration, the level of heterologous polynucleotide expression required to achieve a therapeutic effect, the specific disease treated, any host immune response to the viral vector, a host immune response to the heterologous polynucleotide or expression product (e.g., RNA or protein), and the stability of the expressed molecule. One skilled in the art can readily determine a virion dose range to treat a patient having a particular disease or disorder based on the aforementioned factors, as well as other factors. Generally, doses will range from at least about, or more, for example, 1X109, 1X1 O10, 1 X1011, 1 X1012, 1 X1013,or 1X1014, or more, vector genomes per kilogram (vg/kg) of the weight of the subject, to achieve a therapeutic effect.
[00154] An effective amount or a sufficient amount can, but need not be, provided in a single administration, may require multiple administrations, and, can but need not be, administered alone or in combination with another composition (e.g., agent), treatment, protocol or therapeutic regimen. For example, the amount may be proportionally increased as indicated by the need of the subject, type, status and severity of the disease treated or side effects (if any) of treatment. In addition, an effective amount or a sufficient amount need not be effective or sufficient if given in single or multiple doses without a second composition (e.g., another drug or agent), treatment, protocol or therapeutic regimen, since additional doses, amounts or duration above and beyond such doses, or additional compositions (e.g., drugs or agents), treatments, protocols or therapeutic regimens may be included in order to be considered effective or sufficient in a given subject. Amounts considered effective also include amounts that result in a reduction of the use of another treatment, therapeutic regimen or protocol.
[00155] An effective amount or a sufficient amount need not be effective in each and every subject treated, or a majority of treated subjects in a given group or population. An effective amount or a sufficient amount means effectiveness or sufficiency in a particular subject, not a group or the general population. As is typical for such methods, some subjects will exhibit a greater response, or less or no response to a given treatment method or use. Thus, appropriate amounts will depend upon the condition treated, the therapeutic effect desired, as well as the individual subject (e.g., the bioavailability within the subject, gender, age, etc.).
[00156] With regard to a disease or symptom thereof, or an underlying cellular response, a detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limit or control in the occurrence, frequency, severity, progression, or duration of the disease, or complication caused by or associated with the disease, or an improvement in a symptom or an underlying cause or a consequence of the disease, or a reversal of the disease.
[00157] Thus, a successful treatment outcome can lead to a "therapeutic effect," or "benefit" of decreasing, reducing, inhibiting, suppressing, limiting, controlling or preventing the occurrence, frequency, severity, progression, or duration of a disease, or one or more adverse symptoms or underlying causes or consequences of the disease in a subject. Treatment methods and uses affecting one or more underlying causes of the disease or adverse symptoms are therefore considered to be beneficial. A decrease or reduction in worsening, such as stabilizing the disease, or an adverse symptom thereof, is also a successful treatment outcome.
[00158] A therapeutic benefit or improvement therefore need not be complete ablation of the disease, or any one, most or all adverse symptoms, complications, consequences or underlying causes associated with the disease. Thus, a satisfactory endpoint is achieved when there is an incremental improvement in a subject's disease, or a partial decrease, reduction, inhibition, suppression, limit, control or prevention in the occurrence, frequency, severity, progression, or duration, or inhibition or reversal, of the disease (e.g., stabilizing one or more symptoms or complications), over a short or long duration of time (hours, days, weeks, months, etc.). Effectiveness of a method or use, such as a treatment that provides a potential therapeutic benefit or improvement of a disease, can be ascertained by various methods.
[00159] Disclosed methods and uses can be combined with any compound, agent, drug, treatment or other therapeutic regimen or protocol having a desired therapeutic, beneficial, additive, synergistic or complementary activity or effect. Exemplary combination compositions and treatments include second actives, such as, biologies (proteins), agents and drugs. Such biologies (proteins), agents, drugs, treatments and therapies can be administered or performed prior to, substantially contemporaneously with or following any other method or use of the disclosure.
[00160] The compound, agent, drug, treatment or other therapeutic regimen or protocol can be administered as a combination composition, or administered separately, such as concurrently or in series or sequentially (prior to or following) delivery or administration of an AAV vector or AAV virion as described herein. The disclosure therefore provides combinations where a method or use of the disclosure is in a combination with any compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition, set forth herein or known to one of skill in the art. The compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition can be administered or performed prior to, substantially contemporaneously with or following administration of an AAV vector or AAV virion as described herein, to a subject. Specific non-limiting examples of combination embodiments therefore include the foregoing or other compound, agent, drug, therapeutic regimen, treatment protocol, process, remedy or composition.
[00161] Methods and uses of the disclosure also include, among other things, methods and uses that result in a reduced need or use of another compound, agent, drug, therapeutic regimen, treatment protocol, process, or remedy. For example, for a blood clotting disease, a method or use of the disclosure has a therapeutic benefit if in a given subject a less frequent or reduced dose or elimination of administration of a recombinant clotting factor protein to supplement for the deficient or defective (abnormal or mutant) endogenous clotting factor in the subject. Thus, in accordance with the disclosure, methods and uses of reducing need or use of another treatment or therapy are provided.
[00162] The disclosure is useful in animals including veterinary medical applications. Suitable subjects therefore include mammals, such as humans, as well as non-human mammals such as non-human primates. The term "subject" refers to an animal, typically a mammal, such as humans, non-human primates (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), a domestic animal (dogs and cats), a farm animal (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and experimental animals (mouse, rat, rabbit, guinea pig). Human subjects include fetal, neonatal, infant, juvenile and adult subjects. Subjects include animal disease models, for example, mouse and other animal models of blood clotting diseases and others known to those of skill in the art.
[00163] In some embodiments, a method or use of the disclosure includes: (a) providing an AAV virion whose capsid comprises a variant AAV capsid polypeptide (e.g., prepared as described herein), wherein the AAV virion comprises a heterologous nucleic acid sequence (e.g., in some cases operably linked to an expression control element conferring transcription of said nucleic acid sequence); and (b) administering an amount of the AAV virion to the mammal such that said heterologous nucleic acid is expressed in the mammal.
[00164] In some embodiments, a method or use of the disclosure includes: (a) providing a therapeutic molecule packaged by variant AAV capsid polypeptides (e.g., prepared as described herein), wherein the therapeutic molecule comprises a heterologous nucleic acid sequence (e.g., which can in some cases be operably linked to an expression control element conferring transcription of said nucleic acid sequence); and (b) administering an amount of the therapeutic molecule (including, for example, a vaccine) packaged by variant AAV capsid polypeptides to the mammal such that said heterologous nucleic acid is expressed in the mammal.
[00165] In some embodiments, a method or use of the disclosure includes delivering or transferring a heterologous polynucleotide sequence into a mammal or a cell of a mammal, by administering a heterologous polynucleotide packaged by the variant AAV capsid polypeptides, a plurality of heterologous polynucleotides packaged by variant AAV capsid polypeptides, an AAV virion prepared as described herein, or a plurality of AAV virions comprising the heterologous nucleic acid sequence to a mammal or a cell of a mammal, thereby delivering or transferring the heterologous polynucleotide sequence into the mammal or cell of the mammal. In some embodiments, the heterologous nucleic acid sequence encodes a protein expressed in the mammal, or where the heterologous nucleic acid sequence encodes an inhibitory sequence or protein that reduces expression of an endogenous protein in the mammal.
[00166] In some embodiments, a method or use of the disclosure includes is a method of delivering a payload of interest to the central nervous system of an individual, and includes administering to the individual a nucleic acid or a recombinant AAV (rAAV) particle as described herein (e.g., where the nucleic acid is a viral vector that encodes a variant AAV capsid protein and includes a nucleotides sequence of interest, where the rAAV particle comprises a variant AAV particle and a payload nucleic acid that includes a nucleotides sequence of interest).
[00167] Provided are methods of delivering a payload of interest to a target cell. Such a method can include contacting the target cell with a subject recombinant AAV particle. In some cases, the target cell is in vivo and said contacting comprises administering the recombinant AAV particle to an individual. [00168] Reagents, compositions, and kits/systems that find use in practicing the subject methods are provided.
Example AAV capsid Sequences
[00169] A triple asterisks (***) denotes a TAG stop codon (DNA nucleotides) inserted onto the AAV capsid sequence in order to replace the amino acid at that position with an unnatural amino acid. The replacement is at the position reported in the name of the sequence (e.g., in “AAV8-T457” a stop TAG codon replaces the Threonine at amino acid position 457 in the AAV8 amino acid sequence - such that an unnatural amino acid (used for conjugation as described elsewhere herein) was instead placed at position 457).
DJ
MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDK GEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAK KRLLEPLGLVEEAAKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVP DPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITT STRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLIN NNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPP FPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAH SQSLDRLMNPLIDQYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQR VSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSE KTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRD VYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYS TGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRN (SEQ ID NO: 1 )
DJR/A (R587A and R590A)
MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDK GEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAK KRLLEPLGLVEEAAKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVP DPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITT STRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLIN NNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPP FPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAH SQSLDRLMNPLIDQYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQR VSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSE KTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQ[A]GN[A]QAATADVNTQGVLPGMVWQD RDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQ YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 2)
DJR/A-T456
MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDK GEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAK KRLLEPLGLVEEAAKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVP DPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITT STRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLIN
NNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPP FPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAH SQSLDRLMNPLIDQYLYYLSRTQTTGG***TNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQ
RVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGS
EKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQ[A]GN[A]QAATADVNTQGVLPGMVWQ DRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFIT QYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTR
NL
(SEQ ID NO: 3)
DJR/A-D555
MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDK
GEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAK KRLLEPLGLVEEAAKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVP DPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITT
STRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLIN NNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPP FPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAH
SQSLDRLMNPLIDQYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQR VSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSE KTNV***IEKVMITDEEEIRTTNPVATEQYGSVSTNLQ[A]GN[A]QAATADVNTQGVLPGMVWQ
DRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFIT QYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTR NL
(SEQ ID NO: 4)
DJR/A-N589
MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDK
GEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAK KRLLEPLGLVEEAAKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVP DPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITT
STRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLIN NNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPP FPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAH
SQSLDRLMNPLIDQYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQR VSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSE KTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQ[A]G***[A]QAATADVNTQGVLPGMVWQ
DRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFIT QYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTR NL
(SEQ ID NO: 5)
DJR/A-A587
MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDK
GEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAK KRLLEPLGLVEEAAKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVP DPQPIGEPPAAPSGVGSLTMAAGGGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITT STRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLIN NNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPP FPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAH
SQSLDRLMNPLIDQYLYYLSRTQTTGGTTNTQTLGFSQGGPNTMANQAKNWLPGPCYRQQR VSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSE KTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQ***GN[A]QAATADVNTQGVLPGMVWQD
RDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQ YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 6)
AAV8
MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSE
SVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV ITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCL
PPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSY AHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQ QRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQNA
ARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNR DVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQY STGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL
(SEQ ID NO: 7)
AAV8-E330
MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSE
SVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV ITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLSFKLFNIQVKEVTQN***GTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGC
LPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSS YAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYR QQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQN
AARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQN RDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQ YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL
(SEQ ID NO: 8)
AAV8-N499
MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSE
SVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV ITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCL
PPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSY AHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQ QRVSTTTGQN***NSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQN AARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQN RDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQ YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 9)
AAV8-N590
MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSE SVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV ITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCL PPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSY AHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQ QRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQNA ARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQ***TAPQIGTVNSQGALPGMVWQN RDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQ YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 10)
AAV8-N590+1
MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSE SVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV ITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCL PPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSY AHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQ QRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQNA ARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQN***TAPQIGTVNSQGALPGMVWQ
NRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFIT QYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTR NL
(SEQ ID NO: 11 )
AAV8-T457
MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLD KGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQ AKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSE SVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRV ITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQR LINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCL PPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSY AHSQSLDRLMNPLIDQYLYYLSRTQTTGG***ANTQTLGFSQGGPNTMANQAKNWLPGPCYR QQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQN AARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQN RDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQ YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 12) LK03
MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQA KKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESV
PDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVIT TSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINN NWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPF
PADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAH SQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQ RLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGT
TASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDR DVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQY STGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL
(SEQ ID N0: 13)
LK03-N588
MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLD
KGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQA KKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESV PDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVIT
TSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINN NWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPF PADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAH
SQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQ RLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGT TASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSS***TAPTTRTVNDQGALPGMVWQD
RDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQ YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRP L
(SEQ ID N0: 14)
LK03-T455
MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQA KKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESV
PDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVIT TSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINN NWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPF
PADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAH SQSLDRLMNPLIDQYLYYLNRTQGTTSG***TNQSRLLFSQAGPQSMSLQARNWLPGPCYRQ QRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEG
TTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQD RDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQ YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRP
L
(SEQ ID N0: 15) LK03-T456
MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLD KGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQA KKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESV PDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVIT TSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINN NWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPF PADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAH SQSLDRLMNPLIDQYLYYLNRTQGTTSGT***NQSRLLFSQAGPQSMSLQARNWLPGPCYRQ QRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEG TTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQD RDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQ YSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRP L
(SEQ ID NO: 16)
Exemplary Non-Limiting Aspects of the Disclosure
[00170] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of ordinary skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below. It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention.
1 . A variant adeno-associated virus (AAV) capsid protein comprising: (a) an azide-bearing unnatural amino acid substitution at a position corresponding to:
T456, D555, R587/A587, or N589 of the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2; or
E330, T457, N499, or N590 of the AAV8 amino acid sequence set forth as SEQ ID NO: 7; or
T455, T456, or N588 of the AAV- LK03 amino acid sequence set forth as SEQ ID NO: 13; or (b) an azide-bearing unnatural amino acid insertion between positions corresponding to N590 and T591 of the AAV8 amino acid sequence set forth as SEQ ID NO: 7.
2. The variant AAV capsid protein of 1 , comprising an amino acid sequence having:
(i) 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, wherein said azide- bearing unnatural amino acid substitution is at position N589 or R587/A587;
(ii) 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV8 amino acid sequence set forth as SEQ ID NO: 7, wherein said azide-bearing unnatural amino acid substitution is at position T457; or
(iii) 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV- LK03 amino acid sequence set forth as SEQ ID NO: 13, wherein said azide-bearing unnatural amino acid substitution is at position N588.
3. The variant AAV capsid protein of 1 , comprising an amino acid sequence having 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, wherein said azide-bearing unnatural amino acid substitution is at position N589 or R587/A587.
4. The variant AAV capsid protein of 1 , comprising an amino acid sequence having 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 , 2, 7, and 13.
5. The variant AAV capsid protein of 1 , comprising an amino acid sequence having 90% or more (e.g., 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 , 2, 7, and 13.
6. The variant AAV capsid protein of 1 , comprising an amino acid sequence having 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16. . The variant AAV capsid protein of 1 , comprising an amino acid sequence having 90% or more (e.g., 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16. . The variant AAV capsid protein of 1 , comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16. . The variant AAV capsid protein of 1 , comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5-6, 12, or 14. 0. The variant AAV capsid protein of any one of 1 -9, wherein the unnatural amino acid is an azido-lysine. 1 . The variant AAV capsid protein of any one of 1 -7 and 10, comprising at least one arginine to alanine mutation at a heparan sulfate proteoglycan (HSPG) binding site. 2. The variant AAV capsid protein of 11 , wherein the HSPG binding site is at an amino acid position corresponding to position 587 and/or 590 of SEQ ID NO: 1. 3. The variant AAV capsid protein of any one of 1 -12, wherein the variant AAV capsid protein is conjugated to folic acid via the azide-bearing unnatural amino acid. 4. The variant AAV capsid protein of any one of 1 -12, wherein the variant AAV capsid protein is conjugated to a DNA or RNA aptamer via the azide-bearing unnatural amino acid. 5. The variant AAV capsid protein of 14, wherein the variant AAV capsid protein is conjugated to the DNA aptamer AS1411 . 6. The variant AAV capsid protein of 14, wherein the variant AAV capsid protein is conjugated to the RNA aptamer E3. 7. The variant AAV capsid protein of any one of 13-16, where the variant AAV capsid protein is conjugated via a linker. 8. The variant AAV capsid protein of 17, wherein the linker is a Polyethylene glycol (PEG) linker. 9. The variant AAV capsid protein of 18, wherein the PEG linker length is less than 5 kDa. 0. The variant AAV capsid protein of 18, wherein the PEG linker length is 4 kDa or less (e.g., 3 kDa or less, 2 kDa or less). 21 . The variant AAV capsid protein of 18, wherein the PEG linker length is about 2 kDa.
22. A chemically modified variant adeno-associated virus (AAV) capsid protein comprising an azide-bearing unnatural amino acid substitution, wherein the capsid protein is conjugated to folic acid, a DNA aptamer, or an RNA aptamer via the azide-bearing unnatural amino acid.
23. A nucleic acid comprising a nucleotide sequence encoding the variant AAV capsid protein of any one of 1 -12, wherein said nucleotide sequence comprises a TAG stop codon at the position encoding said unnatural amino acid substitution.
24. A recombinant AAV particle comprising:
(a) the variant AAV capsid protein of any one of 1 -22; and
(b) a nucleic acid payload of interest.
25. The recombinant AAV particle of 24, wherein the nucleic acid payload of interest: encodes a polypeptide (e.g., a genome-editing enzyme such as a CRISPR/Cas effector protein, a zinc finger nuclease, or a TALEN), or is a non-coding RNA (e.g., a CRISPR/Cas guide RNA, an shRNA, an siRNA, a miRNA, an aptamer) or encodes said non-coding RNA, or is an mRNA.
26. The recombinant AAV particle of 25, wherein said polypeptide is encoded by a nucleotide sequence that is operably linked to the small ubiquitous INS84 promoter.
27. A method of delivering a payload of interest to a target cell, the method comprising contacting the target cell with the recombinant AAV particle of any one of 25-26.
28. The method of 27, wherein the target cell is in vivo and said contacting comprises administering the recombinant AAV particle to an individual.
EXPERIMENTAL EXAMPLES
[00171] The following examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. [00172] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.
[00173] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for methods referred to in, or related to, this disclosure are available from commercial vendors such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and the like, as well as repositories such as e.g., Addgene, Inc., American Type Culture Collection (ATCC), and the like
Examples below: Aptamer-programmable adeno-associated viral vectors as a platform for cell-specific gene transfer
[00174] Combining synthetic, molecular, and chemical biology, a novel class of programmable AAV vectors, Ne-AAV, was created by utilizing single unnatural amino acid insertion. The different mutant capsids of NE-AAV vectors were characterized and the NE-AAV vectors were successfully conjugated by click chemistry. As illustrative examples, NE- AAVs were successfully programmed to target specific cancer cells via folic acid, or DNA aptamer, or RNA aptamer conjugation. In vivo studies in xenograft animal models confirmed that the folic acid conjugated AAV vectors and the DNA aptamer AS141 1 conjugated AAV vectors led to highly specific uptake in the intended target cells. Example 1 : Production of chemically modified AAV and click chemistry conjugation [00175] The AAV-DJ capsid was first chemically modified. AAV-DJ was previously isolated because of its efficiency in transducing many cells in vitro as well as its high titer production.
[00176] Similar to what has been reported for AAV2 and AAVDJ/8, the heparan sulfate proteoglycan (HSPG) binding sites were ablated by substituting two alanines for two arginines at the 587 and 590 positions (hereafter referred as AAV-DJR/A (see SEQ ID NO: 2) in order to reduce the infectivity of AAV-DJ (see SEQ ID NO: 1 ) in vitro (Figure 7A). Next, a rational design based on the crystal structure of AAV-DJ was employed (see htt followed by ps: followed by //ww followed by w.rc followed by sb. followed by org) to introduce an amber stop codon (TAG) in different regions of the AAV-DJR/A capsid sequence (DJR/A-N589 (see SEQ ID NO: 5), DJR/A-D555 (see SEQ ID NO: 4), DJR/A-A587 (see SEQ ID NO: 6), and DJR/A-T456 (see SEQ ID NO: 3)) as sites to replace the endogenous amino acid with the unnatural amino acid.
[00177] Consequently, the newly made capsids were used for AAV production using a CAG promoter-firefly luciferase expression cassette for in vitro experiments. The pAcBad ,tR4-MbPyl plasmid, which expresses a pyrrolysyl-tRNA and a pyrrolysyl- tRNA synthase, both derived from the Methanosarcina barkeri, was added to the classical triple transfection protocol for AAV production. Finally, the unnatural amino acid, azido-lysine, was supplemented into the cell media during the vector preparation (Figure 1A).
[00178] In the end, this created an AAV-producing system where the expression of the prokaryotic tRNA/t-RNA synthase forced the mammalian translational system to recognize the UAG stop codon as a regular codon and incorporate the azido-lysine on the AAV capsid during the production of the chemically modified vector. This novel AAV, referred to herein as Ne-AAV, carrying a single azido-amino acid insertion on a specific capsid position allowed the conjugation of any molecule containing a dibenzocyclooctyne chemical group (DBCO) to the capsid by simply performing a click chemistry reaction (Figure 1 A). However, before the vector conjugation, transduction of the newly produced Ne-DJR/A mutants was assessed in HeLa cells. The Ne-DJR/A- N589 and Ne-DJR/A-A587 vectors resulted in slightly higher luciferase activity compared to the Ne-DJR/A-D555 and Ne-DJR/A-T456 vectors (Figure 7B).
[00179] Next, using a copper-free cycloaddition reaction, Ne-DJR/A-N589 vector was conjugated with a DBCO-biotin to detect the biotin-AAV by western blot using a streptavidin-labeled antibody. As shown in Figure 1 B, both the unconjugated and conjugated NE-AAV vectors were detected by the anti-AAV antibody AAVB1 (red signal), while the streptavidin antibody (green signal) recognized only the biotin- conjugated Ne-AAV. Notably, the streptavidin signal overlapped with the AAVB1 antibody (yellow signal) demonstrating the specific nature of the biotin conjugation on the AAV capsid proteins VP1/2/3 (Figure 1 B).
[00180] These data confirm that the azido-lysine was successfully incorporated on the AAV capsid and that the DBCO-biotin was specifically conjugated to the Ne-AAV.
Example 2: Design and characterization of Ns-AAVs upon folic acid conjugation [00181] Since the Ne-AAV capsid incorporated the azido-lysine on each of the VP1 , VP2, and VP3 proteins (Figure 2A), an attempt was also made to specifically target the insertion of the unnatural amino acid onto only the VP2 (Figure 2B) or VP3 proteins (Figure 2C).
[00182] To insert the azido-lysine on VP2, a strategy was used described for other AAV vectors by making a single nucleotide mutation at the DJR/A sequence to circumvent VP2 expression (Ns-VP2-AAV, Figure 2B). Next, another rep/cap DJR/A plasmid was generated to eliminate VP3 expression, and an additional plasmid that only expressed VP3 (NE-VP3-AAV, Figure 2C).
[00183] Western blot analyses show that the NE-VP2-AAV and NE-VP3-AAV can be complemented with the plasmids expressing either VP2 (Figure 7C) or VP3 (Figure 7D). To functionalize the new chemically modified AAV vectors, a click chemistry reaction was performed using a DBCO-folic acid molecule with a 2KDa PEG linker (DBCO-FA) (Figure 2D). As a negative control unconjugated NE-AAV vectors were used. Western blot analysis showed that the DBCO-FA conjugation resulted in a shift in the specific VP capsid proteins bands where the folic acid conjugation took place as highlighted by the red asterisks in Figure 2E. This was further confirmed and shown in a silver-stained gel of folic acid-conjugated vector (Figure 7E). These data demonstrate that the unnatural amino acid insertion and consequent folic acid conjugation occurred either on all the three VP1/2/3 proteins or specifically on VP2 and VP3 depending on the plasmid combination used for the vector production.
[00184] Based on these results, NE-AAVS vectors were produced expressing luciferase and the folic acid-conjugated AAV vectors were tested on cancer cell lines that are reported to express high levels of human folic acid receptor (hFOLRI ) (see htt followed by ps:/ followed by /ww followed by w.p followed by roteinatlas followed by .org/ followed by ENSG00000110195-FOLR1].; Figure 7F). In HeLa cells, the folic acid-conjugated Ne-AAV and Ne-VP3-AAV (DBCO-FA) showed significant higher luciferase activity compared to the unconjugated Ne-AAV control vectors (mock) (Figure 2F). Notably, the FA-conjugated Ne-AAV displayed remarkably enhanced transduction efficiency compared to both the FA-conjugated Ne-VP2-AAV and Ne- VP3-AAV (Figure 2F). Moreover, the Ne-VP2-AAV and Ne-VP3-AAV vectors showed significant decreased infectivity even when transduced at higher multiplicity of infection (MOI) (Figure 7G). Because the Ne-VP2-AAV and Ne-VP3-AAV displayed an overall reduced ability to transduce cells, the Ne-AAV vectors were used in consequent studies (Figure 2A).
Example 3: Rational muta enesis screen to identify novel Ne-AAV mutant capsids [00185] After the successful conjugation of the AAV-DJR/A, the unnatural amino acid was introduced on AAV8 and AAVLK03 capsids. Thus, using the same rational design used for the Ne-AAVDJR/A mutants, new vectors were generated with the azidolysine displayed on the capsid surface. Eight new Ne-AAV variants were produced (five AAV8 and three LK03 mutants, where the capital letter and the number next to the AAV serotype indicate the position and amino acid replaced by the azido-lysine on the vector capsid). Notably, all the new Ne-AAV vectors showed a lower titer compared to their wild-type counterpart (Figure 8A).
[00186] To test the infectivity of the new Ne-AAV mutants, luciferase-expressing vectors were made using either regular media, where no vector is expected to be produced, or supplemented with the azido-lysine and their infectivity on HeLa cells was assessed. For a quick assessment of transduction, crude AAV lysates were used. Among the new vectors only Ne-AAV8-T457 and Ne-LK03-N588 retained the ability to transduce cells when produced in presence of azido-lysine, while Ne-DJR/A-N589 was used as control (Figure 3A). These three vectors were conjugated with the DBCO-PEG (2K)- FA and tested on three different cancer cell lines, HeLa, MCF-7, and A549. The FA- DJR/A-N589 and FA-AAV8-T457 showed a significantly higher transduction efficiency compared to the unconjugated Ne-AAVs controls (Figure 3B, 3C and Figure 8B, 8C). Conversely, the FA-LK03-N588 failed to transduce HeLa, MCF-7 and A549 cells, possibly because the conjugation may have disrupted the vector infectivity (Figure 3D and Figure 8D). Thus, it is possible that the AAV modification through unnatural amino acid incorporation depends on the capsid region where the unnatural amino acid is introduced and that other regions might not be permissive to this modification.
[00187] Introduction of the unnatural amino acid and the folic acid conjugation did not show any structural differences based on transmission electron microscopy in the Ne- DJR/A-N589 and FA- DJR/A-N589 compared to the wild-type AAV-DJR/A vector (Figure 3E).
[00188] To evaluate whether the length of the PEG linker would affect the infectivity of the FA- AAVs vectors, Ne-DJR/A-N589 and Ne-AAV8-T457 were conjugated with DBCO-FA containing different lengths of PEG (2kDa, 5kDa and 10kDa). In MCF-7 cells, both the Ne-DJR/A-N589 and Ne-AAV8-T457 conjugated with the DBCO-FA-2k demonstrated a significant increase in luciferase activity compared to the vectors with longer PEG linkers, DBCO-FA-5k and DBCO-FA-10k and the unconjugated NE-AAV. Remarkably, the FA-AAVs also showed an increased infectivity compared to their wild-type counterpart, DJR/A and AAV8, respectively (Figure 8E, 8F). Interestingly, an inverse correlation was observed between the length of the PEG linker and FA-AAV infectivity regardless of the NE-AAV capsid utilized, where the increasing length of the linkers significantly reduced the AAV transduction (Figure 3F). These data demonstrate that an optimal distance between the AAV capsid and the conjugated molecule (the folic acid in this case) can be found to optimally maintain the infectivity of the NE-AAV vectors.
[00189] To rule out the possibility that the PEG, perse, might influence the AAV infectivity, NE- DJR/A-N589 was conjugated with a DBCO-PEG moiety devoid of folic acid. Of note, the DBCO-PEG-conjugated AAV showed a significant reduction in transduction compared to both the unconjugated NE-DJR/A-N589 and DJR/A AAV vectors (Figure 8G). Finally, it was tested whether the efficiency of DBCO-FA conjugation onto AAV could be further improved by increasing its concentration during the click rection step. To this end, incubating the NE-DJR/A-N589 capsid with 2mM vs 0.5mM DBCO-FA increased the incorporation of FA into VP3 capsid protein (Figure 8H). Nevertheless, as previously reported (Lee et al. 2005), the increase in the ratio between PEGylated molecules and AAV vector resulted in a loss of specific transduction efficiency as observed for FA-DJR/A-N589 (Figure 81). Example 4: FA-AAV specific uptake in vitro
[00190] To further characterize the FA- AAV vectors, an in vitro assay was set up where the cells were co-cultured with an excess of free folic acid, which should compete with the FA-AAVs for the hFOLRI on the cell surface, and eventually reduce the FA-AAV infectivity (Figure 4A). Indeed, the FA-DJR/A-N589 showed a dramatic 8-fold reduction in luciferase activity when HeLa cells were co-cultured with an excess of folic acid compared to cells without folic acid. Conversely, the unconjugated Ne- DJR/A-N589 and the DJR/A, used as controls, displayed a similar level of transduction regardless of whether the folic acid was added onto cells (Figure 4B). Furthermore, similar results were obtained with two different Ne-AAV vectors, FA- AAV8-T457 and FA-DJR/A-A587, (Figure 4C, D). Notably, when the same assay was performed on MCF-7 using the FA-DJR/A-N589 vector, the luciferase activity was massively reduced by ~30-fold while the unconjugated Ne-DJR/A-N589 showed similar transduction levels with or without folic acid incubation (Figure 4E).
[00191] To further verify the specificity of the folic acid-receptor mediated uptake, the cells were treated with an antibody directed against the hFOLRI (hFOLRI -Ab) to block its activity and in turn FA-AAV transduction (Figure 4F). As shown in figure 4G, the FA- DJR/A-N589 displayed a significant 2-fold decrease in luciferase levels when HeLa cells were pre-incubated with the hFOLRI -Ab compared to cells that were not treated with the antibody. In contrast, the unconjugated Ne-DJR/A-N589 and the DJR/A showed similar levels of transduction regardless of whether the cells were treated with the hFOLRI -Ab. Similar results were obtained using the FA-AAV8-T457 showing again that the FA-AAV transduction was folic acid-dependent (Figure 4H). Of note, incubation with hFOLRI -Ab significantly decreased transduction by 3-fold in MCF-7 cells (Figure 41).
[00192] Overall, the use of two independent assays and different Ne-AAVs, corroborate that the uptake of the FA-AAV vectors was remarkably receptor- and FA-specific and independent of the AAV serotype.
Example 5: In vitro characterization of aptamer-conjugated NE-AA VS
[00193] In contrast to folic acid peptide-derived molecules, the Ne-AAV vectors were next conjugated with nucleic acid ligands such as aptamers since they have shown binding properties comparable to antibodies. As proof of concept, it was decided to evaluate one of the most characterized DNA aptamers utilized in cancer research, AS1411 . To conjugate the AS1411 aptamer to the Nc-AAV, a conjugable DBCO-AS1411 molecule was first made. To this end, a DBCO-PEG-NHS was combined with an amine (NH2) modified AS1411 aptamer to create DBCO-PEG-AS1411 (Figure 5A).
[00194] Subsequentially, the DBCO-PEG-AS1411 was conjugated to the Ne-AAV vectors. To assess the AS1411 -AAV conjugation a biotinylated oligonucleotide antidote was designed that would specifically bind to the AS141 1 aptamer. The AS1411-AAV would be detected by western blot using a streptavidin-conjugated antibody. Indeed, western blot analysis showed that only the AS1411 -AAV was detected by the streptavidin antibody (green signal) upon incubation with the biotinylated probe (Figure 5B). Notably, the overlapping signal (yellow) of streptavidin (green) and AAVB1 (red) antibodies showed that the aptamer conjugation was specific to the VP2 capsid proteins on the AS1411 -AAV (Figure 5B). The reason of this apparent VP2 specific conjugation is still unknown. However, the possibility cannot yet be ruled out that the conjugated aptamer was not fully recognized by the biotinylated oligo probe and that the denaturing conditions of the SDS-page could have masked or changed the aptamer availability on VP1 and VP3.
[00195] Moreover, as previously demonstrated with the FA-AAVs, no significant structural changes in the Ne-AAV capsid were observed upon AS1411 aptamer conjugation (Figure 9A). Next, the Ne-DJR/A-N589 vector expressing luciferase was conjugated to the DBCO-PEG-AS1411 aptamer and the MCF-7 breast cancer cell line was transduced. The cells were infected with different amounts of AS1411 -DJR/A-N589 and the unconjugated Ne-DJR/A-N589 was used as a control. Notably, the AS1411 - DJR/A-N589 exhibited a remarkably enhanced infectivity compared to the unconjugated Ne-DJR/A-N589 at all tested multiplicity of infections (MOI) (Figure 5C).
[00196] For specific uptake assessment, an antidote oligonucleotide against the AS1411 aptamer was synthesized and an in vitro assay was set up to demonstrate the specificity of the AS1411 -AAV transduction (Bompiani et al., Curr. Pharm. Biotechnol. 13, 1924-1934 (2012)). By treating the cells with different concentrations of the antidote, it was predicted that transduction of the AS141 1 -AAV would be inhibited along with the luciferase activity (Figure 5D). Three different cancer cell lines were used (MCF-7, A549, and HeLa), which have been extensively employed for AS1411 aptamer characterization. Notably, the luciferase activity from the AS1411 -DJR/A- N589 was massively reduced at the dose of 10|xM of antidote in all three cell lines, while the unconjugated NE-DJR/A-N589 did not show any inhibition (Figure 5E-G). In addition, as little as 1 gM of antidote inhibited AS141 1 -DJR/A-N589 transduction in MCF-7 (Figure 5E) and HeLa cells (Figure 5G). Remarkably, in the absence of antidote, the AS1411 -AAV resulted in a significant 9- ,10-, and 5-fold increase in transduction compared to the unconjugated vector in MCF-7, A549 and Hela cells, respectively (Figure 5E-G). Furthermore, the antidote significantly prevented the transduction of the AS1411 -DJR/A-A587 vector in both HeLa and MCF-7 cells (Figure 9B, C). Moreover, to further demonstrate the specific nature of the AS1411 - AAV uptake, the NE-AAV was conjugated with a different aptamer (Zhao et al., Biomaterials 67, 42-51 (2015)), referred to herein as CD-AAV, originally isolated for uptake in AML cells (Figure 9D). Indeed, Hela cells infected with CD-AAV and in presence of AS141 1 antidote did not show any inhibition of transduction similarly to the unconjugated NE-AAV vector. In contrast, the AS1411 -AAV showed a dose dependent decrease in luciferase activity (Figure 5H). AS141 1 binding to cells has been shown to be blocked in the presence of salmon sperm DNA (ssDNA). To this end, both MCF-7 and A549 cells were treated with 1 mg/mL of ssDNA and transduced with either the AS1411 -DJR/A-N589 or its unconjugated counterpart, NE-DJR/A-N589. The AS1411 -DJR/A-N589 showed a massive ~20-fold decrease infectivity in cells treated with the ssDNA, while the unconjugated NE-DJR/A-N589 displayed similar transduction levels in cells regardless of the ssDNA (Figure 9E, F). Taken together, transduction by AS141 1-AAV is strictly dependent on the AS141 1 aptamer regardless of the NE-AAV capsid used similarly to FA receptor-dependent FA-AAV transduction.
[00197] To further demonstrate the versatility of this system beyond the use of DNA aptamers the AAV was conjugated with the E3 RNA aptamer (Gray et al., Proc. Natl. Acad. Sci. U. S. A. 115, 4761-4766 (2018)). This aptamer has been reported to specifically target various tumor cells and was successfully conjugated with anti-cancer drugs to treat prostate cancer using a xenograft mouse model. To this end, upon vector conjugation, the western blot membrane was probed with a biotinylated E3 oligonucleotide antidote. Similar to the DNA aptamer conjugation, this showed that the E3 aptamer was specifically conjugated to the AAV. In contrast, both the unconjugated and the C36, non-specific control RNA aptamer-conjugated vectors, did not show any specific signal when incubated with the E3 specific probe (Figure 5I). In vitro assessment of E3-AAV showed a significantly enhanced transduction in liver hepatoma cells compared to the unconjugated Ne-AAV or the C36-AAV vectors (Figure 9G). To quickly evaluate the E3 specific uptake in vitro, the cells were incubated with the clathrin inhibitor dynasore, known to inhibit E3 uptake (Figure 5L). The E3-AAV uptake was significantly inhibited by treating Huh7 (Figure 5M) and MCF-7 (Figure 5N) cells with dynasore, whereas the C36-AAV control vector was not inhibited. Interestingly, the addition of salmon sperm DNA (ssDNA) onto cells to block non-specific charged-based aptamer binding inhibited uptake of the AS1411 -AAV (Figure 9E, 9F) but not of the E3-AAV vector.
Example 6: Highly specific targeting of FA-AA V and AS1411 -AA V vectors in vivo [00198] To evaluate the Ne-AAV vectors in vivo, their yield was first improved. Previously, researchers had found that the GAG promoter element in the AAV cassette might negatively impact AAV production due to vector genome truncation. The large GAG promoter was thus replaced with a newly characterized small ubiquitous promoter, INS84. The new Ne-AAV expressing firefly luciferase under the small INS84 promoter resulted in significantly higher vector titers compared to the AAV cassette carrying the GAG promoter (Figure 10A).
[00199] Moreover, the Ne-DJR/A-A587 (Ne-AAV hereafter) containing the INS84-Fluc cassette was conjugated with either folic acid (FA-AA V), or with the AS1411 aptamer (AS1411- AAV) and was tested in HeLa cells. The FA-AAV and AS141 1-AAVs resulted in significantly higher luciferase activity compared to their unconjugated counterpart but with similar activity compared to the Ne-AAV containing the CAG promoter (Figure 10B, C).
[00200] To assess these new Ne-AAV vectors in vivo, HeLa cells were subcutaneously transplanted into immunodeficient nude mice and the AAV vectors were subcutaneously injected once the HeLa cells had formed palpable tumors. The luciferase expression of the unconjugated Ne-AAV, FA-AAV and AS1411 -AAV vectors was assessed three, seven and fourteen days after AAV treatment by in vivo imaging (Figure 6A). All three vectors, Ne-AAV, FA-AAV and AS1411 -AAV, were able to infect the tumor cells, and by day fourteen reached maximal expression, albeit expression was detectable at earlier timepoints (Figure 6B; Figure 10D, E). Ne-AAV and FA-AAV displayed a slightly higher luciferase signal in tumor cells compared to AS141 1 -AAV (Figure 6C). However, a major difference was the off-target expression in the liver with Ne-AAV exhibiting the most luciferase activity, followed by the FA-AAV, and finally the AS1411 -AAV which did not show any activity in the liver (Figure 6D, E). [00201] Interestingly, luciferase measurement in the harvested tissues after fourteen days from the AAV treatment confirmed that the liver was transduced by the Ne-AAV and FA-AAV vectors but not by the AS1411 -AAV. Conversely, no significant difference in luciferase activity was detected on the explanted tumor cells among the mice treated with the Ne-AAV, FA-AAV and AS1411 -AAV vectors (Figure 10F).
[00202] These results corroborated in vivo the previous in vitro findings demonstrating the cell-specific transduction of FA-AAV and AS1411 - AAV.
Discussion of the above examples
[00203] Recently, the gene therapy field has seen a massive increase in the development of novel AAV capsids. However, AAV vectors can still result in off-target transduction and, when used at high doses, raise the risk of acute AAV-related toxicity. Thus, designing vectors able to transduce specific cells and, at the same time, minimize their off-target uptake is an important medical need in the context of AAV in vivo gene transfer. In the above studies, new chemically engineered AAVs were developed which were programmed by single molecule conjugation to re-target vector tropism toward specific cells.
[00204] Different strategies were perused to incorporate the unnatural amino acid onto the AAV capsid, by: 1 ) introducing the modification in all three VP capsid proteins and by 2) inserting the unnatural amino acid only on the VP2 or VP3 proteins. Nevertheless, the latter approach resulted in a significant reduction of vector infectivity. This might have occurred due to the low efficiency of the modified VP2 and VP3 complementation when provided in trans during the vector production. In this case, the final AAV preparation might possibly contain particles devoid of either VP2 or VP3 protein which is known to result in reduced AAV transduction.
[00205] Although the exact number of capsid VP proteins that have been successfully conjugated in the vector preparation is unknow, the data generated in vitro and in vivo clearly support that the ligand-vector conjugation was able to specifically modify the AAV tropism.
[00206] It is noted that the placement of the unnatural amino acid within the capsid affected both AAV production and transduction efficiency, and transduction efficiency was also affected by the size of the linker.
[00207] Biorthogonal copper-free click chemistry for AAV conjugation has been successfully exploited to label the capsid with fluorophores in order to track the vector in live cells and different chemical moieties have been utilized trying to shield the vector from anti- AAV antibodies.
[00208] In the experiments described here, to further improve vector production, the regulatory elements were replaced within the AAV expression cassette. Additionally, a copper-free cycloaddition reaction reported to be more suitable for use in living systems was used for subsequent vector conjugation (the click chemistry reaction) instead of employing copper as catalyst.
[00209] Tumor cells usually undergo genomic and metabolic dysregulation, which leads to the expression of tumor-specific cellular surface molecules. Hence, researchers have been trying to develop ligands to specifically target cancer cells. To this end, folic acid and aptamers have been conjugated to drugs, nucleic acids, and lipid nanoparticles. Nevertheless, nanoparticles have been associated with acute cell toxicity depending on their formulation. Moreover, like most oligonucleotides, aptamers possess very poor and limited endosomal escape that may hamper their development as therapeutics. By contrast, AAV vectors use their intrinsic phospholipase A2 activity which allow their effective release from the endosomal compartment and very efficient nuclear trafficking.
[00210] The studies described herein show the first successful conjugation of aptamers to AAV capsids in order to efficiently enhance and re-direct their tropism. The transduction specificity of AAV conjugated to a peptide molecule (FA), an RNA aptamer (E3), and a DNA aptamer (AS141 1) were characterized through a series of in vitro uptake assays and the use of different cell lines. The ligand’s characteristics were demonstrated to be transferable, since the NE-AAV serotype did not influence the vector uptake.
[00211] In vivo studies in a xenograft animal model demonstrated that FA- and AS1411 -AAVs can be efficiently and specifically taken up by tumor cells unlike the unconjugated vector, which showed off-target expression in liver. Nevertheless, the FA-AAV liver transduction in some animals could likely be due to the ectopic expression of folic acid receptors on the surface of murine hepatocytes.
[00212] One of the limitations of this study could be represented by the lower yield of NE-AAVS compared to their wild-type counterpart. However, engineering the producer cells to constitutively express the prokaryotic tRNA/tRNA synthases combined to the discovery of novel orthogonal tRNAs/tRNA synthases that possess an enhanced ability to incorporate unnatural ammino acid, might dramatically improve the manufacturing of Ne-AAVs.
[00213] The use of aptamers to modulate the AAV tropism presents apparent advantages: a) large scale production of nucleic acids is less expensive than protein- or antibodies- based drugs, b) the possible use of a universal vector will further reduce the costs of manufacturing, c) the aptamer selection for a specific type of cells is more simple than screening libraries of antibodies or AAVs and d) chemical synthesis of oligonucleotides facilitates straight forward attachment of moieties that support their subsequent conjugation to the modified AAV capsids described herein.
[00214] Recently aptamer selection has been expanded in vivo through direct injection of nucleic acid-based libraries in animal models. Moreover, organs-on-chip technology and organoids could be valuable resources for the isolation of specific aptamers for human cells. These advances in combination with machine learning have allowed researchers to predict and optimize an aptamer sequence with an improved affinity for the desired target protein. A further evolution to this approach could be the use of Alpha Fold for precise protein structure prediction which, in the future, might pave the way for a total in silico design of aptamers that bind specific cell surface receptors.
[00215] Thus, the chemically modified Ne-AAVs disclosed herein combined with wide aptamer versatility creates a novel class of easy programmable cell type specific vectors.
Materials and methods for the above examples
Plasmid construction
[00216] All the plasmids generated in this study have been produced by Gibson assembly using NEBuilder® HiFi DNA Assembly Master Mix (NEB, cat. E2621 S). Single nucleotide mutations were introduced by QuickChange II Site-Directed Mutagenesis Kit (Agilent Technologies, cat. 200523). A pcDNA3.1 plasmid backbone was used for the expression of VP2 or VP3 under the control of a CMV promoter. The pAcBad .tR4-MbPyl plasmid was a gift from Peter Schultz (Addgene plasmid cat. 50832)27. The pAAV plasmid with the ubiquitous CAG promoter upstream the firefly luciferase transgene for the in vitro experiments was generated in the Kay lab and available at Addgene (catalog number 83281 ). For the in vivo studies the CAG promoter was swapped with the minimal ubiquitous INS84 promoter. NE-AA V vector production and titration
[00217] Cell transfection for AAV production was performed as previously reported with few modifications. Briefly, the Ns-2-Azidoethyloxycarbonyl-L-lysine (Toronto research, cat. A848920) was added to the transfection media at the final concentration of 1 .5mM for the NE-AAVS production. Transfection was carried out using the following plasmids: pRep/Cap (5|ig/225cm2 flask), pXX6-Ad5 (10p.g/225cm2 flask), pAAV plasmid with the gene of interest (5pg/225cm2 flask), pAcBad ,tR4-MbPyl (10pg/225cm2 flask). The AAV vectors were purified using AAVpro® Purification Kit (Takara, cat. 6666) following manufacturer instructions. For crude lysate AAVs, the cells were harvested and underwent three cycles of freeze/thaw for lysis. They were spun at 5000g at 4°C for 15 minutes to pellet cell debris. Supernatant was collected and treated with 1 pL of benzonase for one hour at 37°C. Cells were spun at 5000g for 10 minutes to pellet debris and the supernatant collected. The supernatant was incubated for one hour at 4°C in ice. Another centrifugation step at 7000g at 4°C for 30 minutes was carried out. Supernatant was collected and stored at -80°C until the day of the AAV transduction.
[00218] Vector titers were obtained by qPCR on the CFSX384 instrument (Biorad) using Brilliant III Ultra-Fast SYBR QPCR MM (Agilent Technologies, cat. 600882).
[00219] Primers: Forward (Luciferase) 5’- cttcgaggctaaggtggtgg-3’ (SEQ ID NO: 17) [00220] Reverse (Luciferase) 5’-tcggggttgttaacgtagcc-3’ (SEQ ID NO: 18)
DBCO chemicals preparation and AAV conjugation
[00221] NE-AAV were conjugated with the DBCO PEG Folic acid or DBCO PEG Biotin (NANOCS, cat. PG2-DBFA, PG2-BNDB). DBCO-PEG chemicals were resuspended in 20% DMSO to a final concentration of 2mM used as stock solution. Before the click reaction, the stock was further diluted to 500mM in 20% of DMSO solution. DBCO- PEG moieties were added to NE-AAV to a final concentration of 10OmM and incubated overnight on a tube rotor at 4°C. The NE-AAV unconjugated control vectors were incubated in the same 20% DMSO solution used for the DBCO-PEG solubilization. DBCO-PEG-Aptamer preparation was carried out as previously reported71. Briefly, the DNA aptamers, AS1411 and CD, carrying an ammino (NH2) modified 5’ end were synthesized from Integrated DNA Technology:
[00222] AS141 1 : 5’-5AmMC6-TTTTTTTTTTTTGGTGGTGGTGGTGTTGGTGGTGGTGG-3’ (SEQ ID NO: 19) [00223] CD:5’-5AmMC6- TTTTTTTTTTTTGGGGCCGGGGCAAGGGGGGGGTACCGTGGTAGGAC-3’ (SEQ ID NO: 20)
[00224] The E3 and C36 RNA aptamers were synthesized as previously reported22. Briefly, the E3 (GGCUUUCGGGCUUUCGGCAACAUCAGCCCCUCAGCC) (SEQ ID NO: 21 ) and C36 (GGCGUAGUGAUUAUGAAUCGUGUGCUAAUACACGCC) (SEQ ID NO: 22) aptamers were synthesized by solid phase synthesis on a MerMade 12 Synthesizer (Biosearch Technologies, as previously described). Briefly, the aptamers were synthesized using 2'-F-modified pyrimidines and 2'OH purines on an inverted dT CPG column. Synthesis reagents were purchased from Glen Research (Sterling, VA) and Chemgenes Corp (Wilmington, MA). All aptamers were synthesized bearing a 5' amine using a C6 phosphoramidite (Glen Research, Sterling, VA).
[00225] The aptamers were incubated with DBCO-PEG4-NHS Ester (Click Chemistry Tool, cat. A134-10) at room temperature for two hours. The resulted DBCO-PEG-Aptamer was finally purified using Cytiva illustra™ MicroSpin™ G-25 Columns (Cytiva life sciences, cat. 27532501) following manufacturer instructions. DBCO-PEG-Aptamer was added to the NE-AAV to a final concentration of 125 pM and incubated overnight on a tube rotor at 4°C.
[00226] The conjugated-AAV, both aptamer- and FA-conjugated, were dialyzed using Amicon Ultra-0.5 Centrifugal Filter Unit 100k (Sigma, cat. UFC510024). The columns were washed by centrifugation at 10000g for ten minutes at 4°C using 450pL of PBS- 0.0001 % Pluronic F-68 (Pluronic F-68 Polyol, 100mL, MP Biomedicals, Fisher cat. ICN2750049). The conjugated AAV was added to 450|iL of PBS-0.0001% Pluronic F- 68 and washed three times with 450|iL of PBS-0.0001 % Pluronic F-68 solution by three centrifugations at 10000g for ten minutes at 4°C. After the last washing step, the conjugated-AAV was transferred into a new 1 ,5mL sterile tube.
[00227] Silver stain analysis of AAV vectors was performed using SYPRO™ Ruby Protein Gel Stain (Thermo Fisher cat. S12000) following manufacturer instructions.
Cell lines
[00228] The cells used for the folic acid experiments were grown in RPMI 1640 Medium (Fisher, 27016021 ) supplemented with 10% fetal bovine serum (FBS), 100 lU/ml pen icillin/streptomycin . The rest of the experiments were carried out using DMEM Medium (Thermo Fisher, cat. 15017CV) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 lll/ml penicillin/streptomycin. All the cells were maintained in a humidified incubator at 37°C with 5% CO2. Total mRNA was extracted from HeLa, MCF-7 and A549 using RNeasy mini kit (QIAGEN) following manufacturer instructions. mRNA was converted to cDNA using Maxima First Strand cDNA Synthesis Kit for RT-qPCR (Thermo Fisher, cat. K1671 ).
[00229] qPCR was performed in the CFSX384 instrument (Biorad) using Brilliant III Ultra-Fast SYBR QPCR MM (Agilent Technologies, cat. 600882).
[00230] Primers: Forward (hFOLRI ) 5’- acaaggattgcatgggccag-3’ (SEQ ID NO: 23) Reverse (hFOLRI ) 5’- aggtgccatctctccacagtg-3’ (SEQ ID NO: 24) Forward (hACTB) 5’- gtcaccaactgggacgacat-3’ (SEQ ID NO: 25) Reverse (hACTB) 5’- gtacatggctggggtgttga-3’ (SEQ ID NO: 26)
AAV transduction
[00231] Cells were incubated with AAV vectors for 48 hours using a multiplicity of infection (MOI) of 5000 unless specified otherwise in the figure legend. Cell lysis and luciferase assay were performed using the Promega Luciferase 1000 Assay System (Promega, cat. E4550) following manufacturer instructions. For the uptake experiments 1.5 ug per well of anti-hFOLR1 antibody (Thermo Fisher, cat. MA5-23917), 200|iM/well of Folic acid (Sigma, cat. F8758-25G), 1 mg/mL per well of ssDNA (Sigma, cat. 262012- 1 GM), 10piM/well of the clathrin inhibitor Dynasore hydrate (Millipore Sigma, cat. D7693-5MG), and different concentrations of AS141 1 aptamer antidote (sequence, 5’- CCACCACCACCACAACCACC-3’) (SEQ ID NO: 27) acquired from Integrated DNA Technology, were used.
Western blot
[00232] AAV samples were loaded on a 4-15% gradient polyacrylamide gel (Fisher, cat. 34028). Protein transfer was performed using the iBIot system (Thermo Fisher, cat. IB23002). The membrane was blocked with Odyssey buffer (Fisher, cat. NC0730870) and incubated with the anti-AAV antibody (Gene Tex, cat. GTX44495) or a streptavidin-conjugated antibody (Fisher, cat. NC9386176) both diluted 1 :1000 in odyssey buffer. The membrane was washed and incubated with a secondary antibody (Fisher, cat. 92532210) diluted 1 :100000 and visualized by Odyssey imaging system (Li-Cor Biosciences). For the AAV-Aptamer detection, the membrane, after the blocking step, was incubated with 1 gM of AS1411 -(5’- CCACCACCACCACAACCACCACCACC-Biotin-3’) (SEQ ID NO: 28), CD-(5’- CCCCCCATGGCACCATCCTG-Biotin-3’) (SEQ ID NO: 29),E3-(5’- GATGTTGCCGAAAGCCCGAA-Biotin-3’) (SEQ ID NO: 30) probes overnight at room temperature, and subsequently incubated with a streptavidin-conjugated antibody for two hours at room temperature (Fisher, cat. NC9386176).
Transmission electronic microscopy
[00233] The AAV vectors were placed on a 300-mesh carbon/formvar coated Cu grids and allow to settle 3 minutes. Samples were washed two times with MilliQ-l-kO and stained 1 minute with 1% Uranyl Acetate in mQ-H2O. AAV samples were allowed to dry. Images acquisition was performed on the JEOL-JEM1400 microscope at 120kV.
In vivo study
[00234] Mouse experiments were conducted and approved by the Administrative Panel on Laboratory Animal Care of Stanford University. Nude mice (NU/J) were acquired from Jackson Lab (cat. 002019). The animals were kept in the animal facility with a normal night/day cycle and on autoclaved chow ad libitum. HeLa cells were growth in 225cm2 flasks at -90% confluency and >90% of viability before transplantation. The day of transplantation cells were harvested and resuspended in PBS. 5x106/cells were injected subcutaneously in the animal flank in 200 j L of sterile PBS. AAV injection were performed subcutaneously using 5x109 vg/mouse. In vivo luciferase imaging was performed using the Lago optimal imaging system (Spectral Instruments Imaging). Luciferase imagines were analyzed using Aura Software (Spectral Instruments Imaging). Explanted liver and HeLa tumor were freshly homogenized in PBS using a Bullet Blender (Next Advance). The luciferase assay was performed using the Promega Luciferase 1000 Assay System (Promega, cat. E4550) following manufacturer instructions. Protein concentration in tissue samples was measured with Pierce BCA protein assay kit (Thermo Fisher, cat. 23227) following manufacturer instructions.
Statistics
[00235] GraphPad Prism was used for statistical analysis. Data sets were compared by ANOVA with a proper post hoc correction (see figure legends). Statistical significance was assumed with P value <0.05 (*) (#), <0.01 (**) (##), <0.001 (***) (###) and <0.0001 (****) (####). Bars in graphs represent standard deviation for each group. [00236] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
[00237] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.
[00238] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

CLAIMS What is claimed is:
1 . A variant adeno-associated virus (AAV) capsid protein comprising:
(c) an azide-bearing unnatural amino acid substitution at a position corresponding to:
T456, D555, R587/A587, or N589 of the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2; or
E330, T457, N499, or N590 of the AAV8 amino acid sequence set forth as SEQ ID NO: 7; or
T455, T456, or N588 of the AAV- LK03 amino acid sequence set forth as SEQ ID NO: 13; or
(d) an azide-bearing unnatural amino acid insertion between positions corresponding to N590 and T591 of the AAV8 amino acid sequence set forth as SEQ ID NO: 7.
2. The variant AAV capsid protein of claim 1 , comprising an amino acid sequence having:
(iv) 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, wherein said azide-bearing unnatural amino acid substitution is at position N589 or R587/A587;
(v) 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV8 amino acid sequence set forth as SEQ ID NO: 7, wherein said azide-bearing unnatural amino acid substitution is at position T457; or
(vi) 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV- LK03 amino acid sequence set forth as SEQ ID NO: 13, wherein said azide-bearing unnatural amino acid substitution is at position N588.
3. The variant AAV capsid protein of claim 1 , comprising an amino acid sequence having 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the AAV-DJ amino acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 2, wherein said azide-bearing unnatural amino acid substitution is at position N589 or R587/A587. The variant AAV capsid protein of claim 1 , comprising an amino acid sequence having 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 , 2, 7, and 13. The variant AAV capsid protein of claim 1 , comprising an amino acid sequence having 90% or more (e.g., 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 1 , 2, 7, and 13. The variant AAV capsid protein of claim 1 , comprising an amino acid sequence having 75% or more (e.g., 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16. The variant AAV capsid protein of claim 1 , comprising an amino acid sequence having 90% or more (e.g., 95% or more, 98% or more, 99% or more) sequence identity with the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16. The variant AAV capsid protein of claim 1 , comprising the amino acid sequence set forth in any one of SEQ ID NOs: 3-6, 8-12, or 14-16. The variant AAV capsid protein of claim 1 , comprising the amino acid sequence set forth in any one of SEQ ID NOs: 5-6, 12, or 14. The variant AAV capsid protein of any one of claims 1 -9, wherein the unnatural amino acid is an azido-lysine. The variant AAV capsid protein of any one of claims 1 -7 and 10, comprising at least one arginine to alanine mutation at a heparan sulfate proteoglycan (HSPG) binding site. The variant AAV capsid protein of claim 1 1 , wherein the HSPG binding site is at an amino acid position corresponding to position 587 and/or 590 of SEQ ID NO: 1 . The variant AAV capsid protein of any one of claims 1 -12, wherein the variant AAV capsid protein is conjugated to folic acid via the azide-bearing unnatural amino acid. The variant AAV capsid protein of any one of claims 1 -12, wherein the variant AAV capsid protein is conjugated to a DNA or RNA aptamer via the azide-bearing unnatural amino acid. The variant AAV capsid protein of claim 14, wherein the variant AAV capsid protein is conjugated to the DNA aptamer AS141 1 . The variant AAV capsid protein of claim 14, wherein the variant AAV capsid protein is conjugated to the RNA aptamer E3. The variant AAV capsid protein of any one of claims 13-16, where the variant AAV capsid protein is conjugated via a linker. The variant AAV capsid protein of claim 17, wherein the linker is a Polyethylene glycol (PEG) linker. The variant AAV capsid protein of claim 18, wherein the PEG linker length is less than 5 kDa. The variant AAV capsid protein of claim 18, wherein the PEG linker length is 4 kDa or less (e.g., 3 kDa or less, 2 kDa or less). The variant AAV capsid protein of claim 18, wherein the PEG linker length is about 2 kDa. A chemically modified variant adeno-associated virus (AAV) capsid protein comprising an azide-bearing unnatural amino acid substitution, wherein the capsid protein is conjugated to folic acid, a DNA aptamer, or an RNA aptamer via the azide-bearing unnatural amino acid. A nucleic acid comprising a nucleotide sequence encoding the variant AAV capsid protein of any one of claims 1 -12, wherein said nucleotide sequence comprises a TAG stop codon at the position encoding said unnatural amino acid substitution. A recombinant AAV particle comprising:
(a) the variant AAV capsid protein of any one of claims 1-22; and
(b) a nucleic acid payload of interest. The recombinant AAV particle of claim 24, wherein the nucleic acid payload of interest: encodes a polypeptide (e.g., a genome-editing enzyme such as a CRISPR/Cas effector protein, a zinc finger nuclease, or a TALEN), or is a non-coding RNA (e.g., a CRISPR/Cas guide RNA, an shRNA, an siRNA, a miRNA, an aptamer) or encodes said non-coding RNA, or is an mRNA. The recombinant AAV particle of claim 25, wherein said polypeptide is encoded by a nucleotide sequence that is operably linked to the small ubiquitous INS84 promoter. A method of delivering a payload of interest to a target cell, the method comprising contacting the target cell with the recombinant AAV particle of any one of claims 25-26. The method of claim 27, wherein the target cell is in vivo and said contacting comprises administering the recombinant AAV particle to an individual.
PCT/US2023/084058 2022-12-14 2023-12-14 Chemically modified aavs WO2024129990A2 (en)

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