AAV Capsids and Uses Thereof

Abstract

Described herein are capsid peptides, compositions (including AAV) that mediate efficient AAV transduction of brain vasculature, primarily endothelial cells and pericytes, and smooth muscle cells, as well as methods of using the same.Embodiments of the present invention include AAV capsid proteins that include an amino acid sequence that includes at least 4, at least 5, or at least 6 consecutive amino acids from the sequence PRPPSTH (SEQ ID NO: 1); MAEPGAR (SEQ ID NO: 2); SQDPSTL (SEQ ID NO: 3); or MLYADNT (SEQ ID NO: 4).

Description

[Technical field]

[0001] Priority claim This application claims the benefit of U.S. Provisional Patent Application No. 63 / 180,320, filed April 27, 2021. The entire contents of the foregoing documents are incorporated herein by reference.

[0002] Federally sponsored research or development This invention was made with Government support under Grant No. DC017117 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] Described herein are capsid peptides, compositions (including AAV) that mediate efficient AAV transduction of the brain vasculature, primarily endothelial and pericytes, as well as smooth muscle cells, and methods of using the same. [Background technology]

[0004] Neurodegenerative and cerebrovascular diseases share fundamental microvascular dysfunction, and selective transgene delivery to brain endothelium, smooth muscle cells, and pericytes may enable gene-targeted therapeutic intervention. Due to its high density throughout the central nervous system, the cerebral vasculature can also serve as a source of secreted therapeutic proteins to neurons and glial cells. Summary of the Invention

[0005] To isolate capsids that are transported to the brain after intravenous (i.v.) delivery, two rounds of in vivo sorting with adeno-associated virus serotype 9 (AAV9) capsid scaffolds representing a heptameric peptide library were performed. In contrast to the parental capsid AAV9, which primarily transduces neurons and astrocytes, several capsids were identified that mediate efficient transduction of the brain vasculature, primarily endothelial and pericytes, as well as smooth muscle cells.

[0006] Provided herein is an AAV capsid protein comprising an amino acid sequence comprising at least four consecutive amino acids from the sequence PRPPSTH (SEQ ID NO: 1); MAEPGAR (SEQ ID NO: 2); SQDPSTL (SEQ ID NO: 3); or MLYADNT (SEQ ID NO: 4). In some embodiments, the AAV capsid protein comprises an amino acid sequence comprising at least five consecutive amino acids from the sequence PRPPSTH (SEQ ID NO: 1); MAEPGAR (SEQ ID NO: 2); SQDPSTL (SEQ ID NO: 3); or MLYADNT (SEQ ID NO: 4).

[0007] In some embodiments, the AAV capsid protein comprises an amino acid sequence comprising at least 6 consecutive amino acids from the sequence PRPPSTH (SEQ ID NO:1); MAEPGAR (SEQ ID NO:2); SQDPSTL (SEQ ID NO:3); or MLYADNT (SEQ ID NO:4).

[0008] In some embodiments, the AAV is AAV9.

[0009] In some embodiments, the AAV capsid protein comprises AAV9 VP1. In some embodiments, the targeting sequence is inserted at positions corresponding to amino acids 588 and 589 of SEQ ID NO:14.

[0010] Also provided herein are nucleic acids encoding the AAV capsid proteins described herein.

[0011] Further provided is an AAV comprising the capsid protein described herein. In some embodiments, the AAV further comprises a transgene, preferably a therapeutic transgene.

[0012] Further provided herein is a targeting sequence comprising [D / P]PST (SEQ ID NO:9). In some embodiments, the targeting sequence comprises at least 4 consecutive amino acids from the sequence PRPPSTH (SEQ ID NO:1); MAEPGAR (SEQ ID NO:2); SQDPSTL (SEQ ID NO:3); or MLYADNT (SEQ ID NO:4). Further provided is a fusion protein comprising the targeting sequence described herein and a heterologous sequence. Also provided is an AAV capsid protein comprising the targeting sequence, for example, in this case, the capsid protein comprises AAV9 VP1. In some embodiments, the targeting sequence is inserted at a position corresponding to amino acids 588 and 589 of SEQ ID NO:14. Also provided is an AAV comprising the targeting sequence, fusion protein, or nucleic acid encoding the AAV capsid protein, and the capsid protein. In some embodiments, the AAV further comprises a transgene, preferably a therapeutic transgene.In some embodiments, the transgene is selected from the group consisting of neurturin; brain-derived neurotrophic factor (BDNF); brain dopamine neurotrophic factor (CDNF); mesencephalic astrocyte-derived neurotrophic factor (MANF); vascular endothelial growth factor (VEGF); glial cell line-derived neurotrophic factor (GDNF); aromatic L-amino acid decarboxylase (AADC); Tau antibody; amyloid precursor protein (APP) antibody; type IV collagen A1 or A2 (COL4A1 / A2); ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1); ATP-binding cassette subfamily C member 6 (ABCC6); 3 prime repair exonuclease 1 (TREX1); forkhead box C1 (FOXC1); paired-like homeodomain 2 (PITX2); SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1); endoglin (ENG); SMAD family member 4 (SMAD4); activin A receptor-like type 1 (ACVRL1); RAS encoding p21 protein activator 1 (RASA1); Notch receptor 3 (NOTCH3); HtrA serine peptidase 1 (HTRA1); Zinc finger CCHC type containing 14 (ZCCHC14), or Apolipoprotein E epsilon 4 (APOE ε4). Other exemplary transgenes include those described herein.

[0013] Also provided herein is a method of delivering a sequence, e.g., a transgene, to a cell, the method comprising contacting the cell with an AAV as described herein. Also provided is an AAV as described herein for use in delivering a sequence to a cell. In some embodiments, the cell is a vascular endothelial cell or a smooth muscle cell. In some embodiments, the cell is a pericyte. In some embodiments, the cell is present in a living subject, e.g., a mammalian subject.

[0014] In some embodiments, the cells are present in a tissue selected from the brain, spinal cord, dorsal root ganglion, heart, liver, or smooth muscle, and combinations thereof.

[0015] In some embodiments, the subject is diagnosed with a disease affecting the vasculature of the central nervous system, optionally with a gene encoding type IV collagen A1 or A2 (COL4A1 / A2); ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1); ATP-binding cassette subfamily C member 6 (ABCC6); 3 prime repair exonuclease 1 (TREX1); forkhead box C1 (FOXC1); paired-like homeodomain 2 (PITX2); SAM- and HD-domain-containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1); endoglin (ENG); SMAD family member 4 (SMAD4); activin A receptor-like type 1 (ACVRL1); RAS p21 protein activator 1 (RASA1); notch receptor 3 (NOTCH3); HtrA serine peptidase 1 (HTRA1); zinc finger CCHC type-containing 14 (ZCCHC14), or apolipoprotein E epsilon 4 (APOE They suffer from a disease associated with a mutation in ε4).

[0016] In some embodiments, the subject suffers from a neurodegenerative disease, eg, Parkinson's disease or Alzheimer's disease.

[0017] In some embodiments, the cells are present in the brain of the subject and the AAV is administered by parenteral delivery. In some embodiments, the parenteral delivery is by intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular delivery.

[0018] In some embodiments, the cells are present in the subject's brain and the AAV is administered by intravenous delivery.

[0019] In some embodiments, the transgene is selected from the group consisting of neurturin; brain-derived neurotrophic factor (BDNF); brain dopamine neurotrophic factor (CDNF); mesencephalic astrocyte-derived neurotrophic factor (MANF); vascular endothelial growth factor (VEGF); glial cell line-derived neurotrophic factor (GDNF); aromatic L-amino acid decarboxylase (AADC); Tau antibody; amyloid precursor protein (APP) antibody; type IV collagen A1 or A2 (COL4A1 / A2); ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1); ATP-binding cassette subfamily C member 6 (ABCC6); 3 prime repair exonuclease 1 (TREX1); forkhead box C1 (FOXC1); paired-like homeodomain 2 (PITX2); SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1); endoglin (ENG); SMAD family member 4 (SMAD4); activin A receptor-like type 1 (ACVRL1); RAS encoding p21 protein activator 1 (RASA1); Notch receptor 3 (NOTCH3); HtrA serine peptidase 1 (HTRA1); Zinc finger CCHC type containing 14 (ZCCHC14), or Apolipoprotein E epsilon 4 (APOE ε4). Other exemplary transgenes include those described herein.

[0020] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials are described herein for use in the present invention; other suitable methods and materials known in the art can also be used.Materials, methods, and examples are illustrative only and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety.In the event of any conflict with this specification, this specification, including definitions, will take precedence.

[0021] Other features and advantages of the invention will become apparent from the following detailed description and drawings, and from the claims. [Brief description of the drawings]

[0022] [Figure 1-1] AAV-PR-CBA-Cre mediates vasculature-tropic transduction in transgenic Ai9 mice (CAG-floxed-STOP-tdTomato). Mice were injected systemically with AAV-PR-CBA-Cre and sacrificed 3 weeks later. (A) Whole hemisphere images of tdTomato immunofluorescence showing vasculature transduction by AAV-PR. (B) Images of cortical regions depicting transduced vasculature and DAPI. (C-D) Higher magnification images of boxed areas from (b) showing transduced vasculature. [Figure 1-2] AAV-PR-CBA-Cre mediates vasculature-tropic transduction in transgenic Ai9 mice (CAG-floxed-STOP-tdTomato). Mice were injected systemically with AAV-PR-CBA-Cre and sacrificed 3 weeks later. (E) Image (4x) from a mouse injected with AAV-MA-CBA-Cre. [Figure 1-3] AAV-PR-CBA-Cre mediates vasculature-tropic transduction in transgenic Ai9 mice (CAG-floxed-STOP-tdTomato). Mice were injected systemically with AAV-PR-CBA-Cre and sacrificed 3 weeks later. (F) Image (10x) from a mouse injected with AAV-MA-CBA-Cre. [Figure 1-4]AAV-PR-CBA-Cre mediates vasculature-tropic transduction in transgenic Ai9 mice (CAG-floxed-STOP-tdTomato). Mice were injected systemically with AAV-PR-CBA-Cre and sacrificed 3 weeks later. (G) Image (10x) from a mouse injected with AAV-SQ-CBA-Cre. [Figure 1-5] AAV-PR-CBA-Cre mediates vasculature-tropic transduction in transgenic Ai9 mice (CAG-floxed-STOP-tdTomato). Mice were injected systemically with AAV-PR-CBA-Cre and sacrificed 3 weeks later. (H) Image (4x) from a mouse injected with AAV-ML-CBA-Cre. [Figure 2-1] AAV-PR-CBA-GFP-mediated vascular transduction decreases over time. Adult male C57BL / 6 mice were injected intravenously with 3x1010vg / mouse of self-complementary AAV-PR-CBA-GFP and sacrificed on days 5 and 28 for GFP analysis. A. Representative 20x images of 20 μm coronal sections of somatosensory cortex, striatum, and hypothalamus showing transduced blood vessels. The area of ​​analysis is represented by a square image of the brain drawing to the right of the image. [Figure 2-2] A. AAV-PR-CBA-GFP-mediated vascular transduction decreases over time. Adult male C57BL / 6 mice were intravenously injected with 3x1010vg / mouse of self-complementary AAV-PR-CBA-GFP and sacrificed on days 5 and 28 for GFP analysis. B. Quantification of GFP fluorescence intensity in the three analyzed regions on days 5 and 28 (cortex, *** represents p=0.0004; striatum, * represents p=0.0135; hypothalamus, * represents p=0.0006, respectively). [Diagram 3]Figure 1 shows that AAV genomes in whole brain do not decrease significantly over time.Adult male C57BL / 6 mice were intravenously injected with 2.2x1010vg / mouse of self-complementary AAV-PR-CBA-GFP and sacrificed on days 5 (n=3) and 28 (n=3) for AAV genome quantification by Taqman qPCR analysis. [Figure 4] Figure 1: AAV-PR transduces brain endothelium with high efficiency after systemic injection. Representative images of GFP signal in the somatosensory cortex in 20 μm coronal brain sections of mice injected with AAV-PR-sc-CBA-GFP showing transduced brain endothelial cells (GFP) colocalizing with the brain endothelial marker PCAM1 (CD31: red) in capillaries, arterioles, and arteries at 5 and 28 days after injection. At higher magnification (boxed image at 40x), partial colocalization between GFP and CD31 is shown (arrows). [Diagram 5] Figure 1: AAV-PR transduces brain pericytes with high efficiency after systemic injection. Representative images of GFP signal in the somatosensory cortex in 20 μm coronal brain sections of mice injected with AAV-PR-sc-CBA-GFP showing transduced brain blood vessels (GFP) with significant colocalization with pericytes identified by PDGFR-β (red) positivity at 5 and 28 days after injection. At higher magnification (40x), partial colocalization between GFP and PDGFR-β is shown (arrows). [Figure 6] Scattered glial and neuronal cells transduced by AAV-PR after systemic injection. Representative image of GFP signal in the somatosensory cortex in a 20 μm coronal brain section from a mouse injected with AAV-PR-sc-CBA-GFP showing transduced brain endothelium (GFP) showing perivascular glial progenitors (yellow arrows) and a single pyramidal neuron (red arrow) as well as very few astrocyte-shaped cells negative for GFAP, MBP, and NEUN. [Figure 7]AAV-PR transduction in the liver after systemic injection. Representative images of GFP signal in 20 μm liver sections from mice injected with AAV-PR-sc-CBA-GFP showing transduced hepatocytes (GFP) but not Kupffer cells, stellate cells, or vascular cells (endothelial CD31+ cells or VSMC SMA and SM22+ cells). [Figure 8-1] Exemplary schematic of experimental design. A. Binary system of library constructs. iTransduce libraries (represented by different colors) composed of different peptide inserts expressed on capsids are administered intravenously (iv) by injection into Ai9 transgenic mice with a loxP-flanked STOP cassette inserted into the Gt(ROSA)26Sor locus upstream of the tdTomato reporter gene. AAV capsids that are able to enter cells of interest but do not functionally transduce the cells (no Cre expression) will not enable tdTomato expression. Capsids that are able to mediate functional transduction (expressing Cre) will enable tdTomato expression. [Figure 8-2] Exemplary schematic of the experimental design. (B) Cells are isolated from the organ of interest (e.g., brain) and transduced cells are sorted for tdTomato expression and optionally a cell marker. [Figure 8-3] Exemplary schematic of experimental design. C. Exemplary images from Ai9 mice administered AAV-CBA-Cre (left) or C57BL.6 mice administered scAAV-CBA-GFP (right). See Hanlon et al., Mol Ther Methods Clin Dev. 2019 Dec 13; 15: 320-332. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Many common neurodegenerative diseases involve the cerebral vasculature, including Alzheimer's and Parkinson's diseases. In addition, the following proteins may be involved in the expression of collagen IV A1 or A2 (COL4A1 / A2); ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1); ATP-binding cassette subfamily C member 6 (ABCC6); 3 prime repair exonuclease 1 (TREX1); forkhead box C1 (FOXC1); paired-like homeodomain 2 (PITX2); SAM- and HD-domain-containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1); endoglin (ENG); SMAD family member 4 (SMAD4); activin A receptor-like type 1 (ACVRL1); RAS p21 protein activator 1 (RASA1); notch receptor 3 (NOTCH3); HtrA serine peptidase 1 (HTRA1); zinc finger CCHC type-containing 14 (ZCCHC14), or apolipoprotein E epsilon 4 (APOE There are several rare genetic diseases that affect the vascular system, including those associated with mutations in ε4).

[0024] Gene therapy using AAV vector has the potential to treat diseases that affect the vasculature.AAV9, the gold standard in CNS gene therapy, approved by FDA for the treatment of spinal muscular atrophy, mainly transduces neurons and astrocytes, with only some limited vasculature transduction.Therefore, there is a need for improvements in the therapy that targets the vasculature.

[0025] Here, novel capsid targeting peptides were identified by an in vivo selection process in mice using an AAV9 peptide display library. These peptides, when inserted into AAV9 between amino acids 588 and 589, showed tropism for the CNS vasculature after intravenous systemic delivery. The peptides are listed in Table 1.

[0026] [Table 1]

[0027] Targeting Sequence The method has identified a number of potential targeting peptides that enhance targeting of the CNS vasculature when, for example, inserted into the capsid of an AAV, e.g., AAV1, AAV2, AAV5, AAV8, or AAV9, or conjugated, chemically or via expression as a fusion protein, to a biologic, e.g., an antibody or other large biomolecule.

[0028] In some embodiments, the targeting peptide comprises a sequence of at least five amino acids. In some embodiments, the amino acid sequence comprises at least four, for example, five, six, or seven consecutive amino acids of the sequence PRPPSTH (SEQ ID NO: 1); MAEPGAR (SEQ ID NO: 2); SQDPSTL (SEQ ID NO: 3); or MLYADNT (SEQ ID NO: 4). In some embodiments, the amino acid sequence comprises at least [D / P]PST (SEQ ID NO: 9).

[0029] Targeting peptides containing L- and D-amino acids can also be used, such as, for example, L-HTSPPRP (SEQ ID NO:10); L-RAGPEAM (SEQ ID NO:11); L-LTSPDQS (SEQ ID NO:12); L-TNDAYLM (SEQ ID NO:13); D-HTSPPRP (SEQ ID NO:10); D-RAGPEAM (SEQ ID NO:11); D-LTSPDQS (SEQ ID NO:12); or D-TNDAYLM (SEQ ID NO:13).

[0030] Targeting peptides containing reversed sequences may also be used, for example HTSPPRP (SEQ ID NO:10); RAGPEAM (SEQ ID NO:11); LTSPDQS (SEQ ID NO:12); or TNDAYLM (SEQ ID NO:13).

[0031] The targeting peptides disclosed herein can be modified according to methods known in the art to create peptidomimetics. For example, Qvit et al., Drug Discov Today. 2017 Feb; 22(2): 454-462;Farhadi and Hashemian, Drug Des Devel Ther. 2018; 12: 1239-1254;Avan et al., Chem. Soc. Rev., 2014,43, 3575-3594;Pathak, et al., Indo American Journal of Pharmaceutical Research, 2015. 8; Kazmierski, WM, ed., Peptidomimetics Protocols, Human Press (Totowa NJ 1998); Goodman et al., eds., Houben-Weyl Methods of Organic Chemistry: Synthesis of Peptides and Peptidomimetics, Thiele Verlag (New York 2003); and Mayo et al., J. Biol. Chem., 278:45746 (2003). In some cases, these modified peptidomimetic versions of the peptides and fragments disclosed herein exhibit enhanced in vivo stability compared to the non-peptidomimetic peptides.

[0032] The method of making peptidomimetics includes replacing one or more, for example all, of the amino acids of a peptide sequence with D-amino acid enantiomers.Such sequences are referred to herein as "retro" sequences.In another method, the order of amino acid residues from N-terminus to C-terminus is reversed, so that the order of amino acid residues from N-terminus to C-terminus of the original peptide becomes the order of amino acid residues from C-terminus to N-terminus in the modified peptidomimetic.Such sequences can be referred to as "inverso" sequences.

[0033] Peptide mimetics can be both retro and inverso versions, i.e. "retro-inverso" versions of the peptides disclosed in the present invention. The new peptidomimetics can be composed of D-amino acids arranged such that the order of amino acid residues from N-terminus to C-terminus in the peptidomimetic corresponds to the order of amino acid residues from C-terminus to N-terminus in the original peptide.

[0034] Another method of making a peptide mimetic involves replacing one or more amino acid residues in a peptide with a chemically distinct but recognized functional analogue of that amino acid, i.e., an artificial amino acid analogue. Artificial amino acid analogues include β-amino acids, β-substituted β-amino acids ("β"). 3 Artificial amino acids include ∀-amino acids, phosphorus analogs of amino acids such as ∀-aminophosphonic acids and ∀-aminophosphinic acids, and amino acids with non-peptide bonds. Artificial amino acids can be used to create peptidomimetics, such as peptoid oligomers (e.g., peptoid amide or ester analogs), β-peptides, cyclic peptides, oligourea or oligocarbamate peptides, or heterocyclic molecules. Exemplary retro-inverso targeting peptidomimetics include HTSPPRP (SEQ ID NO: 19); RAGPEAM (SEQ ID NO: 20); LTSPDQS (SEQ ID NO: 21); or TNDAYLM (SEQ ID NO: 22), where the sequence includes all D-amino acids. These sequences can be modified, for example, by biotinylation of the amino terminus and amidation of the carboxy terminus.

[0035] AAV Viral vectors for use in the present methods and compositions include recombinant retroviruses, adenoviruses, adeno-associated viruses, alphaviruses, and lentiviruses that contain a targeting peptide as described herein and optionally a transgene for expression in the target tissue.

[0036] A preferred viral vector system useful for delivery of nucleic acid in the present method is adeno-associated virus (AAV). AAV is a small, non-enveloped virus with a 25 nm capsid. There are no diseases known or proven to be associated with the wild-type virus. AAV has a single-stranded DNA (ssDNA) genome. AAV has been found to exhibit long-term episomal transgene expression, and AAV has shown excellent transgene expression in the brain, especially neurons. The space for exogenous DNA in AAV is generally limited to the amount of nucleic acid that can physically fit inside the particle. For example, AAV types 1-5 can package up to 6 kb of DNA, and in some reports, AAV5 has been found to package up to 8.9 kb of DNA. DNA can be introduced into cells by using AAV vectors such as those described in Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985). A variety of nucleic acids have been introduced into different cell types using AAV vectors (see, e.g., Hermonat et al., Proc. Natl. Acad. Sci. USA 81:6466-6470 (1984); Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1985); Wondisford et al., Mol. Endocrinol. 2:32-39 (1988); Tratschin et al., J. Virol. 51:611-619 (1984); and Flotte et al., J. Biol. Chem. 268:3781-3790 (1993)). Numerous alternative AAV variants exist (over 100 clones have been generated), and AAV variants have been identified based on desirable properties.In some embodiments, the AAV is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AV6.2, AAV7, AAV8, rh.8, AAV9, rh.10, rh.39, rh.43, or CSp3; for CNS applications, in some embodiments, the AAV is AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, or AAV9. Suitable AAV vectors can be designed to accommodate large amounts of DNA. As an example, AAV9 has been shown to be somewhat efficient at crossing the blood-brain barrier. Using this method, AAV capsids can be engineered to increase vascular penetration by inserting the targeting sequence described herein into the capsid protein, for example, the AAV9 capsid protein VP1 between amino acids 588 and 589.

[0037] An exemplary wild-type AAV9 capsid protein VP1 (Q6JC40-1) sequence is as follows:

[0038] [ka]

[0039] Thus, provided herein is an AAV comprising one or more of the targeting peptide sequences described herein, e.g., a capsid protein comprising a targeting sequence described herein, e.g., an AAV comprising a capsid protein comprising SEQ ID NO:1, with the targeting peptide sequence inserted, e.g., in the sequence between amino acids 588 and 589.

[0040] An exemplary amino acid sequence of AAV9 VP1, including the AAV-PR targeting sequence (shown in bold lowercase), is as follows:

[0041] [ka]

[0042] An exemplary amino acid sequence of AAV9 VP1, including the AAV-MA targeting sequence (shown in bold lowercase), is as follows:

[0043] [ka]

[0044] An exemplary amino acid sequence of AAV9 VP1, including the AAV-SQ targeting sequence (shown in bold lowercase), is as follows:

[0045] [ka]

[0046] An exemplary amino acid sequence of AAV9 VP1, including the AAV-ML targeting sequence (shown in bold lowercase), is as follows:

[0047] [ka]

[0048] Exemplary sequences encoding these VPI variants are provided below.

[0049] In some embodiments, the AAV also includes a transgene sequence (i.e., a heterologous sequence), such as a transgene encoding a therapeutic agent, as described herein or known in the art, or a reporter protein, e.g., an enzyme that catalyzes a reaction that results in a detectable product, such as a fluorescent protein, or a cell surface antigen. The transgene is preferably linked to a sequence that facilitates / drives / regulates expression of the transgene in the target tissue.

[0050] Exemplary transgenes for use as therapeutics include, but are not limited to, neurturin; brain-derived neurotrophic factor (BDNF); brain dopamine neurotrophic factor (CDNF); mesencephalic astrocyte-derived neurotrophic factor (MANF); vascular endothelial growth factor (VEGF); glial cell line-derived neurotrophic factor (GDNF); aromatic L-amino acid decarboxylase (AADC); Tau antibody; amyloid precursor protein (APP) antibody; type IV collagen A1 or A2 (COL4A1 / A2); ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1); ATP-binding cassette subfamily C member 6 (ABCC6); 3 prime repair exonuclease 1 (TREX1); forkhead box C1 (FOXC1); paired-like homeodomain 2 (PITX2); SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1); endoglin (ENG); SMAD family member 4 (SMAD4); activin A receptor-like type 1 (ACVRL1); RAS p21 protein activator 1 (RASA1); Notch receptor 3 (NOTCH3); HtrA serine peptidase 1 (HTRA1); Zinc finger CCHC type containing 14 (ZCCHC14), or Apolipoprotein E epsilon 4 (APOE ε4). Other exemplary transgenes include, for example, neuronal apoptosis inhibitor protein (NAIP), nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), tyrosine hydroxylase (TH), GTP cyclohydrolase (GTPCH), amino acid decarboxylase (AADC), aspartoacylase (ASPA), blood factors such as β-globin, hemoglobin, tissue plasminogen activator, and blood clotting factors; colony stimulating factor (CSF); interleukins such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, and the like;Growth factors, such as keratinocyte growth factor (KGF), stem cell factor (SCF), fibroblast growth factors (FGFs, such as basic FGF and acidic FGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), bone morphogenetic proteins (BMPs), epidermal growth factor (EGF), growth differentiation factor-9 (GDF-9), hepatoma-derived growth factor (HDGF), myostatin (GDF-8), nerve growth factor (NGF), neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), and the like; soluble receptors, such as soluble TNF-α receptor, soluble VEGF receptor, soluble interleukin receptors (such as soluble IL-1 receptor and soluble type II receptor). IL-1 receptor), soluble gamma / delta T cell receptor, ligand-binding fragments of soluble receptors, etc.; enzymes, e.g., alpha-glucosidase, imiglucerase, beta-glucocerebrosidase, etc.; enzyme activators, e.g., tissue plasminogen activator, etc.; chemokines, e.g., IP-10, interferon gamma-inducing monokine (Mig), Groa / IL-8, RANTES, MIP-1alpha, MIP-1beta, MCP-1, PF-4, etc.; angiogenic agents, e.g., vascular endothelial growth factor (VEGF, e.g., VEGF121, VEGF165, VEGF-C, VEGF-2), transforming growth factor beta, basic fibroblast growth factor, glioma-derived growth factor, angiogenin, angiogenin-2, etc.; angiogenesis inhibitors, e.g., soluble VEGF receptor receptors, etc.; protein vaccines; neuroactive peptides, such as nerve growth factor (NGF), bradykinin, cholecystokinin, gastin, secretin, oxytocin, gonadotropin-releasing hormone, beta-endorphin, enkephalin, substance P, somatostatin, prolactin, galanin, growth hormone-releasing hormone, bombesin, dynorphin, warfarin, neurotensin, motilin, thyrotropin, neuropeptide Y, luteinizing hormone, calcitonin, insulin, glucagon, vasopressin, angiotensin II, thyrotropin-releasing hormone, vasoactive intestinal peptide, sleep peptides, etc.; thrombolytic agents; atrial natriuretic peptide; relaxin; glial fibrillary acidic protein;These include those that code for the expression of follicle-stimulating hormone (FSH); human alpha-1 antitrypsin; leukemia inhibitory factor (LIF); transforming growth factor (TGF); tissue factor, luteinizing hormone; macrophage-activating factor; tumor necrosis factor (TNF); neutrophil chemotactic factor (NCF); nerve growth factor; tissue inhibitor of metalloproteinases; vasoactive intestinal peptide; angiogenin; angiotropin; fibrin; hirudin; and IL-1 receptor antagonist. Some other examples of proteins of interest include ciliary neurotrophic factor (CNTF); neurotrophin 3 and 4 / 5 (NT-3 and 4 / 5); glial cell line-derived neurotrophic factor (GDNF); aromatic amino acid decarboxylase (AADC); hemophilia-associated coagulation proteins, such as factor VIII, factor IX, factor X; dystrophin or mini-dystrophin; lysosomal acid lipase; phenylalanine hydroxylase (PAH); glycogen storage disorder-associated enzymes, such as glucose-6-phosphatase, acid maltase, glycogen debranching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase (e.g., PHKA2), glucose transporters (e.g., GLUT2), aldolase A, β-enolase, and glycogen synthase; lysosomal enzymes (e.g., β-N-acetylhexosaminidase A); and any variants thereof.

[0051] The transgene can also encode an antibody, e.g., an immune checkpoint inhibitory antibody, to, for example, PD-L1, PD-1, CTLA-4 (cytotoxic T-lymphocyte-associated protein-4; CD152); LAG-3 (lymphocyte activation gene 3; CD223); TIM-3 (T-cell immunoglobulin and mucin domains 3; HAVCR2); TIGIT (T-cell immunoreceptor with Ig and ITIM domains); B7-H3 (CD276); VSIR (V-set immunoregulatory receptor commonly known as Vista, B7H5, C10orf54); BTLA30 (B- and T-lymphocyte attenuator, CD272); GARP (glycoprotein A repeat; dominant); PVRIG (PVR-related immunoglobulin domain containing); or VTCN1 (V-set domain-containing T-cell activation inhibitor 1, commonly known as B7-H4).

[0052] Other transgenes can include small or inhibitory nucleic acids that alter / reduce expression of target genes, such as siRNAs, shRNAs, miRNAs, antisense oligos, suppressor tRNAs (Wang et al., Nature volume 604, pages 343-348 (2022)) or long non-coding RNAs that alter gene expression (see, for example, WO2012087983 pamphlet and US20140142160), or CRISPR Cas9 / cas12a and guide RNAs. Genome editing reagents (e.g., CRISPR Cas nucleases, base editors, or prime editors, and optionally associated guide RNAs) can also be delivered. For example, various CRISPR systems can be engineered to cleave optimal target sequences. See, e.g., Ledford, Nature. 2021 Sep 10. doi:10.1038 / d41586-021-02461-2. Epub ahead of print; Ramirez-Phillips, AAPS J. 2021 Jun 2;23(4):80; Liu et al., Mol Cell. 2022 Jan 20;82(2):333-347. In some cases, a construct encoding a CRISPR enzyme and a gRNA targeting a particular region are incorporated into the AAV vector as part of the transgene.

[0053] The virus may also include one or more sequences that facilitate the expression of the transgene, such as one or more promoter sequences; enhancer sequences, such as 5' untranslated region (UTR) or 3' UTR; polyadenylation site; and / or insulator sequences. In some embodiments, the promoter is a vascular endothelial cell specific promoter, such as a VE-cadherin promoter, Fms-like tyrosine kinase-1 (FLT-1), intercellular adhesion molecule-2 (ICAM-2), von Willebrand factor (vWF) promoter, TIE2 promoter, or a synthetic EC specific promoter (see, for example, Dai et al., J Virol. 2004 Jun; 78(12): 6209-6221). In some embodiments, the promoter is a pan-cell type promoter, e.g., a "ubiquitous" promoter that drives expression in most cell types, e.g., the cytomegalovirus (CMV) promoter (optionally with a CMV enhancer), chicken beta actin promoter, Rous sarcoma virus (RSV) LTR promoter (optionally with a RSV enhancer), SV40 promoter, dihydrofolate reductase promoter, phosphoglycerol kinase promoter, phosphoglycerol kinase (PGK) promoter, EF1 alpha promoter, ubiquitin C (UBC), B-glucuronidase (GUSB), and CMV immediate / early gene enhancer / CBA promoter, or a steroid promoter or metallothionein promoter. Woodchuck hepatitis virus post-transcriptional response element (WPRE) can also be used.

[0054] In some embodiments, the AAV also has one or more additional mutations that increase delivery to a target tissue, e.g., the CNS, or decrease off-tissue targeting, e.g., liver delivery when CNS, heart, muscle delivery is intended (e.g., as described in Pulicherla et al. (2011) Mol Ther 19:1070-1078); or decrease the addition of other targeting peptides, e.g., as described in Chen et al. (2008) Nat Med 15:1215-1218 or Xu et al., (2005) Virology 341:203-214, or U.S. Pat. Nos. 9,102,949; 9,585,971; and U.S. Patent Publication No. 20170166926. See also Gray and Samulski (2011) "Vector design and considerations for CNS applications," in Gene Vector Design and Application to Treat Nervous System Disorders ed. Glorioso J., editor. (Washington, DC: Society for Neuroscience) 1-9, available at sfn.org / ~ / media / SfN / Documents / Short%20Courses / 2011%20Short%20Course%20I / 2011_SC1_Gray.ashx.

[0055] Targeting peptides as tags / fusions The targeting peptides described herein can also be used to increase targeting of other (heterologous) molecules to endothelial cells in the CNS vasculature, for example by conjugation to a molecule or by expression as part of a fusion protein, for example with an antibody or other large biological molecule. These can include, in addition to therapeutic agents or reporters, genome editing proteins or complexes (e.g., CRISPR RNPs, including TALEs, ZFNs, base editors, and gene editing proteins, such as Cas9 or Cas12a, fused to the peptides described herein (e.g., at the N-terminus, C-terminus, or internally) and guide RNA). The fusions / complexes do not include any other sequences derived from Ku70, for example, heterologous non-Ku70 sequences, not occurring in nature.

[0056] How to use The methods and compositions described herein can be used to deliver any composition, e.g., a transgene or sequence of interest, to tissues, e.g., the central nervous system (brain), e.g., the vasculature, such as endothelial cells and pericytes, and smooth muscle cells of the vasculature. In some embodiments, the methods include delivery to specific brain regions, e.g., the cortex, cerebellum, hippocampus, substantia nigra, amygdala.

[0057] In some embodiments, methods and compositions, e.g., AAV, are used to deliver nucleic acid sequences to a subject suffering from a disease, e.g., a CNS disease; see, e.g., U.S. Pat. No. 9,102,949; U.S. Pat. No. 9,585,971; and U.S. Patent Publication No. 20170166926. In some embodiments, the subject has Parkinson's disease and the vector is used to deliver neurturin; brain-derived neurotrophic factor (BDNF); brain dopamine neurotrophic factor (CDNF); mesencephalic astrocyte-derived neurotrophic factor (MANF); vascular endothelial growth factor (VEGF); glial cell line-derived neurotrophic factor (GDNF), or aromatic L-amino acid decarboxylase (AADC) (see, e.g., Axelsen and Woldbye, J Parkinsons Dis. 2018; 8(2): 195-215; Qin et al., Med Sci Monit. 2022 Mar 16; 28: e935026; Elabi et al., Sci Rep. 2021 Jan 13; 11(1): 1120; Yu et al., Front Neurosci. 2020 Apr 29; 14: 334). In some embodiments, the subject has Alzheimer's disease and the vector is used to deliver a Tau antibody or an amyloid precursor protein (APP) antibody (e.g., Yang et al, Nature. 2022 Mar;603(7903):885-892; Bohannon et al., Cells. 2021 Apr 14;10(4):890; Kimbrough et al., Brain. 2015 Dec;138(Pt 12):3716-33; Sagare et al., Nat Commun. 2013;4:2932; Fisher et al., Brain Pathol. 2022 Mar 14;e13061; Zhang et al., Natl Sci Rev. 2019 Nov;6(6):1223-1238; Agyare et al., Mol Pharm.See 2013 May 6;10(5):1557-65). In some embodiments, the subject is diagnosed with a genetic disease affecting the vasculature, e.g., type IV collagen A1 or A2 (COL4A1 / A2) (Vahedi and Alamowitch, Curr Opin Neurol. 2011 Feb;24(1):63-8; Mao et al. Dis Model Mech. 2017 Apr 1;10(4):475-485); ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1) (Ferreira et al., Genet Med. 2021 Feb;23(2):396-407; Maulding et al., Bone. 2021 Jan;142:115656); ATP-binding cassette subfamily C member 6 (ABCC6) (Shimada et al., Int J Mol Sci. 2021 Apr 27;22(9):4555), 3 prime repair exonuclease 1 (TREX1) (Hoogeveen et al., AJNR Am J Neuroradiol. 2021 Sep;42(9):1604-1609;Rice et al., J Clin Immunol. 2015 Apr;35(3):235-43); Forkhead box C1 (FOXC1) (Prasitsak et al., Dev Dyn. 2015 May;244(5):703-11;French et al., J Clin Invest. 2014 Nov;124(11):4877-81); Paired-like homeodomain 2 (PITX2) (French et al., J Clin Invest. 2014 Nov;124(11):4877-81);SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1) (Li et al., Biomed Res Int. 2015;2015:739586);Endoglin (ENG) (Lozano Sanchez et al., J Pers Med. 2022 Mar 25;12(4):528);SMAD family member 4 (SMAD4) (Nie et al., Immun Inflamm Dis. 2021 Dec;9(4):1306-1320);activin A receptor-like type 1 (ACVRL1)(Walsh et al., J Med Case Rep. 2022 Mar 1;16(1):99);RAS p21 protein activator 1 (RASA1)(Chen et al., JCI Insight. 2022 Feb 22;7(4):e156928);or notch receptor 3 (NOTCH3)(Joutel et al., J Clin Invest. 2000 Mar;105(5):597-605;Opherk et al., Hum Mol Genet. 2009 Aug 1;18(15):2761-7);HtrA serine peptidase 1 (HTRA1)(Shiga et al., Hum Mol Genet. 2011 May 1;20(9):1800-10); zinc finger CCHC type containing 14 (ZCCHC14) (Traylor et al., Ann Neurol. 2017 Mar;81(3):383-394), apolipoprotein E epsilon 4 (APOE ε4) (Khera et al., Circulation. 2019 Mar 26;139(13):1593-1602), and others. The vectors described herein can be used to deliver wild-type transgenes or gene editing reagents (e.g., CRISPR Cas nucleases, base editors, or prime editors, and associated guide RNAs) that correct the mutations in the genome of the targeted cells.

[0058] The therapeutic agent can be delivered as a nucleic acid, e.g., by a viral vector, where the nucleic acid encodes a therapeutic protein or other nucleic acid, e.g., antisense oligos, siRNA, shRNA, etc., or the therapeutic agent can be delivered as a fusion protein / complex with a targeting peptide as described herein.

[0059] Pharmaceutical Compositions and Methods of Administration The methods described herein involve the use of pharmaceutical compositions that include a targeting peptide as an active ingredient.

[0060] Pharmaceutical compositions typically include a pharma- ceutically acceptable carrier. As used herein, the phrase "pharma- ceutically acceptable carrier" includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.

[0061] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intraarterial, subcutaneous, intraperitoneal, intramuscular injection or infusion. Thus, delivery can be systemic or local.

[0062] Methods for formulating suitable pharmaceutical compositions are known in the art, see, for example, Remington: The Science and Practice of Pharmacy, 21st ed., 2005; Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY). For example, the solution or suspension used for parenteral administration can contain the following components: sterile diluent, such as water for injection, saline, non-volatile oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvent; antibacterial agent, such as benzyl alcohol or methyl paraben; antioxidant, such as ascorbic acid or sodium bisulfite; chelating agent, such as ethylenediaminetetraacetic acid; buffer, such as acetate, citrate, or phosphate, and agent for adjusting tonicity, such as sodium chloride or dextrose. pH can be adjusted by acid or base, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0063] Pharmaceutical compositions suitable for injectable use may include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the compositions must be sterile and fluid to the extent that easy syringability exists. They should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride, in the composition. Prolonged absorption of the injectable composition can be brought about by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0064] Sterile injection can be prepared by incorporating the required amount of active compound into a suitable solvent with one or combination of the above-listed components, and then optionally sterilizing by filtration.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other components required from above-listed.For the preparation of sterile powder for sterile injection, the preferred preparation method is vacuum drying and freeze-drying, which produce a powder of active ingredient and any additional desired ingredients from its solution that has been previously sterilized and filtered.

[0065] In one embodiment, the therapeutic compound is prepared with a carrier that protects the therapeutic compound from rapid elimination from the body, such as, for example, a controlled release formulation, including implants and microencapsulated delivery systems. For example, biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Such formulations can be prepared using standard techniques or can be obtained commercially, for example, from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to selected cells with monoclonal antibodies against cellular antigens) can also be used as pharma-ceutically acceptable carriers. These can be prepared by methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0066] The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration. EXAMPLES

[0067] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0068] material and method The following materials and methods were used in the examples that follow.

[0069] Animals: All animal studies were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care and conformed to the guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The inventors used adult (8-10 weeks old) Ai9(B6.Cg-Gt(ROSA)26Sor tm9(CAG-tdTomato)Hze C57BL / J (strain #007909) and C57BL / 6J (strain #000664) mice were used, all of which were obtained from The Jackson Laboratory, Bar Harbor, ME.

[0070] Construction of AAV-PR capsid. To generate a rep / cap plasmid encoding an AAV9 capsid expressing the AAV-PR peptide (PRPPSTH) for the generation of a vector encoding a transgene of interest (e.g., GFP or Cre), we digested the AAV9 rep / cap plasmid (pAR9) with BsiWI and BaeI to remove a fragment flanking the VP3 amino acid 588 site for peptide sequence insertion. Next, we ordered a 997 bp dsDNA fragment from Integrated DNA Technologies (IDT, Coralville, IA), which contains overlapping Gibson homology arms with BsiWI / BaeI-cleaved AAV9 as well as a 21-mer nucleotide sequence encoding the peptide of interest in frame after amino acid 588 of VP3. Finally, we performed Gibson assembly using Gibson Assembly® Master Mix (NEB, Ipswich, MA) to ligate the peptide-encoding insert into the AAV9 rep / cap plasmid. We then transformed 2 μl of NEB 5-alpha competent E. coli (NEB) by Gibson assembly and plated the transformed cells on LB-Amp agar plates. To confirm that the correct sequence was inserted, DNA isolated from selected colonies was sent for Sanger sequencing at the MGH Center for Computational and Integrative Biology DNA Core using primers flanking the peptide coding region.

[0071] AAV Vector Production, Purification, and Titering. For transgene expression studies with AAV-PR vectors, we used the following constructs: (1) pAAV-CBA-NLS-Cres, an AAV expression plasmid, a gift from Miguel Sena-Esteves (UMass Medical Center). This plasmid contains AAV inverted terminal repeats (ITRs) flanking a CBA expression cassette consisting of a hybrid CMV-IE enhancer / chicken β-actin (CBA) promoter, an SV40 nuclear localization signal (NLS), a Cre recombinase cDNA, and a bovine growth hormone (BGH) polyA signal sequence. (2) pAAV-sc-CBA-GFP. This construct drives expression of green fluorescent protein (GFP) under a hybrid CMV immediate early / chicken β-actin (CBA) promoter, which was kindly provided by Dr. Miguel Sena-Esteves (UMass Medical Center). AAV-sc-CBA-GFP is a self-complementary (sc) genome.

[0072] AAV production was performed as previously described 6Briefly, 293T cells were transfected (calcium phosphate method) with (1) AAV-PR rep / cap plasmid, (2) adenovirus helper plasmid pAdΔF6, and (3) ITR-flanked AAV transgene expression plasmid. Cell lysates were harvested 68–72 h posttransfection and purified by ultracentrifugation on an iodixanol density gradient. Iodixanol was removed and buffer exchanged into phosphate-buffered saline (PBS) using Zeba desalting columns, 7 kDa molecular weight cutoff (MWCO; Thermo). Vector was concentrated using 2 ml Amicon Ultra 100 kDa MWCO ultrafiltration devices. Vector titers in VG / ml were measured by Taqman qPCR on an ABI Fast 7500 real-time PCR system (Applied Biosystems) using a probe and primers against the BGH polyA sequence and interpolated from a standard curve generated by the AAV plasmids. The vector was pipetted into single-use aliquots and stored at -80°C until use.

[0073] Injection of vectors in mice. In experiments involving AAV-PR-mediated Cre recombination and tdTomato expression in Ai9 mice, we administered the AAV-PR-CBA-Cre vector systemically to adult (both male and female) Ai9 mice by injection through the lateral tail vein. Three to five weeks after injection, the animals were killed and the brains and other organs were processed for cryosectioning, immunofluorescent staining, and imaged.

[0074] In experiments using AAV-PR as a conventional transgene expression vector, male C57BL / 6J mice were systemically administered AAV-PR-sc-CBA-GFP via injection through the lateral tail vein. Mice were killed at two time points, 5 and 28 days after injection. Brains were processed for cryosectioning, then immunofluorescently stained and imaged.

[0075] Immunofluorescence staining and microscopy. Sections were dried at room temperature for 20 minutes and then incubated with PFA for 15 minutes. Each slide was then rinsed three times with PBS for 5 minutes each. This was followed by a 10 minute incubation with ice-cold methanol and then rinsed with additional PBS. Sections were blocked in 5% goat serum and 5% BSA in PBS for 45 minutes. They were then incubated overnight at 4°C with a mixture of primary antibodies. Primary antibodies were used as co-stains, either anti-GFP (G10362; 1:500) and CD31 (BD550274; 1:50) or anti-GFP (ab1218; 1:500) and PDGFr-B (LS C117692; 1:500), based on the host species. The next day, slides were rinsed once more with PBS and then incubated with AlexaFlor488 (A32723 or A32731; 1:500) and Alexaflor555 (A-21428 or A-21434; 1:500) for 90 minutes. Slides were processed with ProLong Antifade Mountant with DAPI (P36931). Images were taken using a Zeiss LSM 800 Airyscan confocal microscope located at the Microscopy Core of the Program in Membrane Biology (PMB) at MGH. Wide-field fluorescent images were taken at 10x, 20x, 40x, and 63x magnifications with standard exposures for all slides. Images were analyzed using ImageJ software (NIH). The Allen Brain Atlas was referenced to ensure consistency in image positioning between different sections and mouse samples. All image processing and quantification was performed using NIH ImageJ software.

[0076] [Example 1] Selection and identification of AAV-PR We performed in vivo selection with an AAV peptide display library to isolate AAV capsids capable of transducing the brain following systemic administration by injection. The method is generally described in Figures 8A-C; we used our previously described iTransduce library, which combines a 7-mer peptide display library with a Cre recombinase cassette to couple transgene expression in mice expressing a Cre-sensitive fluorescent reporter (Ai9 mice) with rescued peptide encoding sequences. 7,8 (AAV-CBA-Cre-p41-Cap) was used. The inventors initially set the goal of isolating capsids that could transduce bone marrow-derived cells in the brain. The inventors performed two rounds of selection with the capsid library. In the first round of selection, 1.27 × 10 11 vector genome (vg) was injected into one adult male and one female Ai9 mouse. Three weeks after injection, DNA was extracted from brain tissue and the CAP region was amplified, which was then recloned into the AAV plasmid backbone and repackaged for a second round of selection ("brain-enriched capsid library"). In the second round, we used a Cre cassette to isolate transduction-competent capsids. Two female Ai9 mice and one male mouse were injected with 1.91 × 10 6 mAbs, respectively, in the tail vein. 10 or 7.64×10 11 After 3 weeks, mice were killed and brain cells were isolated using anti-CD11b / magnetic beads (Magnetic Activated Cell Sorting, MACS, Miltenyi). The cells were then incubated with tdTomato + and tdTomato - As described in the first run, DNA was isolated from each cell pellet and PCR amplification of the peptide insert coding region was submitted to NGS. From the NGS data, the inventors confirmed that tdTomato +Capsid candidates with high read frequency in fractions were selected. DNA was isolated from each cell pellet using Pico Pure™ DNA Extraction Kit (Thermo Fisher Scientific), and the inventors PCR-amplified the cap region containing the insert to identify peptide profiles by next-generation sequencing (NGS). From the NGS results, four capsid clones, PRPPSTH (SEQ ID NO: 1); MAEPGAR (SEQ ID NO: 2); SQDPSTL (SEQ ID NO: 3); or MLYADNT (SEQ ID NO: 4), were selected for evaluation.

[0077] [Example 2] An engineered peptide representing the AAV9 capsid, AAV-PR, mediates a vasculature-selective transduction phenotype in the brain following intravenous delivery We then tested these novel capsids for transduction of adult mouse brains following systemic intravenous delivery. We packaged a single-stranded AAV-CBA-Cre genome into each candidate capsid and transduced these vectors (1×10 12 vg / mouse) was injected into the tail vein of adult Ai9 mice carrying a Cre-sensitive CAG-floxed-STOP-tdTomato reporter in all cells. All cells successfully transduced by the AAV vector and expressing Cre should consequently produce tdTomato expression. Mice were sacrificed 3 weeks after injection, and brains were thinly sliced ​​to detect tdTomato by immunofluorescence staining. For one capsid expressing the peptide PRPPSTH, named AAV-PR, we observed distinct vasculature immunostaining of tdTomato expression throughout the brain (Figure 1A-D). This profile was in striking contrast to the tropism of the parental AAV9-CBA-Cre in adult Ai9 mice, which mediated transduction of most astrocytes and neurons. 1In the case of the other peptides (MAEPGAR (SEQ ID NO: 2); SQDPSTL (SEQ ID NO: 3); or MLYADNT (SEQ ID NO: 4)), the displaying capsid transduced cells within the vasculature as well as cells with neuronal morphology (Figure 1E-H).

[0078] [Example 3] Time-dependent decrease in brain vasculature transduced cells by IV-injected AAV-PR encoding GFP To assess the "full expression" profile of this capsid, we have begun experiments to evaluate AAV-PR in the Cre / lox system. As has been recently demonstrated, traditional fluorescent reporters underestimate AAV-mediated transduction events (e.g., transient expression). 2 The use of the Cre / Lox system allows for the capture of all of these transduction events, which may be useful for different gene delivery applications, such as genome editing. AAV-PR-mediated Cre expression allows for permanent genetic modification of the mouse genome, which results in stable expression of tdTomato. To test the transduction profile of AAV-PR capsids in a more conventional "gene-add" delivery format that relies on the stability of the extrachromosomal AAV genome for long-term expression, we packaged a self-complementary (sc) AAV-CBA-GFP genome into AAV-PR capsids. We transfected 3.5×10 10 vg / mouse (approximately 1.4 × 10 12 vg / kg) and sacrificed mice 5 (n=2) and 28 (n=3) days after injection. Brains were thinly sliced ​​and immunostained for GFP expression. Brains harvested on day 5 revealed transduction predominantly in the vasculature, similar to the AAV-PR-CBA-Cre / Ai9 mouse experiments. This transduction pattern was observed in several brain regions (Figure 2A). Interestingly, the vasculature transduction phenotype was maintained at day 28, while expression levels were considerably reduced (Figure 2A). Quantification of GFP levels in the three brain regions revealed a striking reduction of approximately 60% between the day 5 and day 28 time points (Figure 2B).

[0079] To evaluate the stability of vector genomes in the brain, heart, and liver, we isolated vector and host genomic DNA from all tissues on days 5 and 28 after systemic administration by injection into adult male C57BL / 6 mice. In the brain, vector genomes decreased by as little as 30% between days 5 and 28 (Figure 3), but this was not statistically significant. In the same time interval, liver decreased by 50% and heart increased by 20% (Figure 3), but both did not reach statistical significance. Liver genomes were more than 100-fold higher in liver than in brain, suggesting that AAV-PR capsids are not detargeted in the liver. These data suggest that AAV genomes delivered by AAV-PR are not significantly decreased in this time interval, which may indicate the transient activity of the hybrid CBA promoter in endothelial cells.

[0080] [Example 4] Characterization of brain and peripheral cell types transduced by AAV-PR-CBA-GFP To confirm the vasculature phenotype of transgene expression mediated by AAV-PR-CBA-GFP, brain sections of vector-injected mice were immunostained with various cell markers. First, we evaluated the colocalization of GFP expression with the endothelial marker CD31 at days 5 and 28 after vector injection. At both time points, we readily detected CD31 / GFP colocalization as expected (Figure 4). Next, we evaluated whether AAV-PR could transduce pericytes. Interestingly, at day 5, we detected that up to 50% of pericytes were transduced by AAV-PR (detected by PDGFR-β staining) (Figure 5). Very sparse detection of GFP-positive astrocytes in the somatosensory cortex and neurons in the hippocampus was detected (Figure 6). Because systemically administered AAV vectors often readily transduce the liver, we evaluated this organ for GFP expression at day 28. AAV-PR robustly transduced hepatocytes, as assessed morphologically (Figure 7).

[0081] References

[0082] [Table 2]

[0083] Other embodiments Although the present invention has been described in conjunction with a detailed description thereof, it should be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An AAV capsid protein comprising an amino acid sequence comprising at least four, five, or six consecutive amino acids from the sequence PRPPSTH (SEQ ID NO:1); MAEPGAR (SEQ ID NO:2); SQDPSTL (SEQ ID NO:3); or MLYADNT (SEQ ID NO:4).

2. The AAV capsid protein of claim 1, wherein the AAV is AAV9.

3. The AAV capsid protein of claim 1 , comprising AAV9 VP1.

4. 4. The AAV capsid protein of claim 3, wherein a targeting sequence is inserted at positions corresponding to amino acids 588 and 589 of SEQ ID NO:

14.

5. A nucleic acid encoding the AAV capsid protein of claim 1.

6. An AAV comprising the capsid protein of claim 1.

7. The AAV of claim 6, further comprising a transgene.

8. The AAV of claim 7, wherein the transgene is a therapeutic transgene.

9. A targeting sequence comprising at least four consecutive amino acids from the sequence PRPPSTH (SEQ ID NO:1); MAEPGAR (SEQ ID NO:2); SQDPSTL (SEQ ID NO:3); or MLYADNT (SEQ ID NO:4), or [D / P]PST (SEQ ID NO:9).

10. A fusion protein comprising the targeting sequence of claim 9 and a heterologous sequence.

11. 10. An AAV capsid protein comprising the targeting sequence of claim 9.

12. The AAV capsid protein of claim 11, comprising AAV9 VP1.

13. The AAV capsid protein of claim 11, wherein the targeting sequence is inserted at positions corresponding to amino acids 588 and 589 of SEQ ID NO:

14.

14. A nucleic acid encoding a targeting sequence according to claim 9, a fusion protein according to claim 10, or an AAV capsid protein according to claim 11.

15. An AAV comprising the capsid protein of claim 11.

16. The AAV of claim 15, further comprising a transgene.

17. The AAV of claim 16, wherein the transgene is a therapeutic transgene.

18. A composition for delivering a transgene to a cell, comprising an AAV according to claim 6 or 15.

19. The composition of claim 18 , wherein the cell is a vascular endothelial or smooth muscle cell, or a pericyte.

20. The composition of claim 18 , wherein the cell is present in a living subject.

21. 21. The composition of claim 20, wherein the subject is a mammalian subject.

22. 20. The composition of claim 18, wherein the cell is present in a tissue selected from the brain, spinal cord, dorsal root ganglion, heart, liver, or smooth muscle, and combinations thereof.

23. 21. The composition of claim 20, wherein the subject suffers from a disease affecting the vasculature of the central nervous system.

24. The disease is characterized in that the disease is characterized by the following: type IV collagen A1 or A2 (COL4A1 / A2); ectonucleotide pyrophosphatase / phosphodiesterase 1 (ENPP1); ATP-binding cassette subfamily C member 6 (ABCC6); 3 prime repair exonuclease 1 (TREX1); forkhead box C1 (FOXC1); paired-like homeodomain 2 (PITX2); SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1); endoglin (ENG); SMAD family member 4 (SMAD4); activin A receptor-like type 1 (ACVRL1); RAS The composition of claim 23, wherein the disease is associated with a mutation in p21 protein activator 1 (RASA1); Notch receptor 3 (NOTCH3); HtrA serine peptidase 1 (HTRA1); zinc finger CCHC type-containing 14 (ZCCHC14), or apolipoprotein E epsilon 4 (APOE ε4).

25. The composition of claim 20 , wherein the subject suffers from a neurodegenerative disease.

26. 26. The composition of claim 25, wherein the neurodegenerative disease is Parkinson's disease or Alzheimer's disease.

27. The composition of claim 18, wherein the cells are present in the brain of a subject and the AAV is administered by parenteral delivery.

28. 28. The composition of claim 27, wherein the parenteral delivery is by intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular delivery.

29. The composition of claim 18, wherein the cells are present in the brain of a subject and the AAV is administered by intravenous delivery.

30. The transgene is selected from the group consisting of neurturin, brain-derived neurotrophic factor (BDNF), brain dopamine neurotrophic factor (CDNF), mesencephalic astrocyte-derived neurotrophic factor (MANF), vascular endothelial growth factor (VEGF), glial cell line derived neurotrophic factor (GDNF), aromatic L-amino acid decarboxylase (AADC), Tau antibody, amyloid precursor protein (APP) antibody, type IV collagen A1 or A2 (COL4A1 / A2), ectonucleotide pyrophosphatase / phosphodiesterase 1 (ECTO-1), and phosphodiesterase 2 (PAGE). (ENPP1); ATP-binding cassette subfamily C member 6 (ABCC6); 3 prime repair exonuclease 1 (TREX1); Forkhead box C1 (FOXC1); Paired-like homeodomain 2 (PITX2); SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1); Endoglin (ENG); SMAD family member 4 (SMAD4); Activin A receptor-like type 1 (ACVRL1); RAS 17. The AAV of claim 7 or 16, encoding p21 protein activator 1 (RASA1); Notch receptor 3 (NOTCH3); HtrA serine peptidase 1 (HTRA1); zinc finger CCHC type-containing 14 (ZCCHC14), or apolipoprotein E epsilon 4 (APOE ε4).

31. The transgene is selected from the group consisting of neurturin, brain-derived neurotrophic factor (BDNF), brain dopamine neurotrophic factor (CDNF), mesencephalic astrocyte-derived neurotrophic factor (MANF), vascular endothelial growth factor (VEGF), glial cell line derived neurotrophic factor (GDNF), aromatic L-amino acid decarboxylase (AADC), Tau antibody, amyloid precursor protein (APP) antibody, type IV collagen A1 or A2 (COL4A1 / A2), ectonucleotide pyrophosphatase / phosphodiesterase 1 (ECTO-1), and phosphodiesterase 2 (PAGE). (ENPP1); ATP-binding cassette subfamily C member 6 (ABCC6); 3 prime repair exonuclease 1 (TREX1); Forkhead box C1 (FOXC1); Paired-like homeodomain 2 (PITX2); SAM and HD domain-containing deoxynucleoside triphosphate triphosphohydrolase 1 (SAMHD1); Endoglin (ENG); SMAD family member 4 (SMAD4); Activin A receptor-like type 1 (ACVRL1); RAS 19. The composition of claim 18, encoding p21 protein activator 1 (RASA1); Notch receptor 3 (NOTCH3); HtrA serine peptidase 1 (HTRA1); zinc finger CCHC type containing 14 (ZCCHC14), or apolipoprotein E epsilon 4 (APOE ε4).

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