AAV capsid modifications that reduce immunogenicity

Abstract

Applicants address the challenge of antibodies that recognize AAVs and can prevent transduction into targeted tissues and cells. Binding antibodies can also increase the risk of complement activation and thrombotic microangiopathy (TMA), therefore many subjects with pre-existing immunity are excluded from receiving AAV related therapies. Applicants modify capsids through the introduction of surface amino acid substitutions designed to reduce their recognition by pre-existing antibodies in humans. Applicants describe novel antibody evading (AE) capsids that maintain transduction activity in the presence of human sera that neutralize the natural serotypes, for example, AAV9 and AAV5.

Description

AAV CAPSID MODIFICATIONS THAT REDUCE IMMUNOGENICITYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 720,703, filed November 14, 2024. The entire contents of the above-identified application are hereby fully incorporated herein by reference.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under Grant No. NS111689 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] Reference is made to the electronic sequence listing ("BROD-6045WP_ST26.xml"; Size is 1,608,254 bytes, created on November 14, 2025), which is herein incorporated by reference in its entirety.TECHNICAL FIELD

[0004] The subject matter disclosed herein relates generally to AAV capsids that exhibit improved evasion of antibodies arising from prior AAV exposure while maintaining desirable functional properties. In embodiments described herein, AAV capsid polypeptides comprise mutations that effectively reduce an antibody’s binding affinity for the capsid, wherein each mutation comprises an amino acid substitution. Further examples relate to a vector system having one or more vectors for the expression of the modified AAV capsid proteins in a cell, the packaging of a recombinant nucleic acid cargo into the modified AAV capsids, and a method of delivering the cargo by either administering the engineered AAV virions directly to a host organism in vivo.BACKGROUND

[0005] Adeno-associated virus (AAV) vectors are widely used to deliver human gene therapies. Systemically administered AAVs can achieve broad, uniform delivery throughout organs of interest, which is essential for the treatment of numerous indications including, but notlimited to, Duchenne muscular dystrophy (DMD), hemophilia A and B, spinal muscular atrophy (SMA), Huntington’s disease, Friedreich’s ataxia, and neuronopathic Gaucher disease. AAVs have been successfully engineered via capsid surface modifications to target organs such as the central nervous system (CNS), muscle, lung, and liver following systemic delivery1 l0. However, systemic administration exposes AAVs to circulating antibodies, and the population of patients that can benefit from AAV-based gene therapies is significantly limited by pre-existing neutralizing antibodies (NAbs) to AAV capsids (e.g., 40% of sampled individuals possessed detectable levels of anti-AAV9 NAbs)"H. Recent clinical trials have shown that a patient’s immune response can significantly reduce the effectiveness of AAV-mediated gene therapy and lead to adverse events such as thrombotic microangiopathy15 16.

[0006] Two complementary strategies are currently being used to address the challenge posed by pre-existing immunity: pharmacological interventions designed to modulate the immune response (e.g., immunoglobulin G (IgG)-degrading enzymes to reduce the concentration of circulating NAbs before AAV administration and Cl esterase, C3, and C5 inhibitors to prevent complement system activation)16’17and AAV capsid engineering to evade immune clearance of AAVs by NAbs. For example, AAV capsid engineering groups have achieved limited success by shuffling naturally occurring capsids18’19, altering antibody epitopes via saturation mutagenesis20’21-22, or by using modified alternative parvoviruses with reduced pre-existing immunity in humans. Each approach is limited regarding the sequence space explored by the conservation of NAb epitopes among naturally occurring serotypes and the inability to perform extensive mutagenesis across the capsid without adversely impacting virion assembly and packaging. Therefore, modified AAV capsids are needed to evade NAbs and are compatible with surface loop modifications that confer enhanced in vivo tropisms.SUMMARY

[0007] In one embodiment, an engineered AAV capsid polypeptide comprises one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype.

[0008] In an embodiment, an engineered AAV capsid polypeptide, wherein the amino acid substitutions include N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q. In an embodiment, an engineered AAV capsid polypeptide, wherein the amino acid substitutions further include from 1 to 12 amino acid substitutions chosen from D327N, N328K, N329D, K332Q, K462E / Q, R533Q, D657N, D657N, N663D, K664Q, N665N, and N668K. In an embodiment, an engineered AAV capsid polypeptide, wherein the amino acid substitutions further include D327N, K332Q, and D657N. In an embodiment, an engineered AAV capsid polypeptide, wherein the additional amino acid substitutions include N328K and N329D. In an embodiment, an engineered AAV capsid polypeptide, wherein the additional amino acid substitutions include K462E / Q and N668K. In an embodiment, an engineered AAV capsid polypeptide, wherein the additional amino acid substitution includes N668K. In an embodiment, an engineered AAV capsid polypeptide, wherein the additional amino acid substitution includes N663D.

[0009] In an embodiment, an engineered AAV capsid polypeptide, wherein the amino acid substitutions include D327N, N328K, N329D, K332Q, N452D, E500D, A502S, P504T, A510K, R550Q, D551N, D554N, K557Q, D657N, N663D, and D665N. In an embodiment, an engineered AAV capsid polypeptide, wherein the amino acid substitutions include D327N, N328K, N329D, K332Q, N452D, E500D, A502S, P504T, A510K, R550Q, D551N, D554N, K557Q and D657N. In an embodiment, an engineered AAV capsid polypeptide, wherein the amino acid substitutions include D327N, K332Q, N452D, E500D, A502S, P504T, A510K, R55OQ, D551N, D554N, K557Q, D657N, N663D, and N668K. In an embodiment, an engineered AAV capsid polypeptide, wherein the amino acid substitutions include N452K, K462E, E500D, A502S, P504T, A510K, R550Q, D551N, D554N, K557Q, D657N, and N663D. In an embodiment, an engineered AAV capsid polypeptide, wherein the amino acid substitutions include N452K, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551K, D554N, K557Q, K664Q, and N668K. In an embodiment, an engineered AAV capsid polypeptide, wherein the amino acid substitutions include G455Q / T / K / N, D551N, N552D, K557E / Q, K664E / Q, D665N, andN668K.

[0010] In an embodiment, an engineered AAV capsid polypeptide, wherein amino acid substitutions further include from 1 to 5 amino acid substitutions selected from the group consisting of D554N, D556K / N, N663D, and N716D. In an embodiment, an engineered AAV capsid polypeptide, wherein an additional amino acid substitution includes D556K / N. In an embodiment, an engineered AAV capsid polypeptide, wherein an additional amino acidsubstitution includes D554N. In an embodiment, an engineered AAV capsid polypeptide, wherein the additional amino acid substitution includes N716D.

[0011] In an embodiment, an engineered AAV capsid polypeptide, wherein the amino acid substitutions include G455Q, D551N, N552D, D556K, K557E, K664E, D665N, N668K, and N716D. In an embodiment, an engineered AAV capsid polypeptide, wherein the amino acid substitutions include G455T, D551N, N552D, D556K, K557E, K664E, D665N, N668K, and N716D. In an embodiment, an engineered AAV capsid polypeptide, wherein the amino acid substitutions include G455K, D551N, N552D, D556K, K557E, K664E, D665N, N668K, and N716D. In an embodiment, an engineered AAV capsid polypeptide, wherein the amino acid substitutions include G455N, D551N, N552D, D554N, D556N, K557E, N663D, K664Q, D665N, and N668K. In an embodiment, the engineered AAV capsid polypeptide is selected from SEQ ID NO: 26-34 and 1641-1648

[0012] In an embodiment, an engineered AAV capsid polypeptide, as described herein, further includes a targeting moiety. In an embodiment, an engineered AAV capsid polypeptide, wherein the targeting moiety is an amino acid insertion or substitution in loop IV, loop VIII, or both of the AAV capsid polypeptide. In an embodiment, an engineered AAV capsid polypeptide, wherein the targeting moiety is a central nervous system (CNS) targeting moiety. In an embodiment, an engineered AAV capsid polypeptide, wherein the CNS targeting moiety binds a transferrin receptor (TfRl), a CD59, a CA4, an ALPL, a Ly6a, a Ly6c, or a Car4 protein. In an embodiment, an engineered AAV capsid polypeptide, wherein the targeting moiety is a TfRl binding targeting moiety. In an embodiment, the TfRl binding targeting moiety is selected from SEQ ID NO: 62-189 or 269-1606. In an embodiment, the engineered AAV capsid polypeptide comprises one of the amino acid sequences set forth in 7-21, 40-167, and 1649-1656. In an embodiment, an engineered AAV capsid polypeptide, wherein the targeting moiety is a CD59 targeting moiety. In an embodiment, an engineered AAV capsid polypeptide, wherein the targeting moiety is a CA4 targeting moiety. In an embodiment, the CA4 targeting moiety is selected from SEQ ID NO: 168-169.

[0013] In an embodiment, an engineered AAV capsid polypeptide, wherein the targeting moiety is a muscle-specific targeting moiety. In an embodiment, an engineered AAV capsid polypeptide, wherein the muscle-specific targeting moiety includes a RGD motif. In an embodiment, wherein the RGD motif is selected from SEQ ID NO: 170-191, 1685-1699, 1705,1708-1709. In an embodiment, the engineered AAV capsid polypeptide is selected from SEQ ID NO: 35-39 and 1657-1684. In an embodiment, the muscle-specific targeting moiety is selected from SEQ ID NO: 1700-1704 and 1706-1707.

[0014] In an embodiment, an engineered AAV capsid polypeptide, wherein the AAV capsid polypeptide is a capsid polypeptide of an AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV rh.74, or AAV rh.10.

[0015] In an embodiment, an engineered AAV particle including the engineered AAV capsid polypeptide as described herein and further including a recombinant AAV genome encoding a transgene. In an embodiment, an engineered AAV particle, wherein the transgene encodes a therapeutic polypeptide, an antibody or fragment thereof, a shRNA, miRNA, a CRISPR-Cas system, Transcription Activator-like Effector (TALE)- or Zinc Finger Protein (ZFP)-based transcriptional activator; repressor; or epigenomic silencer, mammalian transposase bMLT, an RNA encoding a partial gene fragment designed for trans-splicing into an endogenous RNA, one or more transfer RNAs, or a component thereof, or an OMEGA system or any component thereof. In an embodiment, an engineered AAV, wherein the transgene is operably linked to a regulatory sequence that promotes tissue-specific gene expression in a CNS or muscle.

[0016] In an embodiment, a pharmaceutical composition including the recombinant engineered AAV particle and an acceptable carrier as described herein.

[0017] In an embodiment, a method of delivering a polypeptide or polynucleotide to the CNS of a subject comprising administering an engineered AAV particle as described herein, wherein the AAV particle includes a CNS targeting moiety and wherein the engineered AAV particle exhibits increased evasion of AAV-neutralizing antibodies relative to a reference AAV particle. In an embodiment, the reference AAV particle is an AAV9 particle. In another embodiment, the reference AAV particle comprises a K449R substitution. In an embodiment, the reference AAV particle is SEQ ID NO: 2. In an embodiment, the CNS targeting moiety binds a TfRl receptor. In an embodiment, the engineered AAV particle is administered systemically. In an embodiment, the subject suffers from a CNS disease or disorder. In an embodiment, the subject has AAV-neutralizing antibodies.

[0018] In an embodiment, an AAV library including a population of engineered recombinant AAV particles wherein each member of the population of engineered recombinant AAV particlescomprises any one or more of the engineered adeno associated virus (AAV) capsid polypeptides of any of those described herein.

[0019] In an embodiment, a method of screening an AAV capsid library comprising recombinant AAV particles for recombinant AAV particles having reduced binding affinity for neutralizing antibodies comprising: (a) contacting the recombinant AAV capsid library with antibodies from the sample; (b) selecting one or more recombinant AAV capsids that have reduced binding affinity for the antibodies relative to a reference AAV capsid.

[0020] In an embodiment, the AAV capsid library comprises AAV capsids having one or more substitutions for increasing antibody evasion. In one embodiment, the AAV capsid library may comprise AAV capsids comprising the modifications disclosed herein. In an embodiment, the AAV capsid library may further comprises AAV capsids comprising one or more modifications for increasing antibody evasion and a targeting moiety insertion, wherein the method may further comprise (c) selecting recombinant AAV capsids with maintained or enhanced receptor-mediated binding or transduction of cells in culture or an organism relative to a reference AAV capsid. In another embodiment, the sample may be from a subject to be treated with a therapeutic delivered using a recombinant AAV capsid and, therefore, used to select for recombinant AAV capsids with an increased ability to evade neutralizing antibodies from the subject to be treated. The enables tailoring of specific recombinant AAVs with modifications that will enhance delivery in the subject to be treated and / or reduce the dosage needed to achieve a therapeutic effect.

[0021] In another aspect, embodiments disclosed herein are directed to a cultured host cell containing a recombinant nucleic acid molecule that encodes an AAV VP1 capsid protein of any of the engineered AAV capsids described herein.

[0022] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those with ordinary skill in the art upon considering the following detailed description of example embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0023] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0024] FIG. 1A-1B - BI-hTFRlv2 and BI-hTFR2v2 are capsids with enhanced CNS targeting and liver de-targeting properties compared to BI-hTFRl. Humanized TFRC KI mice were intravenously injected at 5E10 viral genomes per capsid per animal encoding a dual reporter CAG-NLS-mScarlet-P2A-Luciferase-pA and were assessed three weeks post-injection. Wide-field imaging of the nuclear-localized mScarlet fluorescent protein in whole brain (1A) and liver sections (IB) with matched exposures and settings within each organ.

[0025] FIG. 2A-2B - Generation and screening of antibody evasion (AE) AAV libraries designed for antibody evasion. 2A. Library target residues represented at the surface of an in silico predicted structural model of BI-hTFRlv2. The previously identified TfRl targeting sequence is shown in dark gray, and the library target residues are shown in black. 2B. Overview of the library selection process. (1) Selection of seed capsid variants; (2) Diversification of chosen surface-exposed amino acids; (3) Library selection in human brain endothelial cells (+ / - IVIG), in the brains of mice expressing human TFRC following intravenous administration and in binding assays with capture affinity resins; (4) Cap DNA / mRNA extraction; (5) Next generation sequencing (NGS); (6) Individual variant characterization.

[0026] FIG. 3 - Selected BI-hTFRlv2-AE and BI-hTFR2v2-AE variants exhibit high sequence divergence. Matrix of sequence divergence between naturally occurring and nominated antibody evading TfRl -binding capsids.

[0027] FIG. 4 - Selected BI-hTFRlv2-AE and BI-hTFR2v2-AE variants evade neutralizing antibodies in IVIG. Neutralization curves were measured for BI-hTFRlv2, BL hTFR2v2 (gray), BI-hTFRlv2-AE, and BI-hTFR2v2-AE variants (black) after incubation with various IVIG dilutions. The half-inhibitory IVIG dilutions for each variant are indicated on the plots, with those for parental capsid controls in gray and those for the other variants in black. Error bars indicate the standard deviation of technical replicates. Graph titles indicate different biological experiments.

[0028] FIG.5 - Screen of 60 human sera from individual donors with AAV9. Neutralizing antibodies are present in over 40% of human sera samples from the US. Individual human sera samples from New England or Michigan were evaluated for inhibition of AAV9 transduction in HEK293T / 17 cells. The half inhibitory serum dilutions are reported. Demographic data is provided.

[0029] FIG. 6A-6B - Selected BI-hTFRlv2-AE variants evade neutralizing antibodies in serum samples from 26 individual donors. (6A) Normalized HEK293T / 17 cell transduction levels by selected capsid variants after incubation with 26 human sera that neutralize AAV9. The transduction levels are normalized to the no serum control, indicating the relative efficiency of each variant in the presence of neutralizing antibodies. (6B) Heatmap representation of normalized transduction levels shown in (6A).

[0030] FIG. 7 - BI-hTFRlv2 AE variants have maintained transduction of human brain endothelial cells, a trait that selects for maintained interaction with human TFR1. CMEC transduction log2 enrichment (log2enr) scores measured by NGS. Cells were transduced with the virus library at an MOI of 1E4 and lysed 48h post-transduction. Each dot represents a replicate, with n = 5 biological replicates and 4 nucleotide sequences encoding the same capsid variant.

[0031] FIG. 8 - Function of nominated BI-hTFR2v2 variants in brain endothelial cells. BI-hTFR2v2 AE variants have maintained transduction of human brain endothelial cells, a trait that selects for maintained interaction with human TFR1. CMEC transduction log2 enrichment (log2enr) scores measured by NGS. Cells were transduced with the virus library at an MOI of 1E4 and lysed 48h post-transduction.

[0032] FIG. 9 -Function of nominated BI-hTFRlv2 variants in humanized mouse brain. Mouse brain transduction log2 enrichment (log2enr) scores measured by NGS. Brain samples were harvested 3 weeks post retro-orbital administration of 3xl0nvg Library 1 A.

[0033] FIG. 10A-10C - Selected BI-hTFRlv2-AE variants transduce the whole brain of humanized TFRC KI mice at levels comparable to the parental capsid BI-hTFRlv2, while being detargeted from the liver. 10A-10B. Humanized TFRC KI mice were intravenously injected at 5xl010viral genomes per capsid per animal encoding a dual reporter CAG-NLS-mScarlet-P2A-Luciferase-WPRE-pA and were assessed three weeks post-injection. Tissues were harvested and processed three weeks post-injection. 10A. Quantification of average luminescence at an exposure of 0.1 seconds using IVIS imaging system of dissected whole tissues after intraperitoneal injection of animals with D-luciferin. 10B-10C. Wide field imaging of the nuclear localized mScarlet fluorescent protein in whole brain (10B) and liver sections (10C) with matched exposures and settings within each organ Wide field imaging of the nuclear localized mScarlet fluorescent protein in whole brain (10A) and liver sections (10B) with matched exposures and settings within each organ. 10C. Quantification of average luminescence at an exposure of 0.1seconds using an IVIS imaging system of dissected whole tissues from the same animal cohort as images from (10A-10B) after intraperitoneal injection of animals with D-luciferin.

[0034] FIG. 11A-11C - Selected BI-hTFR2v2-AE variants transduce the whole brain of TFRC KI mice at levels comparable to the parental capsid BI-hTFR2v2, while being detargeted from the liver. 11A-11B. Humanized TFRC KI mice were intravenously injected at 5xlO10viral genomes per capsid per animal encoding a dual reporter CAG-NLS-mScarlet-P2A-Luciferase-WPRE-pA and were assessed three weeks post-injection. Wide field imaging of the nuclear localized mScarlet fluorescent protein in whole brain (HA) and liver sections (1 IB) with matched exposures and settings within each organ. 11C. Quantification of average luminescence of harvested tissues from the same animal cohort as images from (11A-11B) after intraperitoneal injection of animals with D-luciferin.

[0035] FIG. 12 - Selected BI-hTFRlv2, BI-hTFR2v2, and BI-hTFRlv3 AE variants produce within 2-fold of AAV9. Cell lysate production titers of BI-hTFRlv2 AE variants measured by ddPCR. Capsid variants were produced in 6-well plates via triple transfection of HEK293T / 17 cells.

[0036] FIG. 13 - Selected BI-hTFRlv2 AE variants bind to three commercially available capture affinity resins. Pairwise correlation plots of the RPM scores in the unselected AAV library (x-axis) or library variants bound by the indicated affinity resin (y-axis).

[0037] FIG. 14A-14C - BI-hTFRlv2-AEl.ll overcomes neutralization for 33% of human sera with anti-AAV9 capsid activity in a panel of 40 sera from individual donors.14A. Neutralization curves measured for AAV9 (gray squares), BI-hTFRlv2 (gray diamonds), and BI-hTFRlv2-AEl.ll (black circles) after incubation with various dilutions of serum KP62952 (top), KP65780 (middle), and KP65768 (bottom). 14B. Heatmap of half-inhibitory serum dilutions (ID50) measured for AAV9, BI-hTFRl v2, BI-hTFR2v2, BI-hTFRlv2-AEl .11 and AAV5 with 40 human sera. 14C. Dot plot of ID50 values measured for all five capsids for sera with anti-AAV5 or anti-AAV9 capsid activity. The black line for each capsid variant represents the mean ID50 value across all 16 neutralizing sera.

[0038] FIG. 15A-15D - BI-hTFRl v2-AE 1.11 is compatible with scalable clarification and capture affinity methods for AAV manufacturing. 15A. Clarified lysate titers and recovery measured for BI-hTFRl v2, BI-hTFR2v2 and BI-hTFRl v2-AEl .11 following depth filtration with Clarisolve® 40 um filter cassettes. B-C. Summary of key parameters (15B) and percentagerecovery (15C) measured in two independent BI-hTFRlv2-AEl.ll capture affinity runs with POROS AAVx. (15D) 254 and 280 nm UV absorbance chromatograms generated for the two independent purification runs.

[0039] FIG. 16A-16C - Selected AE variants are compatible with other tissue-targeting loop VIII modifications. Function of PHP.eB-AE (16A), BI28-AE (16B), 9P31-AE (16C) variants in C57BL / 6J mouse brain, assessed using log2 enrichment (log2enr) scores measured by NGS. Brain samples were harvested 3 weeks post retro-orbital administration of 3E11 vg Library IB.

[0040] FIG. 17A-17C - The myoAAV2A-AE1.18 variant transduces mouse muscle at levels comparable to the parental capsid myoAAV2A, while evading neutralizing antibodies from human serum samples. 17A-17B. C57 mice were intravenously injected at 5E10 viral genomes per capsid per animal encoding a dual reporter CAG-GFP-P2A-Luciferase-WPRE-pA. They were assessed three weeks post-injection by quantifying the average luminescence of harvested tissues after intraperitoneal injection of D-luciferin into animals. 17C. Heatmap representation of normalized transduction levels of HEK293T / 17 cells by selected capsid variants after incubation with 26 human sera that neutralize AAV9. The transduction levels are normalized to the no serum control, indicating the relative efficiency of each variant in the presence of neutralizing antibodies.

[0041] FIG. 18 - Subjected TfRl -binding capsids to two rounds of diversification.

[0042] FIG. 19 - Selected individual TfRl-binding antibody evading (AE) variants.

[0043] FIG. 20 - AAV9 capsid residues 539-605 aligned to other previously described capsids. (SEQ ID NO: 197-216) The 7-mer insertion site between AAV9 residues 588 and 589 is shown. The black bars above the alignment highlight surrounding residues modified in this study. Corresponding residues in other example capsid sequences are outlined, and residues that differ from AAV9 are shown in gray. Sequences were aligned using MUSCLE (SnapGene).

[0044] FIG. 21 - Example insertion sites between residues 558 and 559 AAV9 VP1 for binding modifications (SEQ ID NO: 217-229).

[0045] FIG. 22 - Example serotype sequence alignment (SEQ ID NO: 230-242).

[0046] FIG. 23 - Example binding moiety insertion site in AAV9 VP1 capsid (SEQ ID NO: 243-246).

[0047] FIG. 24 - Screen of 78 human sera from individual donors with AAV9. Neutralizing antibodies are present in over 40% of human sera samples. Individual human serum samples were evaluated for their ability to inhibit AAV9 transduction in HEK293T / 17 cells. The half inhibitory serum dilutions are reported; the limit of detection (LOD).

[0048] FIG. 25A-25C - Selected BI-hTFRlv2-AE variants transduce the whole brain of humanized TFRC KI mice at levels comparable to the parental capsid BI-hTFRlv2, while being detargeted from the liver. Humanized TFRC KI mice were intravenously injected at 5xl010viral genomes per capsid per animal encoding a dual reporter CAG-NLS-mScarlet-P2A-Luciferase-WPRE-pA and were assessed three weeks post-injection. 25A. Quantification of average luminescence at an exposure of 0.1 seconds using IVIS imaging system of dissected whole tissues after intraperitoneal injection of animals with D-luciferin.25B-25C. Wide field imaging of the nuclear localized m Scarlet fluorescent protein in whole brain (25B) and liver sections (25C) with matched exposures and settings within each organ.

[0049] FIG. 26A-26C - Selected BI-hTFR2v2-AE variants transduce the whole brain of TFRC KI mice at levels comparable to the parental capsid BI-hTFR2v2, while being detargeted from the liver. Humanized TFRC KI mice were intravenously injected at 5xl010viral genomes per capsid per animal encoding a dual reporter CAG-NLS-mScarlet-P2A-Luciferase-WPRE-pA and were assessed three weeks post-injection. 26A. Quantification of average luminescence of harvested tissues after intraperitoneal injection of animals with D-luciferin. 26B-26C. Wide field imaging of the nuclear localized mScarlet fluorescent protein in whole brain (26B) and liver sections (26C) with matched exposures and settings within each organ.

[0050] FIG. 27A-27C - The antibody evading variant BI-hTFRlv2-AEl.ll exhibits transduction and biodistribution profiles comparable to the parental capsid BI-hTFRlv2 in TFRC KI mice. Humanized TFRC KI mice were intravenously injected with 5E10 viral genomes per capsid per animal encoding a dual reporter CAG-NLS-mScarlet-P2A-Luciferase-WPRE-pA. Three weeks post-injection, wide field imaging of the nuclear-localized mScarlet fluorescence in whole brain (27A), average luminescence levels per ug of protein lysates (27B), and viral genome biodistribution (27C) were assessed in the same animal cohort.

[0051] FIG. 28A-28C - BI-hTFRlv2-AEl.ll overcomes neutralization for 40% of human sera with anti-AAV9 capsid activity, as assessed in a panel of 73 individual donors.28A. Neutralization curves measured for AAV5, AAV9, BI-hTFRlv2, and BI-hTFRlv2-AEl.ll,with the median curve shown as a dotted black line. 28B. Heatmap of half-inhibitory serum dilutions (IC50) measured for the four capsids across all 73 human sera. 28C. Box plot of IC50 values for sera with anti-AAV5 or anti-AAV9 capsid activity, with statistical significance indicated by adjusted / ^-values from a Friedman test with Wilcoxon signed-rank post-hoc comparisons (****: p < 0.0001; ***: p < 0.001; **.p < 0.01; * p < 0.05).

[0052] FIG. 29A-29F - BI-hTFRlv2-AEl.ll overcomes neutralization in mice passively immunized with anti-AAV9 IgG. 29A. TFRC KI mice were injected intraperitoneally (IP) with 0, 10 mg, or 30 mg of IgG purified from neutralizing human serum. Twenty-four hours later, mice received an intravenous (IV) cocktail of AAV9, BI-hTFRlv2, and BI-hTFRlv2-AEl.ll (3.5E10 vg per capsid per animal), each packaging serotype-specific barcoded transgene cassettes. Three weeks post- AAV administration, brain tissues were harvested, RNA was extracted, and cDNA was synthesized. Relative capsid function was assessed by qPCR quantification of cDNA using primers and probes targeting serotype-specific barcodes. 29B-29D. Neutralization curves for AAV9, BI-hTFRlv2, and BI-hTFRlv2-AEl.ll were measured following assays with (29B) human serum, (29C) IgG purified from the same serum, and (29D) sera from TFRC KI mice passively immunized with the IgG (error bars: standard error of the mean). (29E) Heatmap of IC50 values measured in sera from passively immunized mice. (29F) Brain AAV cDNA levels, normalized to saline controls.

[0053] FIG. 30A-30B - The RGD1 and myoAAV4A-AE1.18 variants transduce mouse muscle with efficiencies comparable to those of their parental capsids. C57BL / 6J mice were intravenously injected with 5E10 viral genomes per capsid encoding a CAG-GFP-P2A-Luciferase-WPRE-pA dual reporter. Three weeks post-injection, (30A) wide-field imaging of GFP fluorescence in quadriceps and (30B) ex vivo whole-organ luminescence levels (following intraperitoneal D-luciferin administration) were assessed in the same animal cohort.

[0054] FIG. 31 - Selected RGD1 and MyoAAV4A AE variants produce within 2-fold of AAV9. Cell lysate production titers of RGD1 and MyoAAV4A AE variants measured by ddPCR. Capsid variants were produced in 6-well plates via triple transfection of HEK293T / 17 cells.

[0055] FIG. 32A-32B - The RGD1-AE1.18 variant exhibits lower neutralization by human sera compared to its parental capsids, AAV9 and RGD1, as assessed in a panel of 47 individual donors. (22A) Heatmap of half-inhibitory serum dilutions (IC50) for AAV5, AAV9,RGD1, and RGD1-AE1.18 across all sera. (22B) Dot plot of IC50 values for sera with anti-AAV5 or anti-AAV9 capsid activity.

[0056] FIG. 33 - Introduction of additional mutations in the 5-fold symmetry axis region of RGD1-AE1.18 to increase antibody evasion. Library target residues represented at the AAV capsid surface. Divergent residues between AE1.11 or AE1.18 and AAV9 are shown in gray, while additional mutations introduced into AE1.18 are shown in black.

[0057] FIG. 34 - RGD1 AE variants produce within 2- to 4-fold of AAV9. Cell lysate production titers of RGD1 AE variants measured by ddPCR. Capsid variants were produced in 6-well plates via triple transfection ofHEK293T / 17 cells.

[0058] FIG. 35 - Antibody evading RGD1 variants AE1.18.1, AE1.18.2, AE1.18.4, and AE1.18.8 evaluated against serum samples from 8 individual donors. Heatmap representation of normalized transduction levels of HEK293T / 17 cells by selected capsid variants after incubation with 8 human sera that neutralize AAV9. The transduction levels are normalized to the no serum control, indicating the relative efficiency of each variant in the presence of neutralizing antibodies.

[0059] FIG. 36A-36C - Second-generation antibody evading RGD1 variants overcome neutralization for 45-50% of human sera with anti-RGDl capsid activity, as assessed in a panel of 46 individual donors. 36A. Neutralization curves measured for RGD1, RGD1-AE1.18, RGD1-AE1.18.2, and RGD1-AE1.18.8, with the median curve shown as a dotted black line. 36B.Heatmap of half-inhibitory serum dilutions (IC50) measured for the four capsids across all 46 human sera. 36C. Box plot of IC50 values for sera with anti-RGDl capsid activity, with statistical significance indicated by adjusted i-values from a Friedman test with Wilcoxon signed-rank post-hoc comparisons (****: p < 0.0001; ***: p < 0.001; **: p < 0.01; *:p < 0.05).

[0060] FIG. 37 - RGD1-AE1.18.1 transduces C57BL / 6J muscle at levels comparable to the parental capsid RGD1. C57BL / 6J mice were intravenously injected with 5E10 viral genomes per capsid encoding a CAG-GFP-P2A-Luciferase-WPRE-pA dual reporter. Two weeks postinjection, tissue luminescence was quantified after intraperitoneal administration of D-luciferin.

[0061] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

[0062] A hindrance to efficient and safe AAV delivery to human patients is the presence of AAV-neutralizing antibodies. Previous studies have shown that the average human population has antibody titers resistant to naturally occurring AAVs. Accordingly, using naturally occurring AAV capsid polypeptides for in vivo delivery poses challenges related to safety and efficient delivery. The compositions described herein include amino acid substitutions in AAV capsid polypeptides that prevent or reduce the recognition of AAV particles comprising those polypeptides by naturally occurring AAV-neutralizing antibodies, thereby enhancing the efficacy of AAV delivery while maintaining the structural integrity and tropism of the capsid polypeptides. The substitutions described herein may be used with other modifications, such as targeting insertions, without impacting the specificity or stability of the AAV particle. In addition, the described one or more substitutions have been shown to maintain capture with commonly used affinity reagents. Testing any of these combinations will not require the development of new assays. Therefore, testing any other modification with these substitutions will provide reasonable predictability, and the observed effects of the different modifications will not be affected by these substitutions. Moreover, one or more substitutions may require a reduced dosage due to enhanced evasion properties. Since these capsid polypeptides are less susceptible to inhibition by AAV -targeting antibodies, fewer capsids may be needed to achieve the desired therapeutic effect.

[0063] Additional features and advantages of the embodiments above are further described below.Antibody Evading AAV Capsid Polypeptide Modifications

[0064] In one aspect, engineered AAV capsid polypeptides comprising modifications that reduce anti-AAV antibody binding while maintaining other properties of a reference AAV capsid polypeptide are provided. The following modifications are illustrated using the AAV9 VP1 polypeptide but may also be made at analogous positions in VP1 polypeptides of other AAV serotypes. In an embodiment, the AAV9 VP1 polypeptide is SEQ ID NO: 1. In an embodiment, the AAV9 VP1 may comprise a K449R substitution. In an embodiment, the AAV9 comprising a K449R substitution is SEQ ID NO: 2.

[0065] In one embodiment, the antibody evading modifications comprise one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ,D5 1N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide, an engineered AAV9 VP1 polypeptide with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype. In one aspect, engineered AAV capsid polypeptides comprising antibody evading modifications are provided.

[0066] In one embodiment, the antibody evading modifications comprise one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide, an engineered AAV9 VP1 polypeptide optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0067] In an embodiment, a single amino acid substitution at one of the disclosed positions can provide measurable antibody evasion while maintaining transduction function. In an embodiment, combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid substitutions provide enhanced antibody evasion.

[0068] Combinations of amino acid substitutions may provide synergistic or additive effects in antibody evasion. For example, as shown in Example 1, combinations of more than one amino acid substitution provided robust antibody evasion against multiple human sera while maintaining CNS transduction function. The specific combinations disclosed in Example 1 and in the sequence listings represent optimized sets of substitutions identified through high-throughput screening.

[0069] Individual substitutions from the disclosed positions (e.g., D327N, N328K, N329D, K332Q, N452D, E500D, A502S, P504T, A510K, R55OQ, D551N, D554N, K557Q, D657N, N663D, D665N, N668K, G455Q, G455T, G455K, G455N, K462E, K462Q, N552D, D556K, D556N, K557E, K664E, K664Q, and N716D) can be tested individually or in various combinations using the screening methods described herein to determine their contribution to antibody evasion. The neutralizing antibody assays described in Example 1 (e.g., IVIG neutralization assays and individual human sera neutralization assays) provide suitable methods for assessing the antibody-evasion properties of any individual substitution or combination thereof.

[0070] In one embodiment, the antibody evading modifications comprise two or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide, an engineered AAV9 VP1 polypeptide optionally with tropism modifications derived from loop IV or loop Vlll insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0071] In one embodiment, the antibody evading modifications comprise three or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP 1 polypeptide of another AAV serotype.

[0072] In one embodiment, the antibody evading modifications comprise four or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP 1 polypeptide of another AAV serotype.

[0073] In one embodiment, the antibody evading modifications comprise five or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0074] In one embodiment, the antibody evading modifications comprise six or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0075] In one embodiment, the antibody evading modifications comprise seven or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0076] In one embodiment, the antibody evading modifications comprise eight or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0077] In one embodiment, the antibody evading modifications comprise nine or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived fromloop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0078] In one embodiment, the antibody evading modifications comprise ten or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0079] In one embodiment, the antibody evading modifications comprise eleven or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0080] In one embodiment, the antibody evading modifications comprise twelve or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0081] In one embodiment, the antibody evading modifications comprise thirteen or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of aVP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0082] In one embodiment, the antibody evading modifications comprise fourteen or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0083] In one embodiment, the antibody evading modifications comprise fifteen or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0084] In one embodiment, the antibody evading modifications comprise sixteen or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0085] In one embodiment, the antibody evading modifications comprise seventeen or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q,K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP 1 polypeptide of another AAV serotype.

[0086] In one embodiment, the antibody evading modifications comprise eighteen or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0087] In one embodiment, the antibody evading modifications comprise nineteen or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0088] In one embodiment, the antibody evading modifications comprise twenty or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype.

[0089] For ease of reference, all of the following substitutions in this section are described with reference to an AAV9 VP1 polypeptide (see, e.g., SEQ ID: NO 1) but also includemodification made at an analogous position of a VP1 polypeptide of another AAV serotype (see, e.g., FIG. 23).

[0090] In an embodiment, an engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q.

[0091] In an embodiment, the engineered AAV capsid polypeptide further includes one or more amino acid substitutions selected from the group consisting of D327N, N328K, N329D, K332Q, K462E / Q, R533Q, D657N, D657N, N663D, K664Q, N665N, and N668K. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and one or more amino acid substitutions selected from the group consisting of D327N, N328K, N329D, K332Q, K462E / Q, R533Q, D657N, D657N, N663D, K664Q, N665N.

[0092] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions D327N, K332Q, and D657N. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R55OQ, D551N / K, D554N, and K557Q and the amino acid substitutions D327N, K332Q, and D657N.In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions D327N and K332Q. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and the amino acid substitutions D327N, K332Q, and D657N.

[0093] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions N328K andN329D. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and the amino acid substitutions N328K and N329D.

[0094] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions N328K and N329D. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and the amino acid substitutions N328K and N329D.

[0095] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions N329K and K332Q. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and the amino acid substitutions N329K and K332Q.

[0096] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions D327N, N328K, N329D, and K332Q. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and the amino acid substitutions D327N, N328K, N329D, and K332Q.

[0097] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions N452D. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and the amino acid substitutions D327N, N328K, N329D, and K332Q.

[0098] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitution D657N. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and the amino acid substitution D657N.

[0099] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitution N668K. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and the amino acid substitution N668K.

[0100] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions K664Q and N668K. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and the amino acid substitutions K664Q and N668K.

[0101] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions N663D and N668K. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and the amino acid substitutions N663D and N668K.

[0102] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions D657N, N663D and N668K. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R55OQ, D551N / K, D554N, and K557Q and the amino acid substitutions D657N, N663D and N668K.

[0103] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions N663D, K664Q, D665N, and N668K. In an embodiment, the engineered AAVcapsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and the amino acid substitutions N663D, K664Q, D665N, and N668K.

[0104] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions N663K, K664E, and N668K. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R55OQ, D551N / K, D554N, and K557Q and the amino acid substitutions N663K, K664E, and N668K.

[0105] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions D657N, N663D, and N668K. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R55OQ, D551N / K, D554N, and K557Q and the amino acid substitutions D657N, N663D, and N668K.

[0106] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions K664Q andN668K. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and the amino acid substitutions K664Q and N668K.

[0107] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitution N668K. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and the amino acid substitution N668K.

[0108] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitutions K462E / Q and N668K. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R55OQ, D551N / K, D554N, and K557Q and the amino acid substitutions K462E / Q and N668K.

[0109] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitution N663D. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q and the amino acid substitution N663D.

[0110] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitution K664Q. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N668K.

[0111] In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions D327N, N328K, N329D, K332Q, N452D, E500D, A502S, P504T, A510K, R55OQ, D551N, D554N, K557Q, D657N, N663D, and D665N.

[0112] In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions D327N, N328K, N329D, K332Q, N452D, E500D, A502S, P504T, A510K, R55OQ, D551N, D554N, K557Q and D657N.

[0113] In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions D327N, K332Q, N452D, E500D, A502S, P504T, A510K, R550Q, D551N, D554N, K557Q, D657N, N663D, and N668K.

[0114] In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N452K, K462E, E500D, A502S, P504T, A510K, R550Q, D551N, D554N, K557Q, D657N, and N663D.

[0115] In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions N452K, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551K, D554N, K557Q, K664Q, and N668K.

[0116] In an embodiment, an engineered AAV capsid polypeptide includes the amino acid substitutions G455Q / T / K / N, D551N, N552D, K557E / Q, K664E / Q, D665N, and N668K.

[0117] In an embodiment, the engineered AAV capsid polypeptide further includes one or more amino acid substitutions selected from the group consisting of D554N, D556K / N, N663D, and N716D. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions G455Q / T / K / N, D551N, N552D, K557E / Q, K664E / Q, D665N, andN668K, and one or more amino acid substitutions selected from the group consisting of D554N.

[0118] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitution D556K / N. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions G455Q / I7K / N, D551N, N552D, K557E / Q, K664E / Q, D665N, and N668K, as well as the amino acid substitution D556K / N.

[0119] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitution D554N. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions G455Q / T / K / N, D551N, N552D, K557E / Q, K664E / Q, D665N, and N668K, as well as the amino acid substitution D554N.

[0120] In an embodiment, the engineered AAV capsid polypeptide further includes the amino acid substitution N716D. In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions G455Q / T / K / N, D551N, N552D, K557E / Q, K664E / Q, D665N, and N668K and the amino acid substitution N716D.

[0121] In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions G455Q, D551N, N552D, D556K, K557E, K664E, D665N, N668K, and N716D.

[0122] In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions G455T, D551N, N552D, D556K, K557E, K664E, D665N, N668K, and N716D.

[0123] In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions G455K, D551N, N552D, D556K, K557E, K664E, D665N, N668K, and N716D.

[0124] In an embodiment, the engineered AAV capsid polypeptide includes the amino acid substitutions G455N, D551N, N552D, D554N, D556N, K557E, N663D, K664Q, D665N, and N668K.

[0125] In an embodiment, the engineered AAV capsid polypeptide is selected from SEQ ID NO: 26-34 and 1641-1648.

[0126] In an embodiment, the TfRl binding AAV VPI polypeptide is SEQ ID NO: 3

[0127] In an embodiment, the TfRl binding AAV VPI polypeptide is SEQ ID NO: 4

[0128] In an embodiment, the TfRl binding AAV VPI polypeptide is SEQ ID NO: 5

[0129] In an embodiment, the engineered AAV capsid polypeptide includes a TfRl binding AAV VPI selected from SEQ ID NO: 7-21, 40-167, and 1649-1656.

[0130] In an embodiment, the engineered AAV capsid polypeptide includes a muscle binding AAV VPI selected from SEQ ID NO: 35-39 and 1657-1684.

[0131] In an embodiment, the integrin-binding MyoAAV VPI polypeptide is SEQ ID NO: 19.

[0132] In another embodiment, the VPI polypeptide may be a VPI polypeptide of another AAV serotype, including AAV1, AAVrhlO, AAV-DJ, AAV-DJ8, AAV5, AAV-PHP.B (PHP.B), AAV-PHP.A (PHP. A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1- 35, AAV-PHP.B2 (PHP.B2), AAV-PHP.B3 (PHP.B3), AAV-PHP.N / PHP.B-DGT, AAV-PHP.B-EST, AAV-PHP.B-GGT, AAV-PHP.B-ATP, AAV-PHP.B-ATT-T, AAV-PHP.B- DGT-T, AAV-PHP.B-GGT-T, AAV-PHP.B-SGS, AAV-PHP.B-AQP, AAV-PHP.B-QQP, AAV-PHP.B-SNP(3), AAV-PHP.B-SNP, AAV-PHP.B-QGT, AAV-PHP.B-NQT, AAV-PHP.B- EGS, AAV-PHP.B-SGN, AAV-PHP.B-EGT, AAV-PHP.B-DST, AAV-PHP.B-DST, AAV-PHP.B-STP,AAV-PHP.B-PQP, AAV-PHP.B-SQP, AAV-PHP.B-QLP, AAV-PHP.B- TMP, AAV-PHP.B-TTP, AAV-PHP.S / G2A12, AA VG2A 15 / G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV 10, AAV11, AAV 12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42- lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVl-7 / rh.48, AAVl-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.5O, AAV2-5 / rh.5 1, AAV3. l / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-1 l / rh.53, AAV4- 8 / rl 1.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.lO, AAV16. 12 / hu.l 1, AAV29.3 / bb.l, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV1 14.3 / hu.4O, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145. l / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.1O / hu.6O, AAV161.6 / hu.61, AAV33.12 / hu. 17, AAV33.4 / hu.l5, AAV33.8 / hu. 16, AAV52 / hu.l9, AAV52. l / hu.20, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi. 1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVH-l / hu.l, AAVH-5 / hu.3, AAVLG- 10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu. 1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.l 7, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu .t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10,AAVrh.12, AAVrh.13, AAVrh. 13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48. L2, AAVrh.48.2, AAVrh.49, AAVA.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVA.61, AAVrh.64, AAVA.64R1, AAVA.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVA8R, AAVA8R A586R mutant, AAVA8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhEl . 1, AAVhErl .5, AAVhERl . 14, AAVhErl .8, AAVhErl . 16, AAVhErl . 18, AAVhErl .35, AAVhErl .7, AAVhErl .36, AAVhEr2.29, AAVhEr2.4, AAVhEr2. 16, AAVhEr2.30, AAVhEr2.3 1, AAVhEr2.36, AAVhERl .23, AAVhEr3. 1, AAV2.5T , AAV-PAEC, AAV-LK01, AAV-LK02, AAV- LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV-LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC 11, AAV-PAEC 12, AAV-2-pre-miRNA-101 , AAV-8h, AAV- 8b, AAV-h, AAV-b, AAV SM 10-2 , AAV Shuffle 100-1 , AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100- 2, AAV SM 10-1, AAV SM 10-8 , AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu. 19, AAVhu. 11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54. l / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128. l / hu.43, true type AAV (ttAAV), UPEN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7. 1, AAV CBr-7. 10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr- E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6. 1, AAV CHt-6. 10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B 1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-Hl, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd- H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-Fl, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAVCLg-F8, AAV CLv-1, AAV CLvl-1, AAV Clvl-10, AAV CLvl-2, AAV CLv-12, AAV CLvl-3, AAV CLv-1 3, AAV CLvl-4, AAV Clvl-7, AAV Clvl-8, AAV Clvl-9, AAV CLv- 2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-Dl, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLv-Kl, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-Ml, AAV CLv-Ml 1, AAV CLv-M2, AAV CLv-M5, AAV CLv- M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-Rl, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-1 1, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8. 10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9 and comprise substitutions at positions analogous to those of the AAV9 VP1 polypeptide. See e.g., US20210380969, the content of which is incorporated by reference herein in its entirety. In addition, see Figure 7 for the sequence alignment of a representative group of AAV VP1 polypeptides.

[0133] In one embodiment, the VP1 capsid polypeptide comprising the engineered AAV particle may comprise a further substitution at position 449 of an AAV9 VP1 capsid polypeptide or at an analogous position of a VP1 polypeptide of another AAV serotype. In an embodiment, the substitution at position 449 is a K449R substitution.

[0134] In an embodiment, any one or more substitutions described herein that confer antibodyevasion also maintain the other properties of an AAV capsid polypeptide. For example, the substitutions described herein maintain a natural tropism or modified tropism of a reference AAV capsid polypeptide. In another example, any one or more substitutions described herein maintain natural stability or increased stability of a reference AAV capsid polypeptide.Engineered Viral Capsids and Particles

[0135] In another aspect, embodiments directed to AAV capsids, or particles, comprising the engineered AAV capsid polypeptides described above are provided. A capsid will generally refer to the empty capsid and particle to a capsid comprising a cargo for delivery. In the context of theantibody evading modifications, the terms are used interchangeably since both include the AAV capsid. In an embodiment, the engineered AAV capsids described herein can include from 1 to 60 engineered AAV capsid polypeptides, wherein each engineered AAV capsid polypeptide comprises one or more of the disclosed amino acid substitutions. In an embodiment, where a single substitution is present, the substitution may provide sufficient antibody evasion for therapeutic applications, particularly when combined with other properties such as tissue-specific tropism. In an embodiment, where multiple substitutions are present (e.g., 2-30 substitutions), the combined effect may provide enhanced antibody evasion, potentially enabling treatment of patients with high titers of neutralizing antibodies or repeated dosing regimens. In an embodiment, the engineered AAV capsid can include 1-60 engineered AAV capsid polypeptides described herein. In an embodiment, the engineered AAV capsid can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 engineered capsid proteins. In an embodiment, the engineered AAV capsid can contain 0-59 wild-type AAV capsid polypeptides. In an embodiment, the engineered AAV capsid can contain 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59 wildtype AAV capsid polypeptides.

[0136] In an embodiment, the engineered AAV particles may comprise an engineered capsid polypeptide comprising one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype, are provided. In one embodiment, the engineered AAV particle is an engineered AAV9 particle comprising at least one of the engineered AAV9 VP1 polypeptides described herein.

[0137] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising two or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q,E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype, are provided.

[0138] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising three or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype, are provided.

[0139] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising four or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype, are provided.

[0140] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising five or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP I polypeptide of another AAV serotype, are provided.

[0141] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising six or more amino acid substitutions selected from D327N,N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype, are provided.

[0142] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising seven or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP 1 polypeptide of another AAV serotype, are provided.

[0143] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising eight or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP 1 polypeptide of another AAV serotype, are provided.

[0144] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising nine or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP 1 polypeptide of another AAV serotype, are provided.

[0145] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising ten or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP 1 polypeptide of another AAV serotype, are provided.

[0146] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising eleven or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype, are provided.

[0147] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising twelve or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype, are provided.

[0148] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising thirteen or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotypeoptionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype, are provided.

[0149] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising fourteen or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype, are provided.

[0150] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising fifteen or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype, are provided.

[0151] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising sixteen or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype, are provided.

[0152] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising seventeen or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of anAAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP 1 polypeptide of another AAV serotype, are provided.

[0153] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising eighteen or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP 1 polypeptide of another AAV serotype, are provided.

[0154] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising nineteen or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP 1 polypeptide of another AAV serotype, are provided.

[0155] In another aspect, AAV particles comprising the engineered antibody-evading AAV capsid polypeptides comprising twenty or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype optionally with tropism modifications derived from loop IV or loop VIII insertions or substitutions, or an analogous position of a VP1 polypeptide of another AAV serotype, are provided.

[0156] In another embodiment, the engineered AAV particle is an AAV1, AAVrhlO, AAV-DJ, AAV-DJ8, AAV5, AAV-PHP.B (PHP.B), AAV-PHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1- 35, AAV-PHP.B2 (PHP.B2), AAV-PHP.B3 (PHP.B3), AAV-PHP.N / PHP.B-DGT, AAV-PHP.B-EST, AAV-PHP.B-GGT, AAV-PHP.B -ATP, AAV-PHP.B-ATT-T, AAV-PHP.B- DGT-T, AAV-PHP B-GGT-T, AAV-PHP.B-SGS, AAV-PHP. B-AQP, AAV-PHP B-QQP, AAV-PHP.B-SNP(3), AAV-PHP.B-SNP, AAV-PHP.B-QGT, AAV-PHP.B-NQT, AAV-PHP.B- EGS, AAV-PHP.B-SGN, AAV-PHP.B-EGT, AAV-PHP.B-DST, AAV-PHP.B-DST, AAV-PHP.B-STP, AAV-PHP. B-PQP, AAV-PHP.B-SQP, AAV-PHP.B-QLP, AAV-PHP.B- TMP, AAV-PHP.B-TTP, AAV-PHP. S / G2 A 12, AA VG2A 15 / G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV2, AAV2G9, AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV 10, AAV11, AAV 12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42- lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVl-7 / rh.48, AAV1-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.5O, AAV2-5 / rh.5 1, AAV3. l / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-1 l / rh.53, AAV4- 8 / rl 1.64, AAV4-9 / rh.54, AAV4-19 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.lO, AAV16. 12 / hu.l 1, AAV29.3 / bb.l, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV1 14.3 / hu.4O, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145. l / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.1O / hu.6O, AAV161.6 / hu.61, AAV33.12 / hu. 17, AAV33.4 / hu.l5, AAV33.8 / hu. 16, AAV52 / hu.l9, AAV52. l / hu.20, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAV A3.7, AAVC1, AAVC2, AAVC5, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi. 1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVH-l / hu.l, AAVH-5 / hu.3, AAVLG- 10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu. 1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.l 7, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47,AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu .t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh. 13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36, AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48. L2, AAVrh.48.2, AAVrh.49, AAVA.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVA.61, AAVrh.64, AAVA.64R1, AAVA.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVA8R, AAVA8R A586R mutant, AAVA8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhEl. 1, AAVhErl .5, AAVhERl . 14, AAVhErl .8, AAVhErl . 16, AAVhErl.18, AAVhErl .35, AAVhErl .7, AAVhErl .36, AAVhEr2.29, AAVhEr2.4, AAVhEr2. 16, AAVhEr2.30, AAVhEr2.31, AAVhEr2.36, AAVhERl .23, AAVhEr3. 1, AAV2.5T , AAV-PAEC, AAV-LK01, AAV-LK02, AAV- LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV- LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV- LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC 11, AAV-PAEC 12, AAV-2-pre-miRNA-101 , AAV-8h, AAV- 8b, AAV-h, AAV-b, AAV SM 10-2 , AAV Shuffle 100-1 , AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100- 2, AAV SM 10-1, AAV SM 10-8 , AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu.19, AAVhu.11, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54. l / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128. l / hu.43, true type AAV (ttAAV), UPEN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7. 1, AAV CBr-7. 10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr- E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6. 1, AAV CHt-6. 10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B 1, AAV CKd-B2, AAV CKd-B3, AAVCKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-Hl, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd- H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-Fl, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLvl-1, AAV Clvl-10, AAV CLvl-2, AAV CLv-12, AAV CLvl-3, AAV CLv-1 3, AAV CLvl-4, AAV Clvl-7, AAV Clvl-8, AAV Clvl-9, AAV CLv- 2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-Dl, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLv-Kl, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-Ml, AAV CLv-Ml 1, AAV CLv-M2, AAV CLv-M5, AAV CLv-M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-Rl, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-1 1, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8. 10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9 comprising at least one engineered VP1 polypeptide having one or more substitutions at an analogous position to those described above in the context of an AAV9 VP1 polypeptide. In one embodiment, the engineered AAV particle may comprise an engineered VP1 capsid polypeptide from that serotype (e.g., an AAV1 particle comprising an engineered AAV1 VP1 capsid polypeptide) or may comprise an engineered VP1 capsid polypeptide from another serotype (e.g., an AAV1 particle comprising an engineered AAV9 VP1 capsid polypeptide).Table A. Example AAV VP1 amino acid sequences - see FIG.20 for an example alignment of the example virus sequences

[0157] The amino acid substitutions disclosed herein, identified at positions in AAV9 VP1, can be applied to analogous positions in VP1 polypeptides of other AAV serotypes. The principles applied to AAV9-derived capsids can be broadly applied to AAV serotypes due to the capsids' conserved structure.

[0158] Figure 22 shows an exemplary alignment of AAV serotypes 1-13, AAVrh.8, AAVrh.10, AAVrh74, AAV-LK03, AAV-LK19, and AAV-DJ, demonstrating how analogous positions are identified. Table A provides representative VP1 sequences from these AAV serotypes and variants, as well as from additional AAV serotypes. One of ordinary skill in the art can identify analogous positions using sequence alignment, as shown in these figures and tables. The substitutions can be introduced at analogous positions using standard molecular biology techniques, including site-directed mutagenesis.

[0159] To identify analogous positions in a VP1 polypeptide of an AAV serotype not explicitly shown in Figure 22 or Table A, the following protocol can be employed: (A) Retrieve the VP1 amino acid sequence of the target AAV serotype from public databases (e.g., GenBank, UniProt, or published literature); (B) Align the target VP1 sequence with AAV9 VP1 (e.g., SEQ ID NO: 1) using a standard alignment algorithm. For example, the MUSCLE algorithm, ClustalW, or BLAST can be used. Multiple sequence alignment, including additional AAV serotypes from Figure 22, may improve alignment accuracy; (C) Locate the amino acid position in the target serotype that aligns with the desired AAV9 position. Gaps can be placed to maximize alignment score while maintaining biological relevance; (D) Confirm that the identified position is surface-exposed using available crystal structures (if available for the target serotype) or structural prediction tools (e.g., AlphaFold, SWISS-MODEL). This step ensures that the analogous position will be accessible for antibody binding, consistent with the antibody-evasion function; (E) Create the desired amino acid substitution at the identified analogous position using site-directed mutagenesis or gene synthesis techniques known in the art; (F) Test the resulting engineered capsid using the screening methods described herein (e.g., neutralizing antibody assays, transduction assays) to confirm both antibody evasion and maintained transduction function. This protocol references standard techniques that would be understood by one of ordinary skill in the art.

[0160] For example, an alignment of AAV5 to AAV9 shows AAV5 position N327 corresponds to AAV9 N329. Accordingly, the N327D substitution in AAV5 would be theanalogous modification to N329D in AAV9. This methodology enables application of the disclosed antibody-evasion substitutions across the full diversity of AAV serotypes, both naturally occurring and engineered variants.

[0161] The engineered AAV capsid can include one or more cargo polynucleotides to generate engineered AAV particles. Cargo polynucleotides are discussed in greater detail elsewhere herein. Methods of making the engineered AAV particles from viral and non-viral vectors are described elsewhere herein. Formulations containing the engineered virus particles are described elsewhere herein.

[0162] The engineered AAV capsids disclosed herein can be manufactured using standard AAV production methods known in the art, with yields and purification profiles comparable to those of widely used AAV serotypes such as AAV9. For example, the engineered capsids can be produced in HEK293T cells by triple transfection, yielding yields within 2-fold of AAV9 without substantially impairing production fitness. Production can be performed at both small scale (6-well plate format) and larger scale (2L suspension culture), with scale-up maintaining comparable titers.

[0163] The engineered capsids are compatible with common downstream purification methods, including affinity chromatography using commercially available resins. For example, purification can be carried out using POROS AAVx affinity resin, with binding of the engineered capsids to the resin and recovery of the elution fractions with or without optimization. The capsids can also be compatible with depth filtration.

[0164] Additional purification methods applicable to the engineered capsids include, but are not limited to, cesium chloride gradient ultracentrifugation, iodixanol gradient ultracentrifugation, ion exchange chromatography, and size exclusion chromatography. The choice of purification method can be optimized based on the specific engineered capsid, scale of production, and purity requirements. Quality control testing, including capsid titration (e.g., by ddPCR as described herein), empty / full capsid ratio analysis, and potency assays (e.g., in vitro transduction assays), can be performed using standard methods to ensure product quality and consistency across manufacturing batches.AAV Particles Comprising Target Binding Modifications

[0165] The antibody-evading modifications described above may be combined with target binding modifications that change the tropism of the resulting engineered AAV particle. Thesetarget binding modifications may also be located in an AAV capsid protein but are not necessarily limited to the VP1 capsid polypeptide. A target binding modification with an enhanced tropism promotes, increases, or otherwise improves binding to and / or transduction of the targeted system or tissue compared to a capsid lacking the target binding modification.

[0166] In an embodiment, the target binding modification is an n-mer insertion (targeting moiety) between two viral protein amino acids. The targeting moiety is external (i.e., presented on the surface of) to a viral particle. The length of the n-mer may be any necessary length to transduce the targeted system or tissue. In example embodiments, the n-mer is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids in length. In an example embodiment, the n-mer motif has a length of at least seven amino acids. The increase in transduction efficiency (which may correspond to the tropism efficiency) of the n-mer to a cell may be compared to a composition that does not contain the target binding modification; for example, the inclusion of one or more target binding modifications in a composition can result in an increase in transduction and or transduction efficiency by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more. In an exemplary embodiment, the increase in transduction and or transduction efficiency is one-and-a-half fold, two-fold, three-fold, four-fold, five-fold, six-fold, seven-fold, eight-fold, nine-fold, ten-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold or more relative to a composition lacking the n-mer. In one embodiment, the transduction and / or transduction efficiency is increased or enhanced in cells; in one embodiment, it increases in cells of the targeted system or tissue, for example, the CNS or muscle. In an embodiment, the transduction and / or transduction efficiency is increased or enhanced in cells of the targeted system or tissue. In an embodiment, the n-mer composition is selective to a target cell compared to other cell types and / or virus particles. As used herein, ‘selective’ and ‘cell-selective’ refer to preferential targeting for cells compared to other cell types. Preferably, the target binding modification is selective for a desired target (e.g., cell, organ, system, e.g., target tissues) or set of targets by atleast2:l, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1. 10:1 or more; or 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or more, relative to other targets or cells. In an example embodiment, the composition comprising a target binding modification described herein can have an increased uptake, delivery rate, transduction rate, efficiency, amount, or a combination thereof in a target cell (e.g., cells across the system or tissue, e.g., CNS or muscle) as compared to other cells types (e.g. hepatocytes) and / or other virusparticles (e ., AAVs not containing the target binding modification) and other compositions that do not contain the cell-selective n-mer motif of the present invention.

[0167] The antibody-evasion substitutions disclosed herein can be combined with various targeting moieties by introducing both the substitutions and the targeting moiety into the same AAV capsid polypeptide. This combinatorial approach enables the generation of AAV capsids with both enhanced tissue-specific tropism and reduced susceptibility to neutralizing antibodies, thereby potentially expanding the treatable patient population and enabling repeated dosing regimens.

[0168] In an embodiment, the n-mer disclosed herein can be inserted between two consecutive amino acids in the wild-type viral protein (VP) (or capsid protein), including in regions that are surface exposed when incorporated into a viral capsid. In an embodiment, the n-mer can be inserted between two amino acids in a variable region in a viral capsid protein. In an embodiment, the n-mer can be inserted between two amino acids in an AAV capsid protein’s variable amino acid region. In an embodiment, one or more n-mer can be inserted between two amino acids in one or more of the 12 variable regions in the wild-type AVV capsid proteins. In an embodiment, one or more n-mers can each be inserted between two amino acids in VR-I, VR-II, VR-III, VR-IV, VR-V, VR-VI, VR-VII, VR-III, VR-IX, VR-X, VR-XI, VR-XII, or a combination thereof. In an example embodiment, the target binding modification is inserted or substituted in loop IV and / or loop VIII.

[0169] In an embodiment, the n-mer is inserted between two amino acids between 586-588 and 589-592 of a VP1 capsid protein of AAV9 (including insertion of the n-mer, such as a 7-mer, between positions 588-589) or in an analogous position of a capsid protein from AAV1, AAVrhlO, AAV-DJ, AAV-DJ8, AAV5, AAV-PHP.B (PHP.B), AAV-PHP.A (PHP.A), AAVG2B-26, AAVG2B-13, AAVTH1.1-32, AAVTH1.1-35, AAV-PHP.B2 (PHP.B2), AAV-PHP.B3 (PHP.B3), AAV-PHP.N / PHP.B-DGT, AAV-PHP.B-EST, AAV-PHP.B-GGT, AAV-PHP.B-ATP, AAV-PHP.B-ATT-T, AAV-PHP.B- DGT-T, AAV-PHP.B-GGT-T, AAV-PHP.B-SGS, AAV-PHP.B-AQP, AAV-PHP.B-QQP, AAV-PHP.B-SNP(3), AAV-PHP.B -SNP, AAV-PHP.B-QGT, AAV-PHP.B-NQT, AAV-PHP.B- EGS, AAV-PHP.B-SGN, AAV-PHP.B-EGT, AAV-PHP.B-DST, AAV-PHP.B-DST, AAV-PHP.B-STP, AAV-PHP.B-PQP, AAV-PHP.B-SQP, AAV-PHP.B-QLP, AAV-PHP.B- TMP, AAV-PHP.B-TTP, AAV-PHP.S / G2A12, AA VG2A 15 / G2A3 (G2A3), AAVG2B4 (G2B4), AAVG2B5 (G2B5), PHP.S, AAV2, AAV2G9,AAV3, AAV3a, AAV3b, AAV3-3, AAV4, AAV4-4, AAV6, AAV6.1, AAV6.2, AAV6.1.2, AAV7, AAV7.2, AAV8, AAV9.11, AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84, AAV9.9, AAV 10, AAV11, AAV 12, AAV16.3, AAV24.1, AAV27.3, AAV42.12, AAV42-lb, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a, AAV42-5b, AAV42-6b, AAV42-8, AAV42-10, AAV42-11, AAV42-12, AAV42-13, AAV42-15, AAV42-aa, AAV43-1, AAV43-12, AAV43-20, AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV44.1, AAV44.2, AAV44.5, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAVl-7 / rh.48, AAVl-8 / rh.49, AAV2-15 / rh.62, AAV2-3 / rh.61, AAV2-4 / rh.5O, AAV2-5 / rh.5 1, AAV3. l / hu.6, AAV3.1 / hu.9, AAV3-9 / rh.52, AAV3-1 l / rh.53, AAV4-8 / rl 1.64, AAV4-9 / rh.54, AAV449 / rh.55, AAV5-3 / rh.57, AAV5-22 / rh.58, AAV7.3 / hu.7, AAV16.8 / hu.lO, AAV16. 12 / hu.l 1, AAV29.3 / bb.l, AAV29.5 / bb.2, AAV106.1 / hu.37, AAV1 14.3 / hu.4O, AAV127.2 / hu.41, AAV127.5 / hu.42, AAV128.3 / hu.44, AAV130.4 / hu.48, AAV145. l / hu.53, AAV145.5 / hu.54, AAV145.6 / hu.55, AAV161.1O / hu.6O, AAV161.6 / hu.61, AAV33.12 / hu. 17, AAV33.4 / hu.l5, AAV33.8 / hu. 16, AAV52 / hu.l9, AAV52. l / hu.20, AAV58.2 / hu.25, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAVC1, AAVC2, AAVC5, AAVF3, AAVF5, AAVH2, AAVrh.72, AAVhu.8, AAVrh.68, AAVrh.70, AAVpi. 1, AAVpi.3, AAVpi.2, AAVrh.60, AAVrh.44, AAVrh.65, AAVrh.55, AAVrh.47, AAVrh.69, AAVrh.45, AAVrh.59, AAVhu.12, AAVH6, AAVH-l / hu.l, AAVH-5 / hu.3, AAVLG- 10 / rh.40, AAVLG-4 / rh.38, AAVLG-9 / hu.39, AAVN721-8 / rh.43, AAVCh.5, AAVCh.5Rl, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5Rl, AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAVhu. 1, AAVhu.2, AAVhu.3, AAVhu.4, AAVhu.5, AAVhu.6, AAVhu.7, AAVhu.9, AAVhu.10, AAVhu.11, AAVhu.13, AAVhu.15, AAVhu.16, AAVhu.l 7, AAVhu.18, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24, AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.31, AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.40, AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44Rl, AAVhu.44R2, AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48Rl, AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.51, AAVhu.52, AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.60, AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.14 / 9, AAVhu .t 19, AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.lO, AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36,AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.46, AAVrh.48, AAVrh.48.1, AAVrh.48. L2, AAVrh.48.2, AAVrh.49, AAVA.51, AAVrh.52, AAVrh.53, AAVrh.54, AAVrh.56, AAVrh.57, AAVrh.58, AAVA.61, AAVrh.64, AAVA.64R1, AAVA.64R2, AAVrh.67, AAVrh.73, AAVrh.74, AAVA8R, AAVA8R A586R mutant, AAVA8R R533A mutant, AAAV, BAAV, caprine AAV, bovine AAV, AAVhEl . 1, AAVhErl .5, AAVhERl . 14, AAVhErl .8, AAVhErl . 16, AAVhErl . 18, AAVhErl .35, AAVhErl .7, AAVhErl .36, AAVhEr2.29, AAVhEr2.4, AAVhEr2. 16, AAVhEr2.30, AAVhEr2.3 1, AAVhEr2.36, AAVhERl .23, AAVhEr3. 1, AAV2.5T , AAV-PAEC, AAV-LK01, AAV-LK02, AAV- LK03, AAV-LK04, AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV- LK10, AAV-LK11, AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK16, AAV- LK17, AAV-LK18, AAV-LK19, AAV-PAEC2, AAV-PAEC4, AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAV-PAEC 11, AAV-PAEC 12, AAV-2-pre-miRNA-101, AAV-8h, AAV- 8b, AAV-h, AAV-b, AAV SM 10-2, AAV Shuffle 100-1, AAV Shuffle 100-3, AAV Shuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAV Shuffle 100- 2, AAV SM 10-1, AAV SM 10-8, AAV SM 100-3, AAV SM 100-10, BNP61 AAV, BNP62 AAV, BNP63 AAV, AAVrh.50, AAVrh.43, AAVrh.62, AAVrh.48, AAVhu. 19, AAVhu. 1 1, AAVhu.53, AAV4-8 / rh.64, AAVLG-9 / hu.39, AAV54.5 / hu.23, AAV54.2 / hu.22, AAV54.7 / hu.24, AAV54. l / hu.21, AAV54.4R / hu.27, AAV46.2 / hu.28, AAV46.6 / hu.29, AAV128. l / hu.43, true type AAV (ttAAV), UPEN AAV 10, Japanese AAV 10 serotypes, AAV CBr-7. 1, AAV CBr-7. 10, AAV CBr-7.2, AAV CBr-7.3, AAV CBr-7.4, AAV CBr-7.5, AAV CBr-7.7, AAV CBr-7.8, AAV CBr-B7.3, AAV CBr-B7.4, AAV CBr-El, AAV CBr- E2, AAV CBr-E3, AAV CBr-E4, AAV CBr-E5, AAV CBr-e5, AAV CBr-E6, AAV CBr-E7, AAV CBr-E8, AAV CHt-1, AAV CHt-2, AAV CHt-3, AAV CHt-6. 1, AAV CHt-6. 10, AAV CHt-6.5, AAV CHt-6.6, AAV CHt-6.7, AAV CHt-6.8, AAV CHt-Pl, AAV CHt-P2, AAV CHt-P5, AAV CHt-P6, AAV CHt-P8, AAV CHt-P9, AAV CKd-1, AAV CKd-10, AAV CKd-2, AAV CKd-3, AAV CKd-4, AAV CKd-6, AAV CKd-7, AAV CKd-8, AAV CKd-B 1, AAV CKd-B2, AAV CKd-B3, AAV CKd-B4, AAV CKd-B5, AAV CKd-B6, AAV CKd-B7, AAV CKd-B8, AAV CKd-Hl, AAV CKd-H2, AAV CKd-H3, AAV CKd-H4, AAV CKd- H5, AAV CKd-H6, AAV CKd-N3, AAV CKd-N4, AAV CKd-N9, AAV CLg-Fl, AAV CLg-F2, AAV CLg-F3, AAV CLg-F4, AAV CLg-F5, AAV CLg-F6, AAV CLg-F7, AAV CLg-F8, AAV CLv-1, AAV CLvl-1, AAV Clvl-10, AAV CLvl-2, AAV CLv-12, AAV CLvl-3, AAV CLv-1 3, AAV CLvl-4, AAV Clvl-7, AAV Clvl-8,AAV Clvl-9, AAV CLv- 2, AAV CLv-3, AAV CLv-4, AAV CLv-6, AAV CLv-8, AAV CLv-Dl, AAV CLv-D2, AAV CLv-D3, AAV CLv-D4, AAV CLv-D5, AAV CLv-D6, AAV CLv-D7, AAV CLv-D8, AAV CLv-El, AAV CLv-Kl, AAV CLv-K3, AAV CLv-K6, AAV CLv-L4, AAV CLv-L5, AAV CLv-L6, AAV CLv-Ml, AAV CLv-Ml 1, AAV CLv-M2, AAV CLv-M5, AAV CLv- M6, AAV CLv-M7, AAV CLv-M8, AAV CLv-M9, AAV CLv-Rl, AAV CLv-R2, AAV CLv-R3, AAV CLv-R4, AAV CLv-R5, AAV CLv-R6, AAV CLv-R7, AAV CLv-R8, AAV CLv-R9, AAV CSp-1, AAV CSp-10, AAV CSp-1 1, AAV CSp-2, AAV CSp-3, AAV CSp-4, AAV CSp-6, AAV CSp-7, AAV CSp-8, AAV CSp-8. 10, AAV CSp-8.2, AAV CSp-8.4, AAV CSp-8.5, AAV CSp-8.6, AAV CSp-8.7, AAV CSp-8.8, AAV CSp-8.9, AAV CSp-9, AAV.hu.48R3, AAV.VR-355, AAV3B, AAV4, AAV5, AAVF1 / HSC1, AAVF11 / HSC11, AAVF12 / HSC12, AAVF13 / HSC13, AAVF14 / HSC14, AAVF15 / HSC15, AAVF16 / HSC16, AAVF 17 / HSC 17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9 and variants thereof. In an embodiment, any sequential amino acids 449-459 and / or any sequential amino acids 452-463 of a capsid protein of AAV9, or in an analogous position of a capsid protein from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, AAVrh.10, or any of those listed above can be modified.

[0170] In an embodiment, the engineered capsid is a modified AAV1 capsid and can have a n-mer motif inserted after or a neighbor of amino acid 590 (i.e., between amino acids 590 and 591).

[0171] In an embodiment, the engineered capsid is a modified AAV3 capsid and can have a n-mer motif inserted after or a neighbor of amino acid 586.

[0172] In an embodiment, the engineered capsid is a modified AAV4 capsid and can have a n-mer motif inserted after or a neighbor of amino acid 586.

[0173] In an embodiment, the engineered capsid is a modified AAV5 capsid and can have a n-mer motif inserted after or a neighbor of amino acid 575.

[0174] In an embodiment, the engineered capsid is a modified AAV6 capsid that can have an n-mer inserted at or near amino acid 585 and optionally Y705-731, T492V, and K53 IE.

[0175] In an embodiment, the engineered capsid is a modified AAV8 capsid and can have a n-mer inserted after or a neighbor of amino acids 585 and 590.

[0176] In an embodiment, the engineered capsid is a modified AAV9 capsid and can have a n-mer inserted in between amino acids 588 and 589. (Biining, H.; Srivastava, A. CapsidModifications for Targeting and Improving the Efficacy of AAV Vectors. Molecular Therapy -Methods & Clinical Development 2019, 12, 248-265). In an embodiment, the engineered capsid can have a n-mer motif inserted between amino acids 588 and 589 of an AAV9 viral protein. SEQ ID NO: l isa reference AAV9 capsid sequence for at least referencing the insertion sites discussed above. In an embodiment, the engineered capsid can have a 7-mer motif inserted between two consecutive amino acids within amino acids 451-460 of the AAV9 capsid protein. SEQ ID NO: 1 is a reference AAV9 capsid sequence for at least referencing the insertion sites discussed above.

[0177] In an embodiment, the first 1, 2, 3, or 4 amino acids of a target binding modification can replace 1, 2, 3, or 4 amino acids of an AAV capsid polypeptide into which it is inserted and preceding an insertion site, such as those mentioned above. Using an AAV as another nonlimiting example, one or more of the n-mers can be inserted into, e.g., an AAV9 capsid polypeptide between amino acids 588 and 589, and the target binding modification can replace amino acids 586, 587, and 588 such that the amino acid immediately preceding the target binding modification after insertion is residue 585. It will be appreciated that this principle can apply in any other insertion context and is not necessarily limited to insertion between residues 588 and 589 of an AAV9 capsid or equivalent position in another AAV capsid. In an example embodiment, the AAV capsid protein is selected from SEQ ID NO: 1.Central Nervous System (CNS) Targeting Modifications

[0178] In an embodiment, the targeting moiety promotes, increases, or otherwise improves binding to, and in some cases, transduction of central nervous system tissues.TFRC

[0179] In an embodiment, the targeting moiety effectively increases transduction of central nervous system (CNS) tissues by binding to the transferrin receptor (TFRC). In an example embodiment, the targeting moiety binds to one or more of the extracellular domain’s apical, helical, and / or protease-like domains. In an example embodiment, the targeting moiety binds to the apical domain. Design considerations for TFRC targeting moieties are described in International Patent Application Number PCT / US2023 / 070285 and US Patent Application Number 18 / 791,629, each of which is incorporated herein by reference in its entirety. In an example embodiment, the n-mer motif is selected from the group consisting of SEQ ID NO: 10952-20481 and 36241-42428 from International Patent Application Number PCT / US2023 / 070285, which is incorporated herein by reference. In an example embodiment, then-mer is selected from any of the amino acid sequences in Tables 1-22, or any combination thereof, from International Patent Application Number PCT / US2023 / 070285, which is incorporated herein by reference.

[0180] In an embodiment, the TfRl binding targeting moiety is selected from SEQ ID NO:40-167

[0181] In an example embodiment, the n-mer motif is selected from the group consisting of SEQ ID NO: 3-21 from US Patent Application Number 18 / 791,629. In an example embodiment, the n-mer is selected from any amino acid sequence in Tables 1-3 from US Patent Application Number 18 / 791,629.

[0182] In an embodiment, the n-mer is inserted between two amino acids of a VP1 capsid protein of AAV9, and the capsid further includes one or more mutations or amino acid substitutions near, around, or next to a n-mer motif as described in US Provisional Application Number 63 / 621,839, which is hereby incorporated by reference in its entirety. In an example embodiment, the one or more mutations or amino acid substitutions near, around, or next to a n-mer motif are selected from the group consisting of SEQ ID NO: 269-1606 of the instant application.CD59 polypeptide

[0183] In an embodiment, the targeting moiety enhances CNS tropism by binding to the CD59 polypeptide. Example target binding modifications for CD59 can be found in US Provisional Application Number 63 / 631,415, incorporated herein by reference.

[0184] In an example embodiment, the targeting moiety is selected from the group consisting of SEQ ID NO: 89-5983 from US Provisional Application Number 63 / 631,415. In an example embodiment, the targeting moiety is selected from any amino acid sequence in Table A of the US Provisional Application Number 63 / 631,415, which is incorporated herein by reference.Carbonic Anhydrase 4 (Ci 4)

[0185] In an embodiment, the targeting moiety is a Carbonic Anhydrase 4 (CA4) targeting moiety. Carbonic anhydrase IV is a GPI-anchored protein (i.e., carbonic anhydrase 4; CA4; CAIV; Car4; RP17), belonging to a zinc metalloenzyme family. CA4 is the most distributed isoform and maintains homeostasis of carbon dioxide and bicarbonate in the brain. Example CA4 target binding moieties are disclosed in US Provisional Application Number 63 / 631,410, incorporated herein by reference. In an example embodiment, the n-mer is selected from the groupconsisting of SEQ ID NO: 1-7 and 64-4320 from US Provisional Application Number 63 / 631,410. An example embodiment selects the n-mer from any amino acid sequence in Table A from US Provisional Application Number 63 / 631,410, which is incorporated by reference herein. In an embodiment, the CA4 targeting moiety is selected from SEQ ID NO: 168-169. Blood-Brain Barrier

[0186] In an embodiment, the targeting moiety enhances tropism for endothelial cells of the CNS vasculature. A target-binding modification with enhanced tropism for endothelial cells of the CNS vasculature promotes, increases, or otherwise improves binding. In some cases, transduction of the CN S vasculature is compared with that of a natural or wild-type target moiety. Example targeting moieties with enhanced tropism for endothelial cells of the CNS vasculature can be found in US Patent Application Number 18 / 580,716 and International Patent Application Publication Number WO 2024 / 163842 A2, both of which are incorporated herein by reference.

[0187] In an example embodiment, the targeting moiety is selected from any amino acid sequences in Table 1-6 from US Patent Application Number 18 / 580,716, which is incorporated herein by reference.Alkaline Phosphatase (ALPL}

[0188] In an embodiment, the targeting moiety is an Alkaline Phosphatase (ALPL) targetingbinding modification that confers on the capsid enhanced tropism for CNS endothelial cells. In an embodiment, the ALPL targeting moiety binds to an ALPL polypeptide. Design considerations for ALPL targeting moieties (i.e., ligand or peptide) can be found in International Patent Application Publication Number WO 2024030976 A2, which is incorporated herein by reference.

[0189] In an example embodiment, the targeting moiety is selected from the group consisting of SEQ ID NOs 200-940, 1800-2241, 2242-2886, or 2887-3076, as outlined in International Patent Application Publication Number WO 2024030976 A2. In an example embodiment, the targeting moiety is selected from any amino acid sequences in Table 1, 2A, 2B, 2C, or 13-19 from International Patent Application Publication Number WO 2024030976 A2, which is incorporated herein by reference.Muscle Tissue

[0190] In an embodiment, the targeting moiety is a muscle tissue targeting moiety. A target moiety with an enhanced tropism for cells associated with muscle tissue promotes, increases, or otherwise improves binding to, and in some cases, transduction of muscle cells as compared to anatural or wild-type target moiety. In an example embodiment, the muscle tissue targeting moiety binds to skeletal muscle. In an example embodiment, the muscle tissue targeting moiety binds to muscle stem cells. In an example embodiment, the muscle tissue targeting moiety binds to skeletal muscle cells. In an example embodiment, the muscle tissue targeting moiety binds to muscle fibers. In an example embodiment, the muscle tissue targeting moiety binds to cardiomyocytes.

[0191] In an embodiment, the muscle-specific targeting moiety comprises a RGD motif In an embodiment, the n-mer motif contains a second-generation RGD motif. In an embodiment, the RGD motif is selected from SEQ ID NO: 170-191, 1685-1699, 1705, 1708-1709. Design considerations for muscle-targeting moieties can be found in US Patent Application Publication Numbers US 2022 / 0340929 Al and US 2023 / 0159949 Al; US Patent Application Number 18 / 294,594; and International Patent Application Publication Number WO 2024 / 168266, each of which is incorporated herein by reference.

[0192] In an example embodiment, the targeting moiety is selected from the group consisting of SEQ ID NOs 13-50, 1277-2493, 3737-4979, 6647-8313, 8314-8502, or 8692-8889, as disclosed in US Patent Application Publication Number US 2022 / 0340929 Al, which is incorporated herein by reference.

[0193] In an example embodiment, the targeting moiety is selected from the group consisting of Table 2 (SEQ ID NO: 2-7, 20-21, 41-409), Table 3 (SEQ ID NO: 2, 28, 30-32, 55, 76, 96, 103, 135, 158, 207, 214, 252, 306, 316, 398, 410-768), FIG. 14F (SEQ ID NO: 8-12, 14-18), or any combination from US Patent Application Publication Number US 2023 / 0159949 Al, which is incorporated herein by reference.

[0194] In an example embodiment, the targeting moiety is selected from the group consisting of Table 4 (SEQ ID NO: 2-7, 20-21, 41-409), Table 5 (SEQ ID NO: 2, 28, 30-32, 55, 76, 96, 103, 135, 158, 207, 214, 252, 306, 316, 398, 410-768), FIG. 14F (SEQ ID NO: 8-12, 14-18), Table 6 (SEQ ID NO: 774-795), FIG. 13 (SEQ ID NO: 2-12), FIG. 14F (SEQ ID NO: 8-12, 14-18), FIG.19B (2-7, 20-21), FIG. 27B (SEQ ID NO: 2-12), FIG. 28B (28-32), FIG. 28D (SEQ ID NO: 28-30), or any combination from US Patent Application Number 18 / 294,594, which is incorporated herein by reference.

[0195] In an embodiment, the targeting moiety is selected from the group of targeting moieties listed in Tables 1-4 of International Patent Application Publication Number WO 2024 / 168266, which is incorporated herein by reference.

[0196] In an embodiment, the muscle-specific targeting moiety is selected from SEQ ID NO: 1700-1704 and 1706-1707. In an embodiment, the muscle-specific targeting moiety is selected from SEQ ID NO: 1710-1737.AAV Particle Production

[0197] There are two main strategies for producing AAV particles from AAV vectors and systems thereof, such as those described herein, which depend on whether the adenovirus helper factors are provided (helper v. helper-free). In an embodiment, a method of producing AAV particles from AAV vectors and systems thereof can include adenovirus infection into cell lines that stably harbor AAV replication and capsid encoding polynucleotides, along with an AAV vector containing the polynucleotide to be packaged and delivered by the resulting AAV particle (e.g., the engineered AAV capsid polynucleotide(s)). In an embodiment, a method of producing AAV particles from AAV vectors and systems thereof can be a “helper free” method, which includes co-transfection of an appropriate producing cell line with three vectors (e.g., plasmid vectors): (1) an AAV vector that contains a polynucleotide of interest (e.g., a transgene encoding a therapeutic protein or nucleic acid operably linked to a regulatory element that promotes expression in the target tissue) between 2 ITRs; (2) a vector that carries the AAV Rep-Cap encoding polynucleotide, including the engineered capsid protein described herein; and helper polynucleotides. One of ordinary skill in the art will appreciate various methods and variations thereof, both helper- and helper-free, as well as the different advantages of each system.

[0198] A vector (including non-viral carriers) described herein can be introduced into host cells to produce thereby transcripts, proteins, or peptides, including fusion proteins or peptides encoded by nucleic acids as described herein (e.g., engineered AAV capsid system transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc ), and virus particles (such as from viral vectors and systems thereof).

[0199] AAV capsids prepared from one or more engineered AAV capsid polynucleotides can be used to deliver a recombinant AAV (rAAV) genome encoding a therapeutic protein or nucleic acid of interest. Alternatively, adenovirus or other plasmid or viral vector types as previously described, can be used, in particular, using formulations and doses from, for example, US Patents Nos. 8,454,972 (formulations, doses for adenovirus), 8,404,658 (formulations, doses for AAV) and 5,846,946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus. For example, for AAV,the route of administration, formulation and dose can be as in US Patent No. 8,454,972 and as in clinical trials involving AAV. For Adenovirus, the route of administration, formulation and dose can be as in US Patent No. 8,404,658 and as in clinical trials involving adenovirus.

[0200] For plasmid delivery, the route of administration, formulation and dose can be as in US Patent No 5,846,946 and as in clinical studies involving plasmids. In an embodiment, doses can be based on or extrapolated to an average 70 kg individual (e.g., a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed. The viral vectors can be injected into or otherwise delivered to the tissue or cell of interest.

[0201] In terms of in vivo delivery, AAV is advantageous over other viral vectors for a couple of reasons such as low toxicity (this may be due to the purification method not requiring ultracentrifugation of cell particles that can activate the immune response) and a low probability of causing insertional mutagenesis because it does not integrate into the host genome.

[0202] The vector(s) and virus particles described herein can be delivered into a host cell in vitro, in vivo, and or ex vivo. Delivery can occur by any suitable method including, but not limited to, physical methods, chemical methods, and biological methods. Physical delivery methods are those methods that employ physical force to counteract the membrane barrier of the cells to facilitate intracellular delivery of the vector. Suitable physical methods include, but are not limited to, needles (e.g., injections), ballistic polynucleotides (e.g., particle bombardment, micro projectile gene transfer, and gene gun), electroporation, sonoporation, photoporation, magnetofection, hydroporation, and mechanical massage. Chemical methods are those methods that employ a chemical to elicit a change in the cells membrane permeability or other character! stic(s) to facilitate entry of the vector into the cell. For example, the environmental pH can be altered which can elicit a change in the permeability of the cell membrane. Biological methods are those that rely and capitalize on the host cell’s biological processes or biological characteristics to facilitate transport of the vector (with or without a carrier) into a cell. For example, the vector and / or its carrier can stimulate an endocytosis or similar process in the cell to facilitate uptake of the vector into the cell.

[0203] Delivery of engineered AAV capsid system components (e.g., polynucleotides encoding engineered AAV capsid and / or capsid proteins) to cells via particles. The term “particle” as used herein, refers to any suitable sized particles for delivery of the engineered AAV capsid system components described herein. Suitable sizes include macro-, micro-, and nano-sized particles. In an embodiment, any of the of the engineered AAV capsid system components (e.g., polypeptides, polynucleotides, vectors, and combinations thereof described herein) can be attached to, coupled to, integrated with, otherwise associated with one or more particles or component thereof as described herein. The particles described herein can then be administered to a cell or organism by an appropriate route and / or technique. In an embodiment, particle delivery can be selected and be advantageous for delivery of the polynucleotide or vector components. It will be appreciated that in embodiments, particle delivery can also be advantageous for other engineered capsid system molecules and formulations described elsewhere herein.

[0204] In an embodiment, a vector used in producing the rAAVs disclosed herein comprises a rep gene and a cap gene). The rep gene typically encodes Rep78, Rep68, Rep52, and Rep40 from a single ORF. These replication factors aid AAV genome replication and virion assembly. The cap gene typically encodes the three capsid proteins (i.e., virion protein 1 (VP1), VP2, and VP3) from a single ORF. In addition, the three capsid proteins are regulated by transcription from a start codon (ACG) and alternative splicing. The cap gene also encodes, from an in-frameshifted ORF, an assembly-activating protein (AAP). The AAP is essential for capsid assembly.

[0205] In an embodiment, the vector can include an engineered viral (e.g., AAV) capsid polynucleotide having a 3’ polyadenylation signal. In an embodiment, the 3’ polyadenylation is an SV40 polyadenylation signal. In an embodiment, the vector does not have splice regulatory elements. The vector includes one or more minimal splice regulatory elements in an embodiment. In an embodiment, the vector can further include a modified splice regulatory element, wherein the modification inactivates the splice regulatory element. In an embodiment, the modified splice regulatory element is a polynucleotide sequence sufficient to induce splicing between a rep protein polynucleotide and the engineered viral (e.g., AAV) capsid protein variant polynucleotide. In an embodiment, the polynucleotide sequence can be sufficient to induce splicing if it is a splice acceptor or a splice donor. In an embodiment, the viral (e.g., AAV) capsid polynucleotide is an engineered viral (e.g., AAV) capsid polynucleotide as described elsewhere. In someembodiments, the vector does not include one or more minimal splice regulatory elements, modified splice regulatory agent, splice acceptor, and / or splice donor.

[0206] The vectors and / or vector systems can be used, for example, to express one or more of the engineered viral (e.g., AAV) capsid and / or other polynucleotides in a cell, such as a producer cell, to produce engineered viral (e g., AAV) particles and / or other compositions (e.g., polypeptides, particles, etc.) containing an engineered viral (e.g., AAV) capsid or other composition containing an n-mer motif of the present invention described elsewhere herein. Other uses for the vectors and vector systems described herein are also within the scope of this disclosure. In general, and throughout this specification, the term is a tool that allows or facilitates the transfer of an entity from one environment to another. In some contexts that will be appreciated by those of ordinary skill in the art, “vector” can be a term of art to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. A vector can be a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted to replicate the inserted segment. Generally, a vector can replicate when associated with the proper control elements.

[0207] Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g., circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One vector type is a “plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g., retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)). Viral vectors also include polynucleotides a virus carries for transfection into a host cell. Certain vectors can autonomously replicate in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the host cell's genome upon introduction into the host cell and, thereby, are replicated along with the host genome. Moreover, certain vectors can direct the expression of genes to which they are operatively linked. Such vectors are referred to herein as“expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.

[0208] Recombinant expression vectors can be composed of a nucleic acid (e.g., a polynucleotide) of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which can be selected based on the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, “operably linked” and “operatively linked” are used interchangeably herein and further defined elsewhere. In the context of a vector, the term “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for the expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or a host cell when the vector is introduced into the host cell).

[0209] In an embodiment, the vector can be bicistronic. In an embodiment, a bicistronic vector can be used for one or more elements of the engineered viral (e.g., AAV) capsid system described herein. In an embodiment, the expression of elements of the engineered viral (e.g., AAV) capsid system described herein can be driven by a suitable constitutive or tissue-specific promoter. Where the element of the engineered viral (e.g., AAV) capsid system is an RNA, its expression can be driven by a Pol III promoter, such as a U6 promoter. In an embodiment, the two are combined.Polynucleotides Encoding AAVs

[0210] The engineered viral capsid and / or capsid proteins can be encoded by one or more engineered viral capsid polynucleotides. In an embodiment, the engineered viral capsid polynucleotide is an engineered AAV capsid polynucleotide or engineered adenovirus capsid polynucleotide. In an embodiment, an engineered viral capsid polynucleotide (e.g., an engineered AAV capsid polynucleotide or engineered adenovirus capsid polynucleotide) can include a 3’ polyadenylation signal. The polyadenylation signal can be an SV40 polyadenylation signal. In an embodiment, the engineered polynucleotide can be included in a polynucleotide that is configured to express the engineered capsid in a host cell system for production of viral particles. In an embodiment, the engineered AAV capsid encoding polynucleotide can be included in a polynucleotide that is configured to express the engineered capsid in a host cell system for production of AAV viral particles.Cell-based Vector Amplification and Expression

[0211] Vectors comprising the aforementioned polynucleotides can be designed to express one or more elements of the engineered viral (e.g., AAV) capsid in a suitable host cell. Vectors can further be designed for expression of one or more elements of the engineered viral (e.g., AAV) capsid system or other compositions containing a target binding modification of the present disclosure described herein (e.g., nucleic acid transcripts, proteins, enzymes, and combinations thereof) in a suitable host cell.

[0212] In an embodiment, the suitable host cell is prokaryotic. Suitable host cells include, but are not limited to, bacterial cells, yeast cells, insect cells, and mammalian cells. The vectors can be viral-based or non-viral-based. In an embodiment, the suitable host cell is eukaryotic. In an embodiment, the suitable host cell is a suitable bacterial cell. Suitable bacterial cells include but are not limited to, bacterial cells from Escherichia coli bacteria. Many suitable strains of E. coli are known in the art for the expression of vectors. These include but are not limited to, Pirl, Stbl2, Stbl3, Stbl4, TOPIO, XL1 Blue, and XL10 Gold. In an embodiment, the host cell is a suitable insect cell. Suitable insect cells include those from Spodoptera frugiperda. Suitable strains of S. frugiperda cells include but are not limited to, Sf9 and Sf 1. In an embodiment, the host cell is a suitable yeast cell. In an embodiment, the yeast cell can be from Saccharomyces cerevisiae. In an embodiment, the host cell is a suitable mammalian cell. Many types of mammalian cells have been developed to express vectors. Suitable mammalian cells include but are not limited to, HEK293, Chinese Hamster Ovary Cells (CHOs), mouse myeloma cells, HeLa, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO-Rb50, HepG G2, DIKX-X11, J558L, Baby hamster kidney cells (BHK), and chicken embryo fibroblasts (CEFs). Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).

[0213] In an embodiment, the vector can be a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerevisiae include pYepSecl (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.). As used herein, a "yeast expression vector" refers to a nucleic acid that contains one or more sequences encoding an RNA and / or polypeptide and may further contain any desired elements that control the expression of the nucleic acid(s), as well asany elements that enable the replication and maintenance of the expression vector inside the yeast cell. Many suitable yeast expression vectors and features thereof are known in the art; for example, various vectors and techniques are illustrated in Yeast Protocols, 2nd edition, Xiao, W., ed. (Humana Press, New York, 2007) and Buckholz, R.G. and Gleeson, M.A. (1991) Biotechnology (NY) 9(11): 1067-72. Yeast vectors can contain, without limitation, a centromeric (CEN) sequence, an autonomous replication sequence (ARS), a promoter, such as an RNA Polymerase III promoter, operably linked to a sequence or gene of interest, a terminator such as an RNA polymerase III terminator, an origin of replication, and a marker gene (e.g., auxotrophic, antibiotic, or other selectable markers). Examples of expression vectors for use in yeast may include plasmids, yeast artificial chromosomes, 2p plasmids, yeast integrative plasmids, yeast replicative plasmids, shuttle vectors, and episomal plasmids.

[0214] In an embodiment, the vector is a baculovirus or an expression vector and is suitable for expressing polynucleotides and / or proteins in insect cells. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al., 1983, Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989, Virology 170: 31-39). rAAV (recombinant Adeno-associated viral) vectors are preferably produced in insect cells, e.g., Spodoptera frugiperda Sf9 insect cells, grown in serum-free suspension culture. Serum-free insect cells can be purchased from commercial vendors, e.g., Sigma Aldrich (EXCELL 405).

[0215] In an embodiment, the vector is a mammalian expression vector. In an embodiment, the mammalian expression vector can express one or more polynucleotides and / or polypeptides in a mammalian cell. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, 1987, Nature 329: 840) and pMT2PC (Kaufman, et al., 1987, EMBO J. 6: 187-195). The mammalian expression vector can include one or more suitable regulatory elements capable of controlling the expression of one or more polynucleotides and / or proteins in the mammalian cell. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. More details on suitable regulatory elements are described elsewhere herein.

[0216] For other suitable expression vectors and vector systems for both prokaryotic and eukaryotic cells, see, e.g., Chapters 16 and 17 of Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0217] In an embodiment, the recombinant mammalian expression vector can direct the nucleic acid's expression preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43 : 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Set. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary glandspecific promoters (e g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the a-fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546). Regarding these prokaryotic and eukaryotic vectors, mention is made of U.S. Patent 6,750,059, which are incorporated by reference herein in their entirety. Other embodiments can utilize viral vectors, which are mentioned in U.S. Patent application 13 / 092,085 and incorporated by reference herein in their entirety. Tissue-specific regulatory elements are known in the art. In this regard, mention is made of U.S. Patent 7,776,321, which are incorporated by reference herein in their entirety. In an embodiment, a regulatory element can be operably linked to a transgene in a recombinant genome packaged by the engineered AAV capsid system to drive expression of the one or more elements of the transgene delivered by the viral vector as described herein in a tissue-specific manner.

[0218] Vectors may be introduced and propagated in a prokaryote or prokaryotic cell. In an embodiment, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g., amplifying a plasmid as part of a viral vector packaging system). In an embodiment, a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism.

[0219] In an embodiment, the vector can be a fusion expression vector. In an embodiment, fusion vectors add a number of amino acids to a protein encoded therein, such as the amino terminus, carboxy terminus, or both of a recombinant protein. Such fusion vectors can serve one or more purposes, such as (i) to increase the expression of recombinant protein, (ii) to increase the solubility of the recombinant protein, and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. In an embodiment, expression of polynucleotides (such as non-coding polynucleotides) and proteins in prokaryotes can be carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion polynucleotides and / or proteins. In an embodiment, the fusion expression vector can include a proteolytic cleavage site, which can be introduced at the junction of the fusion vector backbone or other fusion moiety and the recombinant polynucleotide or protein to enable separation of the recombinant polynucleotide or protein from the fusion vector backbone or another fusion moiety after purification of the fusion polynucleotide or protein. Such enzymes and their cognate recognition sequences include Factor Xa, thrombin, and enterokinase. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET lid (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0220] In an embodiment, two or more of the elements expressed from the same or different regulatory element(s) can be combined in a single vector, with one or more additional vectors providing any components of the system not included in the first vector. Engineered polynucleotides of the present invention that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element and oriented in the same or opposite direction. In an embodiment, a single promoter drives expression of a transcript encoding one or more engineered viral (e.g., AAV) capsid proteinsVector Features

[0221] The vectors can include additional features that can confer one or more functionalities to the vector, the polynucleotide to be delivered, a virus particle produced therefrom, or a polypeptide expressed thereof. Such features include, but are not limited to, regulatory elements, selectable markers, molecular identifiers (e.g., molecular barcodes), stabilizing elements, and the like. It will be appreciated by those skilled in the art that the design of the expression vector and the additional features included can depend on factors such as the choice of host cell to be transformed, the level of expression desired, etc.Regulatory Elements

[0222] In embodiments, the polynucleotides and / or vectors thereof described herein (including, but not limited to, the engineered AAV capsid polynucleotides of the present disclosure) can include one or more regulatory elements that can be operatively linked to the polynucleotide. The term “regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g., transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). In an embodiment, the engineered AAV capsid encoding polynucleotide can be operably coupled to a polyadenylation tail. In an embodiment, the poly adenylation tail can be an SV40 poly adenylation tail. Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cells and those that direct expression in only certain host cells (e.g., tissue-specific regulatory sequences). The host cell system may also include a construct that expresses a recombinant viral genome that comprises a transgene encoding a polypeptide or nucleic acid operably linked to one or more regulatory sequences that promote expression of the transgene in a target cell, including a recombinant AAV genome where AAV ITR sequences flank the transgene and regulatory sequences. A tissue-specific promoter can direct expression primarily in a desired tissue, such as muscle, neurons, bone, skin, blood, specific organs (e.g., liver, brain), or particular cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a temporally dependent manner, such as cell-cycle- or developmental-stage-dependent expression, which may or may not also be tissue- or cell-type-specific. In an embodiment, a vector comprises one or more pol III promoter (e.g., 1, 2, 3, 4, 5, or more pol IIIpromoters), one or more pol II promoters (e.g., 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g., 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and Hl promoters. Examples of pol II promoters include, but are not limited to, 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 P-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter. Also encompassed by the term “regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5’ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit P-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981).

[0223] In an embodiment, the regulatory sequence can be as described in U.S. Pat. No.7,776,321, U.S. Pat. Pub. No. 2011 / 0027239, and PCT publication WO 2011 / 028929, the contents of which are incorporated by reference. In an embodiment, the vector can contain a minimal promoter. In an embodiment, the minimal promoter is the Mecp2 promoter, tRNA promoter, or U6. In a further embodiment, the minimal promoter is tissue-specific. In an embodiment, the length of the vector polynucleotide, the minimal promoters, and the polynucleotide sequences are less than 4.4Kb.

[0224] To express a polynucleotide, the vector can include one or more transcriptional and / or translational initiation regulatory sequences, e.g., promoters, that direct the transcription of the gene and / or translation of the encoded protein in a cell. In an embodiment, a constitutive promoter may be employed. Suitable constitutive promoters for mammalian cells are generally known in the art and include, but are not limited to, SV40, CAG, CMV, EF-la, P-actin, RSV, and PGK. Suitable constitutive promoters for bacterial, yeast, and fungal cells are generally known in the art, such as a T7 promoter for bacterial expression and an alcohol dehydrogenase promoter for yeast expression.

[0225] In an embodiment, the regulatory element can be a regulated promoter. In an embodiment, the AAV capsid encoding polynucleotide can be operably coupled to a promoter. “Regulated promoter” refers to promoters that direct gene expression not constitutively, but in a temporally- and / or spatially-regulated manner, and includes tissue-specific, tissue-preferred, and inducible promoters. In an embodiment, the regulated promoter is a tissue-specific promoter aspreviously discussed elsewhere herein. In an embodiment, the regulatory sequence that controls the expression of the transgene is a promoter, which can be tissue- or cell-type specific. Regulated promoters include conditional promoters and inducible promoters. In an embodiment, conditional promoters can be employed to direct expression of a polynucleotide in a specific cell type, under specific environmental conditions, and / or during a specific developmental stage. Suitable tissue specific promoters can include, but are not limited to, liver specific promoters (e.g., AP0A2, SERPIN Al (hAAT), CYP3A4, and MIR122), pancreatic cell promoters (e.g., INS, IRS2, Pdxl, Alx3, Ppy), cardiac specific promoters (e.g. Myh6 (alpha MHC), MYL2 (MLC-2v), TNI3 (cTnl), NPPA (ANF), Slc8al (Ncxl)), central nervous system cell promoters (SYN1, GFAP, INA, NES, MOBP, MBP, TH, F0XA2 (HNF3 beta)), skin cell specific promoters (e.g., FLG, K14, TGM3), immune cell specific promoters, (e.g. ITGAM, CD43 promoter, CD14 promoter, CD45 promoter, CD68 promoter), urogenital cell specific promoters (e.g., Pbsn, Upk2, Sbp, Ferll4), endothelial cell specific promoters (e.g., ENG), pluripotent and embryonic germ layer cell specific promoters (e.g. Oct4, NANOG, Synthetic Oct4, T brachyury, NES, SOX17, F0XA2, MIR122), and muscle cell specific promoter (e.g., Desmin). Other tissue and / or cell-specific promoters are discussed elsewhere herein and can be generally known in the art and are within the scope of this disclosure.

[0226] Inducible / conditional promoters can be positively inducible / conditional promoters (e.g., a promoter that activates transcription of the polynucleotide upon appropriate interaction with an activated activator, or an inducer (compound, environmental condition, or other stimulus) or a negative / conditional inducible promoter (e.g., a promoter that is repressed (e.g., bound by a repressor) until the repressor condition of the promotor is removed (e.g., inducer binds a repressor bound to the promoter stimulating release of the promoter by the repressor or removal of a chemical repressor from the promoter environment). The inducer can be a compound, environmental condition, or other stimulus. Thus, inducible / conditional promoters can be responsive to various stimuli, such as chemical, biological, or other molecular agents, temperature, light, and / or pH. Suitable inducible / conditional promoters include, but are not limited to, Tet-On, Tet-Off, Lac promoter, pBad, AlcA, LexA, Hsp70 promoter, Hsp90 promoter, pDawn, XVE / OlexA, GVG, and pOp / LhGR.

[0227] In an embodiment, the tissue-specific promoter is specific for muscle (e.g., cardiac, skeletal, and / or smooth muscle), neurons or other nervous system cells (e.g., astrocytes, glial cells, Schwann cells, ependymal cells, pericyte, oligodendrocyte, oligodendrocyte progenitor), specificneuronal subtype (e.g., dopaminergic neuron; Purkinje Cell; Parvalbumin, somatostatin, VIP inhibitory neuron; medium spiny neuron, Pyramidal neuron, motor neuron, etc.), endothelial cell, fat, spleen, liver, kidney, immune cells, synovial fluid cells, skin cells, cartilage, tendons, connective tissue, bone, pancreas, adrenal gland, blood cell, bone marrow cells, placenta, endothelial cells, and combinations thereof. In an embodiment, the promoter can be constitutive. Suitable tissue-specific promoters and constitutive promoters are discussed elsewhere herein and are generally known in the art and can be commercially available. Suitable neuronal tissue / cell-specific promoters include, but are not limited to, GFAP promoter (astrocytes), SYN1 promoter (neurons), and NSE / RU5’ (mature neurons). In an embodiment, the regulatory sequence that controls the expression of the transgene is a promoter, which can be a cell-state-regulating promoter or a drug-inducible promoter.

[0228] A neuron-specific promoter refers to a promoter that, when administered, e.g., peripherally, directly into the central nervous system (CNS), or delivered to neuronal cells, including in vitro, ex vivo, or in vivo, preferentially drives or regulates expression of an operably-linked transgene in neurons as compared to expression in non-neuronal cells. Non-limiting example of tissue-specific expression elements for neurons include neuron-specific enolase (NSE) (see, e.g., EMBL HSEN02, X51956); an aromatic amino acid decarboxylase (AADC) promoter; a neurofilament promoter (see, e.g., GenBank HUM NFL, L04147); a synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1 promoter (see, e.g., Chen et al„ (1987) Cell, 51 :7-19; Llewellyn et al. (2010) Nat. Med., 16(10):l 161-1 166); a serotonin receptor promoter (see, e.g., GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Oh et al., (2009) Gene Ther., 16:437; Sasaoka et al., (1992) Mol. Brain Res., 16:274; Boundy et al., (1998) J. Neurosci. , 18:9989; and Kaneda et al., (1991) Neuron, 6:583-594); a methyl-CpG binding protein 2 (MeCP2) promoter, an optimized methyl- CpG binding protein 2 (MeCP2) promoter (the published International Patent Application No. W02020180928, the content of which is incorporated by reference herein in its entirety), a Ca2+-calmodulin-dependent protein kinase II-alpha (CaMKIIa) promoter (see, e.g., Mayford et al., (1996) Proc. Natl. Acad. Sci. USA, 93:13250; and Casanova et al., (2001) Genesis, 31 :37); a GnRH promoter (see, e.g., Radovick et al., (1991) Proc. Natl. Acad. Sci. USA, 88:3402- 3406); an L7 promoter (see, e.g., Oberdick et al., (1990) Science, 248:223-226); a DNMT promoter (see, e.g., Badge et al., (1988) Proc. Natl. Acad. Sci. USA, 85:3648-3652); an enkephalin promoter (see, e.g., Comb et al., (1988) EMBOJ., 17:3793-3805); a myelin basic protein (MBP) promoter; a CMV enhancer / platelet-derived growth factor-p promoter (see, e.g., Liu et al., (2004) Gene Ther., 11 :52-60); and the like. In some aspects, a portion of or all the minimal human synapsin 1 promoter (SYN) can be used (Kugler et al., (2003) Gene Ther., 10(4): 337-47; Thiel et al, (1991) Proc. Natl. Acad. Sci. USA, 88(8) 3431 -5; Castle et al., (2016) Methods Mol. Biol., 1382: 133-49; McLean et al., (2014) Neurosci. Lett., 576: 73-78; Kugler et al., (2003) Virology, 311 (1): 89-95). In other aspects, the neural-specific promoter can be mGluR2, NFL, NFH, np2, PPE, Enk and EAAT2 promoters. A non-limiting example of a tissue-specific expression elements for astrocytes include the glial fibrillary acidic protein (GFAP) and EAAT2 promoters. A non-limiting example of a tissuespecific expression element for oligodendrocytes include the myelin basic protein (MBP) promoter. In certain aspects, a neuronal promoter can include a neuronal enhancer to direct expression to specific regions of the brain (see, for example, published U.S. Patent Application No.2019 / 0247516, the content of which is incorporated by reference herein in its entirety). In one aspect, the promoter can be a fugu SST (somatostatin) promoter (Nathanson, et al. Frontiers in Neural Circuits 3: 19). Examples of retinal-specific promoters include, but are not limited to, NA65p (RPE cells), Nefh (ganglion cells), hGRKl (rod and cone photoreceptor cells), hRLBPl (Muller glial cells and RPE cells), human RHO (rhodopsin), human rhodopsin kinase (RH0K / GRK1) (an exemplary list of retina cell-specific promoters can be found in Buck et al. (2020) International Journal of Molecular Sciences 21 (12), the content of which is incorporated by reference in its entirety). Non-limiting examples of liver promoters include hAAT and TBG. Non-limiting examples of skeletal muscle promoters include Desmin, MCK and C5-12. Additional exemplary tissue-specific promoters can be found in the TiProD (Tissue-specific promoter database webpage tiprod.bioinf.med.uni-goettingen.de).

[0229] In other embodiments, a promoter can be an inducible promoter (i.e., a promoter whose activity is controlled by an external stimulus, such as a particular temperature, compound, or protein). In an embodiment, a promoter may be temporally restricted, driving expression only during its temporal window. For example, a temporally restricted promoter may drive expression only during specific stages of a biological process. Prokaryotic (Gossen et al. TIBS 18: 471475, 1993) and insect regulatory systems (No et al. Proc. Natl. Acad. Sci. USA 93: 3346-3351, 1996) have been adapted to construct gene switches that function in mammalian cells. Since inducer molecules are not expected to have targets in mammalian cells, the possibility of interference withcellular processes is reduced. Of the prokaryotic proteins, the repressors from the lac operon (Brown, M., et at. Cell 49: 603- 612, 1987; and Hu, M. C. -T. andN. Davidson Cell 48: 555-566, 1987), the tet operon (e.g., U.S. Patent No. 7,541,446, the content of which is incorporated by reference herein in its entirety) and the cumate operon (e.g., U.S. Patent No. 7,745,592, the content of which is incorporated by reference herein in its entirety) have been shown to function in mammalian cells. Many have been incorporated into eukaryotic inducible expression systems using different strategies to control activation and repression. Activation of expression is mediated by a chimeric trans-activator protein formed by the fusion of the bacterial repressor with an activation domain (Gossen, M. and H. Bujard, Proc. Natl. acad. Sci. USA 89: 5547- 5551, 1992, and Gossen, M., et al. Science 268: 1766-1769, 1995; U.S. Patent No.7,745,592, the contents of which are incorporated by reference herein in their entirety. The trans-activator can activate transcription when bound to its DNA recognition sequence placed upstream of the minimal promoter. The ability of the activator to bind DNA is dependent on the presence / absence of the inducer molecule (e.g., doxycycline or cumate, depending on the inducible system being used). Repression of expression is mediated by the repressor bound to operator sites placed downstream of the minimal promoter in the absence of inducer, and repression is relieved on the addition of the inducer (Brown, M., et al. Cell 49: 603-612, 1987). In one aspect, the promoter may be less than 1 kb. The promoter may have a length of 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 or more than 800. The promoter may have a length between 200-300, 200-400, 200-500, 200-600, 200-700, 200-800, 300-400, 300-500, 300-600, 300-700, 300-800, 400-500, 400-600, 400-700, 400-800, 500-600, 500-700, 500-800, 600-700, 600-800 or 700-800 nucleotides. In one aspect, the promoter can be a pol Ill-dependent promoter, e.g., a U6 snRNA or Hl-RNA promoter, for the expression of noncoding RNAs, including, but not limited to, U snRNAs or miRNAs. In another aspect, the promoter can be a polymerase II U snRNA-dependent promoter, e.g., a human U1 snRNA gene and its promoter and terminator regions (see, for example, published U.S. Patent No.7, 947, 823, the content of which is incorporated by reference herein in its entirety).

[0230] Suitable muscle-specific promoters include, but are not limited to CK8, MHCK7, Myoglobin promoter (Mb), Desmin promoter, muscle creatine kinase promoter (MCK) and variants thereof, and SPc5-12 synthetic promoter.

[0231] Suitable immune cell-specific promoters include, but are not limited to, B29 promoter (B cells), CD 14 promoter (monocytic cells), CD43 promoter (leukocytes and platelets), CD68 (macrophages), and SV40 / CD43 promoter (leukocytes and platelets).

[0232] Suitable blood cell-specific promoters include, but are not limited to, CD43 promoter (leukocytes and platelets), CD45 promoter (hematopoietic cells), IFN-beta (hematopoietic cells), WASP promoter (hematopoietic cells), SV40 / CD43 promoter (leukocytes and platelets), and SV40 / CD45 promoter (hematopoietic cells).

[0233] Suitable pancreatic-specific promoters include, but are not limited to, the Elastase- 1 promoter.

[0234] Suitable endothelial cell-specific promoters include, but are not limited to, Fit-1 promoter and ICAM-2 promoter.

[0235] Suitable neuronal tissue / cell-specific promoters include, but are not limited to, GFAP promoter (astrocytes), SYN1 promoter (neurons), andNSE / RU5’ (mature neurons).

[0236] Suitable kidney-specific promoters include, but are not limited to, the NphsI promoter (podocytes).

[0237] Suitable bone-specific promoters include, but are not limited to, the OG-2 promoter (osteoblasts, odontoblasts).

[0238] Suitable lung-specific promoters include, but are not limited to, the SP-B promoter (lung).

[0239] Suitable liver-specific promoters include, but are not limited to, SV40 / Alb promoter.

[0240] Suitable heart-specific promoters include, but are not limited to, the alpha-MHC promoter.

[0241] Suitable constitutive promoters include, but are not limited to CMV, RSV, SV40, EFl alpha, CAG, and beta-actin.

[0242] Additional promoters can be found in Wang, E. T.-S. & Poukalov, K. K. Methods and compositions to confer regulation to gene therapy cargoes by heterologous use of alternative splicing cassettes. World Patent (2022); Boyne, A. R., Danos, O. F., Voiles, M. J., & Guo, X. Regulation of gene expression by aptamer-mediated modulation of alternative splicing. US Patent(2022); Ranum, P. T., Monteys, A. M., Hundley, A. A. & Davidson, B. L. Compositions and methods for inducible alternative splicing regulation of gene expression. World Patent (2021); Monteys, A. M. et al. Regulated control of gene therapies by drug-induced splicing. Nature 1-5 (2021); Doshi, A., Sadeghi, F., Varadarajan, N. & Cirino, P. C. Small-molecule inducible transcriptional control in mammalian cells. Crit. Rev. Biotechnol. 40, 1131-1150 (2020); Monteys, A. M. et al. Regulated control of gene therapies with a drug-induced switch.2020.02.21.956664 (2020) doi: 10.1101 / 2020.02.21.956664.; Davidson, B. L., Monteys, A. M., Al HUNDLEY, A. & Ranum, P. T. ALTERNATIVE SPLICING REGULATION OF GENE EXPRESSION AND THERAPEUTIC METHODS. CT (2020); Boyne, A. R , Olivier DANOS, F., Michael VOLLES, J. & Xuecui, G. U. O. Regulation of gene expression by aptamer-mediated modulation of alternative splicing. World Patent (2016); REGULATABLE EXPRESSION SYSTEMS. World Patent, BERGLUND, lohn, Andrew DELGADO, Elizabeth IENQUIN, lana, Rose WANG, Eric, Tzy-Shi. GENE THERAPY VECTORS. World Patent, incorporated herein by reference in its entirety.

[0243] In an embodiment, the vector or system thereof can include one or more elements capable of translocating and / or expressing an engineered polynucleotide of the present disclosure (e.g., an engineered viral (e.g., AAV) capsid polynucleotide) to / in a specific cell component or organelle. Such organelles can include, but are not limited to, the nucleus, ribosome, endoplasmic reticulum, Golgi apparatus, chloroplast, mitochondria, vacuole, lysosome, cytoskeleton, plasma membrane, cell wall, peroxisome, centrioles, etc.CpG content

[0244] In one aspect, a rAAV vector, including an rAAV vector genome as described herein, comprises at least one synthetic AAV ITR, wherein one or more CpG islands (a cytosine base followed immediately by a guanine base (a CpG) in which the cytosines in such arrangement tend to be methylated) that typically occur at, or near the transcription start site in an ITR are deleted and / or substituted. In one aspect, a decrease in the number of CpG islands can reduce the immunogenicity of the rAAV vector. This results from reduced or complete inhibition of TLR-9 binding to the rAAV vector DNA sequence at CpG islands. It is also well known that methylation of CpG motifs results in transcriptional silencing. Removal of CpG motifs in the ITR is expected to result in decreased TLR-9 recognition and / or reduced methylation and therefore, decreased transgene silencing. In some aspects, it is the minimal functional ITR in which one or more CpGislands are deleted and / or substituted. In one aspect, AAV ITR2 is known to contain 16 CpG islands, of which one or more, or all 16, can be deleted.

[0245] In some aspects, at least 1 CpG motif is deleted and / or substituted, e.g., at least four or more or eight or more CpG motifs, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 CpG motifs. The phrase “deleted and / or substituted” as used herein means that one or both nucleotides in the CpG motif are deleted, substituted with a different nucleotide, or any combination of deletions and substitutions.

[0246] In certain aspects, the transgene nucleic acid sequence can also be codon-optimized to enhance expression in vivo and / or to reduce the number of CpG islands and to avoid an innate immune response to the vector. An example of CpG depletion can be found in the published International Application No. PCT / US2023 / 067901, the content of which is incorporated by reference herein in its entirety.Dual AA V vectors

[0247] Various strategies have been investigated to overcome the limitation of AAV cargo capacity. Several groups have attempted to “force” large genes into one of the many AAV capsids available by developing the so-called oversized vectors. Although administration of oversize AAV vectors can achieve therapeutically relevant levels of transgene expression in rodent and canine models of human inherited diseases, including the retina of the Abca4~ / ~ and shaker 1 (shl) mouse models of STGD and USH1B, the mechanism underlying oversize AAV-mediated transduction remains elusive. Oversized AAV vectors do not contain a pure population of intact large-size genomes but rather a heterogeneous mixture of mostly truncated genomes<5 kb in length. Following infection, reassembling these truncated genomes in the target cell nucleus has been proposed as a mechanism for oversized AAV vector transduction. Independent of transduction mechanism and in vivo efficacy, the heterogeneity in oversized AAV genome sizes is a major limitation for their application in human gene therapy.

[0248] Alternatively, the inherent ability of AAV genomes to undergo intermolecular concatemerization can be exploited to transfer large genes in vivo by splitting a large gene expression cassette into halves (<5 kb in size), each contained in one of two separate (dual) AAV vectors. In the dual AAV trans-splicing strategy, a splice donor (SD) signal is placed at the 3' end of the 5’ - half vector, and a splice acceptor (SA) signal is placed at the 5' end of the 3 -half vector. Upon coinfection of the same cell with the dual AAV vectors, inverted terminal repeat (ITR)-mediated head-to-tail concatemerization of the two halves results in trans-splicing, producing a mature mRNA and a full-size protein. Trans-splicing has been successfully used to express large genes in muscle and retina.

[0249] Alternatively, the two halves of a large transgene expression cassette contained in dual AAV vectors may contain homologous overlapping sequences (at the 3' end of the 5 ’-half vector and at the 5' end of the 3 '-half vector, dual AAV overlapping), which will mediate reconstitution of a single large genome by homologous recombination. This strategy depends on the recombinogenic properties of the transgene overlapping sequences.

[0250] A third dual AAV strategy (hybrid) involves adding a highly recombinogenic region from an exogenous gene (e.g., alkaline phosphatase, AP) to the trans-splicing vector. The added region is placed downstream of the SD signal in the 5'-half vector and upstream of the SA signal in the 3 ’-half vector to increase recombination between the dual AAVs. The published US patent application No. 2010 / 003218 and US Patent No. 10,494,645, both of which are incorporated by reference herein in their entireties, provide additional examples of dual vector systems.Cell-free Vector and Polynucleotide Expression

[0251] In an embodiment, the polynucleotide(s) encoding an engineered AAV capsid polypeptide comprising one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, andN716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, optionally one or more target binding modifications, of the present disclosure can be expressed from a vector or suitable polynucleotide in a cell-free in vitro system. In an embodiment, the polynucleotide encoding one or more features of the engineered AAV capsid system can be expressed in a cell-free in vitro system from a vector or a suitable polynucleotide. In other words, the polynucleotide can be transcribed and optionally translated in vitro. In vitro transcription / translation systems and appropriate vectors are generally known in the art and commercially available. Generally, in vitro transcription and translation systems replicate the processes of RNA and protein synthesis, respectively, outside the cellular environment. Vectors and suitable polynucleotides for in vitro transcription can include T7, SP6, T3, and other promoters, as well as regulatory sequencesrecognized and acted upon by an appropriate polymerase to transcribe the polynucleotide or vector.

[0252] In vitro translation can be performed as a stand-alone process (e.g., translation of a purified polyribonucleotide) or coupled to transcription. In an embodiment, the cell-free (or in vitro) translation system can include extracts from rabbit reticulocytes, wheat germ, and / or E. coli. The extracts can include various macromolecular components needed for the translation of exogenous RNA (e.g., 70S or 80S ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation, elongation, and termination factors). Other components can be included or added during the translation reaction, including but not limited to amino acids, energy sources (ATP, GTP), energy regenerating systems (creatine phosphate and creatine phosphokinase (eukaryotic systems)) (phosphoenol pyruvate and pyruvate kinase for bacterial systems), and other cofactors (Mg2+, K+, etc.). As previously mentioned, in vitro translation can be performed using RNA or DNA as the starting material. Some translation systems can utilize an RNA template as starting material (e.g., reticulocyte lysates and wheat germ extracts). Some translation systems can use a DNA template as starting material (e.g., E. coli-based systems). In these systems, transcription and translation are coupled: DNA is first transcribed into RNA, which is then translated. Suitable standard and coupled cell-free translation systems are generally known in the art and are commercially available.Vectors Encoding the Transsene

[0253] In an embodiment, the vector encoding the transgene (also referred to as “an artificial genome”) comprises the transgene to be delivered flanked on either side by AAV ITRs. Only -145 bp AAV ITRs are required for recombinant AAV (rAAV) propagation because they participate in vector production, induce transgene expression, and ensure continual cell transduction. Accordingly, -96% of the AAV genome can be removed for gene therapy. For example, the rep and cap genes can be substituted for the expression cassette containing a promoter (such as those described herein), a therapeutic transgene (for example, IDS), and a poly(A) tail, which forms the essence of all AAV vectors.

[0254] In an embodiment, additional modifications may be implemented to increase the efficacy of the AAV further. For example, the AAV ITRs may be modified to increase the expression of the rAAV vector upon transduction, which may allow the transgene to be expressed without second-strand DNA synthesis; the promoter may be modified to increase transcription;and the codons in the transgene may be engineered to modify mRNA production and / or translation.

[0255] In an embodiment, the ITRs are modified to overcome second-strand synthesis after infection. The AAV transduction rate is restricted by the synthesis of dsDNA from the singlestranded AAV genome. ITRs initiate second-strand synthesis. In an example embodiment, modified ITRs are no longer suitable substrates for the Rep68 and Rep78 proteins. As a result, the terminal resolution of replication is obviated, and specific self-complementary AAV (scAAV) replication intermediates are produced. The scAAV intermediates comprise plus and minus strands of DNA, fused by the modified ITRs, which are encapsulated within the virion shell. Wild-type AAVs package either a single plus-strand or a single minus-strand DNA. The modified scAAV intermediates are delivered to the nucleus, and the plus and minus strands anneal instantaneously to form dsDNA.

[0256] In an embodiment, the cis-elements are optimized for targeted delivery. The ciselements are optimized because AAVs have limited packaging capacity. In an example embodiment, small cis-elements replace long promoter sequences to deliver large therapeutic transgenes (e.g., 4.4-4.5 kbs).

[0257] In an embodiment, several strategies may be used to deliver transgenes using AAV vectors. Example approach 1 takes advantage of an AAV genome concatemerized via the homologous recombination of ITR sequences. In this approach, transgene cassettes may be split into two or more vectors, which are then delivered to the same cells. After the virus is uncoated, an intact transgene is formed by the homologous recombination between two or more fragments.

[0258] In example approach 2, truncated transgene fragments of different lengths are packaged into different AAV virions at undefined locations on the vector genome. Either homologous recombination of the overlapping regions of the different AAV vector genomes or annealing of different AAV vector genomes at complementary regions via single- stranded templates produces the transgene cassette. In an embodiment, overlapping fragments may be added to the ends of individual AAV vectors to promote homologous recombination.

[0259] In example approach 3, a hybrid dual-vector incorporates an overlapping region with intron splice sites in the split vector transgenes. Approach 3 uses the concatemerization activity of AAV genomes to bring independent AAV vector genomes together. Recombination (for example, the starting vectors are segregated into two halves, each carrying the 5' and 3' splicingelements, respectively), and splicing provides the appropriate transgene protein. This strategy may increase the expression of a full functional protein.

[0260] In example approach 4, an AAV genome is cross-packaged into the capsids of other parvoviruses, thus creating chimeric vectors.

[0261] In example approach 5, intein-mediated protein trans-splicing is used. Intein catalyzes protein splicing, thereby ligating two polypeptides via trans-splicing (a process similar to intron-mediated RNA splicing). Multiple AAV vectors are delivered to the same cells. Each AAV vector encodes a fragment of a target protein, and short split inteins flank the fragment. The full-length protein forms after protein trans-splicing. See e g., Li, C., Samulski, R.J. Engineering adeno-associated virus vectors for gene therapy — Nat Rev Genet 21, 255-272 (2020), herein incorporated by reference.Vector Construction

[0262] The vectors described herein can be constructed using any suitable process or technique. In an embodiment, one or more suitable recombination and / or cloning methods or techniques can be used on the vector(s) described herein. Suitable recombination and / or cloning techniques and / or methods can include, but are not limited to, those described in U.S. Application publication No. US 2004-0171156 Al. Other suitable methods and techniques are described elsewhere herein.

[0263] Construction of recombinant AAV vectors is described in several publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989). Any of the techniques and / or methods can be used and / or adapted for constructing an AAV or other vector described herein. AAV vectors are discussed elsewhere herein.

[0264] In an embodiment, the vector can have one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”). In an embodiment, one or more insertion sites (e g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and / or downstream of one or more sequence elements of one or more vectors.

[0265] Delivery vehicles, vectors, particles, nanoparticles, formulations, and components thereof for expression of one or more elements of an engineered AAV capsid system describedherein are as used in the foregoing documents, such as International Patent Application Publication WO 2014 / 093622 (PCT / US2013 / 074667), and are discussed in greater detail herein.Example Cargos

[0266] The engineered AAVs disclosed herein may be used to deliver different cargoes. Cargos can include, without limitation, nucleotides, oligonucleotides, polynucleotides, amino acids, peptides, polypeptides, riboproteins, lipids, sugars, pharmaceutically active agents (e.g., drugs, imaging and other diagnostic agents), chemical compounds, and combinations thereof. In an embodiment, the cargo is DNA, RNA, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, guide sequences for ribozymes that inhibit translation or transcription of essential tumor proteins and genes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, antihistamines, anti-infectives, radiation sensitizers, chemotherapeutics, radioactive compounds, imaging agents, and combinations thereof. In an embodiment, the cargo is a recombinant AAV genome comprising a transgene, for example, encoding a therapeutic protein or nucleic acid, operably linked to regulatory sequences that direct expression of the therapeutic protein or nucleic acid in a target tissue, flanked by AAV ITR sequences.

[0267] In an embodiment, the cargo is capable of treating or preventing a disease or disorder, details of which are described herein.

[0268] In an embodiment, the cargo is a morpholino, a peptide-linked morpholino, an antisense oligonucleotide, a phosphorodiamidate morpholino oligomers (PMO), a therapeutic transgene, a polynucleotide encoding a therapeutic polypeptide or peptide, a peptide-conjugated PMO (PPMO), one or more peptides, one or more polynucleotides encoding a CRISPR-Cas protein, a guide RNA, or both, a ribonucleoprotein, wherein the ribonucleoprotein comprises a CRISPR-Cas system molecule, a therapeutic transgene RNA, or other gene modifying or therapeutic RNA and / or protein, or any combination thereof.Cargo Polynucleotides

[0269] Cargos are also described elsewhere herein. In an embodiment, the cargo is a cargo polynucleotide that can be packaged into an engineered viral particle and subsequently delivered to a cell. In an embodiment, delivery is cell selective, e.g., neurons and glial cells of the CNS. In an embodiment, the one or more cargo polynucleotides are part of the engineered viral (e.g., AAV) genome of the viral (e.g., AAV) system and packaged within the engineered capsidcontaining one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, optionally one or more target binding modifications, of the present disclosure. The cargo polynucleotides can be packaged into an engineered viral particle (e.g., AAV), which can be delivered to, e.g., a cell. In an embodiment, the cargo polynucleotide can modify a polynucleotide (e.g., a gene or transcript) of a cell to which it is delivered. As used herein, “gene” can refer to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism. The term gene can refer to translated and / or untranslated regions of a genome. “Gene” can refer to the specific sequence of DNA that is transcribed into an RNA transcript that can be translated into a polypeptide or be a catalytic RNA molecule, including but not limited to tRNA, siRNA, piRNA, miRNA, long-non-coding RNA, and shRNA. Polynucleotide, gene, transcript, etc. modification includes all genetic engineering techniques, including, but not limited to, gene editing, as well as conventional recombinational gene modification techniques (e.g., whole or partial gene insertion, deletion, and mutagenesis (e.g., insertional and deletional mutagenesis)).

[0270] In an embodiment, the cargo molecule is a polynucleotide that encodes a vaccine. In an embodiment, the cargo molecule is a polynucleotide encoding an antibody.RNA Interference Agents

[0271] In an embodiment, the one or more polynucleotides may encode one or more RNA interference agents. RNA interference agents are RNA molecules that suppress gene expression. Examples of RNA interference agents include, but are not limited to, small interfering RNAs (siRNAs), microRNAs (miRNAs), and short hairpin RNAs (shRNAs).

[0272] In an embodiment, the interference RNA may be a siRNA. Small interfering RNA (siRNA) molecules can inhibit target gene expression by interfering with RNA. siRNAs may be chemically synthesized, or may be obtained by in vitro transcription, or may be synthesized in vivo in the target cell. siRNAs may comprise double-stranded RNA from 15 to 40 nucleotides in length and can contain a protuberant region 3' and / or 5' from 1 to 6 nucleotides in length. Length of the protuberant region is independent of the total length of the siRNA molecule. siRNAs mayact by post-transcriptional degradation or silencing of the target messenger. In some cases, the exogenous polynucleotides encode shRNAs. In shRNAs, the antiparallel strands that form siRNA are connected by a loop or hairpin region.

[0273] In an embodiment, the cargo polynucleotide is an RNAi molecule, an antisense molecule, a gene silencing oligonucleotide, or a polynucleotide that encodes an RNAi molecule, an antisense molecule, or a gene silencing oligonucleotide.

[0274] As used herein, “gene silencing oligonucleotide” refers to any oligonucleotide that can alone or with other gene silencing oligonucleotides utilize a cell’s endogenous mechanisms, molecules, proteins, enzymes, and / or other cell machinery or exogenous molecule, agent, protein, enzyme, and / or polynucleotide to cause a global or specific reduction or elimination in gene expression, RNA level(s), RNA translation, RNA transcription, that can lead to a reduction or effective loss of a protein expression and / or function of a non-coding RNA as compared to wildtype or a suitable control. This is synonymous with the phrase “gene knockdown” Reduction in gene expression, RNA level(s), RNA translation, RNA transcription, and / or protein expression can range from about 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 4241, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, to 1% or less reduction. “Gene silencing oligonucleotides” include, but are not limited to, any antisense oligonucleotide, ribozyme, any oligonucleotide (single or double stranded) used to stimulate the RNA interference (RNAi) pathway in a cell (collectively RNAi oligonucleotides), small interfering RNA (siRNA), microRNA, and short-hairpin RNA (shRNA). Commercially available programs and tools are available to design the nucleotide sequence of gene silencing oligonucleotides for a desired gene, based on the gene sequence and other information available to one of ordinary skill in the art.

[0275] In an embodiment, a cargo polynucleotide, such as an encoding polynucleotide, is flanked by at least a retroelement polypeptide encoding polynucleotide 3 ’ UTR or portion thereof, such as the proximal region of about 500 base pairs of the 3’ UTR. In an embodiment a cargo polynucleotide, such as an encoding polynucleotide, is flanked by a (e.g., endogenous or engineered) retroelement polypeptide (such as a retroviral gag protein or gag homolog) 5’ UTR. In an embodiment a cargo polynucleotide, such as an encoding polynucleotide, is flanked by an(e g., endogenous or engineered) retroelement polypeptide encoding polynucleotide 5’ and 3’ UTR. In an embodiment, the flanking retroelement polypeptide encoding polynucleotide UTR(s) are from PNMA, Arc, PEG10 or other Sushi Class polypeptide. In an embodiment, the inclusion of the 3’ UTR, the 5 ’UTR, or both can increase packaging and / or delivery of the cargo that they flank. These and other packaging elements are described in greater detail elsewhere herein.Gene Modification Carso Polynucleotides

[0276] In an embodiment, the cargo molecule can be a polynucleotide encoding a polypeptide that can alone or when delivered as part of a system, whether or not delivered with other components of the system, operate to modify the genome, epigenome, and / or transcriptome of a cell to which it is delivered. Such systems include, but are not limited to, CRISPR-Cas systems. Other gene modification systems, e.g., Transcription Activator-like Effector (TALE)- or Zinc Finger Protein (ZFP)-based transcriptional activator; repressor; or epigenomic silencer, a RNA encoding a partial gene fragment designed for transplacing into an endogenous RNA, one or more transfer RNAs, or a component thereof, or an OMEGA system or any component thereof, Cre-Lox, morpholinos, etc. are other non-limiting examples of gene modification systems whose one or more components can be delivered by the engineered viral (e.g., AAV) particles described herein.

[0277] In an embodiment, the cargo molecule encodes a gene editing system or component thereof. In an embodiment, the cargo molecule encodes a CRISPR-Cas system molecule or a component thereof. In an embodiment, the cargo molecule is a polynucleotide that encodes one or more components of a gene modification system (such as a CRISPR-Cas system). In an embodiment the cargo molecule is or encodes a gRNA. CRISPR-Cas system as used herein is intended to encompass by Class 1 and Class 2 CRISPR-Cas systems and derivatives of CRISPR-Cas systems such as base editors, prime editors, and CRISPR-associated transposases (CAST) systems.

[0278] In an embodiment, the cargo molecule can be a polynucleotide or polynucleotide encoding a polypeptide that can alone or when delivered as part of a system, whether or not delivered with other components of the system, operate to modify the genome, epigenome, and / or transcriptome of a cell to which it is delivered, is such that it treats or prevents a disease, a disorder, or a symptom thereof of a neurologic disease or disorder, and / or viruses (such as single stranded RNA viruses). In an embodiment, the cargo molecule, whether or not delivered withother components of the system, operates to modify the genome, epigenome, and / or transcriptome of a cell to which it is delivered, is such that it treats or prevents a neurological disease or disorder described further herein.

[0279] In an embodiment, the cargo molecule, whether or not delivered with other components of the system, operates to modify the genome, epigenome, and / or transcriptome of a cell to which it is delivered, is such that can modify the GAA gene, such as any of those described in US Pat. App. Pub. 20190284555, the contents of which are incorporated by reference as if expressed in their entirety herein and can be adapted for use with the present disclosure.

[0280] In an embodiment, the cargo molecule is or encodes an antisense oligomer or RNA molecule, such as those described in U.S. Pat. App. Pub. US20160251398, US20150267202, and US20180216111, the contents of which are incorporated by reference as if expressed in their entirety herein and can be adapted for use with the present disclosure.

[0281] In an embodiment, the cargo molecule can be a peptide-oligomer, conjugate as described in e.g., International Patent Application Publication W02017106304A1, the contents of which are incorporated by reference as if expressed in their entirety herein and can be adapted for use with the present disclosure.

[0282] An embodiment of the disclosure encompasses methods of modifying a genomic locus of interest to change gene expression in a cell by introducing into the cell any of the compositions described herein.

[0283] An embodiment of the disclosure is that the above elements are comprised in a single composition or comprised in individual compositions. These compositions may advantageously be applied to a host to elicit a functional effect on the genomic level.Polypeptides

[0284] In an embodiment, the cargo molecule be a nucleic acid (e.g., polynucleotide) encoding a polypeptide. The polypeptide may be a full-length protein or a functional fragment or functional domain thereof, that is a fragment or domain that maintains the desired functionality of the full-length protein. As used within this section “protein” is meant to refer to full-length proteins and functional fragments and domains thereof. A wide array of polypeptides may be delivered (i.e., via a polynucleotide encoding the polypeptide) using the engineered delivery vesicles described herein, including but not limited to, secretory proteins, immunomodulatory proteins, anti-fibrotic proteins, proteins that promote tissue regeneration and / or transplantsurvival functions, hormones, anti-microbial proteins, anti-fibrillating polypeptides, and antibodies. The one or more polypeptides may also comprise combinations of the aforementioned example classes of polypeptides. It will be appreciated that any of the polypeptides described herein can also be delivered via the engineered delivery vesicles and systems described herein via delivery of the corresponding encoding polynucleotide.Antibodies

[0285] In an embodiment, the one or more polypeptides may comprise one or more antibodies. The term “antibody” is used interchangeably with the term “immunoglobulin” herein, and includes intact antibodies, fragments of antibodies, e.g., Fab, F(ab')2 fragments, scFva, and intact antibodies and fragments that have been mutated either in their constant and / or variable region (e.g., mutations to produce chimeric, partially humanized, or fully humanized antibodies, as well as to produce antibodies with a desired trait, e.g., enhanced binding and / or reduced FcR binding). The term "fragment" refers to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain. Fragments can be obtained via chemical or enzymatic’ treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. Exemplary fragments include Fab, Fab', F(ab')2, Fabc, Fd, dAb, VHH and scF and / or Fv fragments. As used herein, a preparation of antibody protein “having less than about 50% of non-antibody protein (also referred to herein as a "contaminating protein"), or of chemical precursors, is considered to be "substantially free." 40%, 30%, 20%, 10% and more preferably 5% (by dry weight) of nonantibody protein, or of chemical precursors is considered to be substantially free. When the antibody protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 30%, preferably less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume or mass of the protein preparation.

[0286] In an embodiment, the antibody is a fragment or portion thereof. In an example embodiment, the antibody is an epitope binding protein or portion thereof. The term "binding portion" of an antibody (or "antibody portion") includes one or more complete domains, e.g., a pair of complete domains, as well as fragments of an antibody that retain the ability to specifically bind to a target molecule. It has been shown that the binding function of an antibody can be performed by fragments of a full-length antibody. Binding fragments are produced byrecombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab', F(ab')2, Fabc, Fd, dAb, Fv, single chains, single-chain antibodies, e.g., scFv, and single domain antibodies.

[0287] In an embodiment, the cargo or antibody is an antibody fragment or portion. In an embodiment, the cargo is an epitope binding protein. Examples of portions of antibodies or epitope-binding proteins encompassed by the present definition include: (i) the Fab fragment, having VL, CL, VH and CHI domains; (ii) the Fab' fragment, which’ is a Fab fragment having one or more cysteine residues at the C-terminus of the CHI domain; (iii) the Fd fragment having VH and CHI domains; (iv) the Fd' fragment having VH and CHI domains and one or more cysteine residues at the C-terminus of the CHI domain; (v) the Fv fragment having the VL and VH domains of a single arm of an antibody; (vi) the dAb fragment (Ward et al., 341 Nature 544 (1989)) which consists of a VH domain or a VL domain that binds antigen; (vii)’ isolated CDR regions or isolated CDR regions presented in a functional framework; (viii) F(ab')2 fragments which are bivalent fragments including two Fab' fragments linked by a disulphide bridge at the hinge region; (ix) single chain antibody molecules (e.g., single chain Fv; scFv) (Bird et al., 242 Science 423 (1988); and Huston et al., 85 PNAS 5879 (1988)); (x) "diabodies" with two antigen binding sites, comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (see, e.g., EP 404,097; WO 93 / 11161; Hollinger et al., 90 PNAS 6444 (1993)); (xi) "linear antibodies" comprising a pair of tandem Fd segments (Vn-Chl-VH-Chl) which, together with complementary light chain oligopeptides, form a pair of “antigen binding regions” (Zapata et al., Protein Eng. 8(10): 1057-62 (1995); and U.S. Patent No. 5,641,870).

[0288] The term "antigen-binding fragment" refers to a polypeptide fragment of an immunoglobulin or antibody that binds antigen or competes with intact antibody (i.e., with the intact antibody from which they were derived) for antigen binding (i.e., specific binding). As such these antibodies or fragments thereof are included in the scope of the disclosure, provided that the antibody or fragment binds specifically to a target molecule.

[0289] In an embodiment, the antibody is a single-chain antibody (scFvs). The term “singlechain variable fragment”, as used herein refers to a fusion protein containing the variable region(s) of the heavy (VH) and light (VL) of an immunoglobulin that are connected via a linker peptide. The linker peptide typically ranges from about 10 to about 25 amino acids. The linker can be flexible and can contain one or more glycine residues for flexibility. The linker can contain oneor more serine or threonine residues to increase or modify solubility. The VH and light (VL) can be linked via the linker in any order. In an embodiment, N terminus of the VH and is coupled, via a linker, C terminus of the (VL). In an embodiment, C terminus of the VH and is coupled, via a linker, N terminus of the (VL). In an embodiment, the scFV is a bivalent or trivalent scFvs. In an embodiment bitrivalent or trivalent scFvs are bi or trispecific, menaing that they can target 2 or 3, respectively, different epitopes. See also e.g., Hollinger, Philipp; Prospero, T; Winter, G (July 1993). “Diabodies": small bivalent and bispecific antibody fragments”. Proceedings of the National Academy of Sciences of the United States of America. 90(14): 6444-8; incq, S; Bosman, F; Buyse, MA; Degrieck, R; Celis, L; De Boer, M; Van Doorsselaere, V; Sablon, E (2001). “Expression and purification of monospecific and bispecific recombinant antibody fragments derived from antibodies that block the CD80 / CD86-CD28 costimulatory pathway”. Protein Expression and Purification. 22 (1): 11-24. doi:10.1006 / prep.2001.1417; Le Gall, F.; Kipriyanov, SM; Moldenhauer, G; Little, M (1999). “Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding”. FEBS Letters. 453 (1): 164- 168. doi:10.1016 / S0014-5793(99)00713-9; Huston, J. S.; Levinson, D.; Mudgett-Hunter, M.; Tai, M. S.; Novotny, J.; Margolies, M. N.; Crea, R. (1988). “Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli”. Proceedings of the National Academy of Sciences of the United States of America. 85 (16): 5879-5883; de Graaf et al., Methods Mol Biol. 2002;178:379-87. doi: 10.1385 / 1-59259-240-6:379; Zhou, H.X., J Mol Biol. 2003 May 23;329(1): 1-8. doi: 10.1016 / s0022-2836(03)00372-3; Bird and Walker. Trends Biotechnol. 1991 Apr;9(4): 132-7. doi: 10.1016 / 0167-7799(91)90044-1; Worn et al., J Mol Biol. 2001 Feb 2;305(5):989-1010. doi: 10.1006 / jmbi.2000.4265.

[0290] As used herein, “heavy chain antibody,” “VHH” or “single-domain antibodies” (sdAbs) refers to an antibody which is composed only of two heavy chains and lacks the two light chains usually found in antibodies (see, e.g., Henry and MacKenzie, Antigen recognition by single-domain antibodies: structural latitudes and constraints. MAbs. 2018 Aug-Sep; 10(6): 815— 826). VHH can refer to an antibody or VHH domain. Single-domain antibodies (sdAb) are also referred to as a “nanobody”, which is defined herein as an antibody fragment composed of a single monomeric variable antibody domain. As used herein “VHH” is used interchangeably with “nanobody .” The -12-15 kDa variable domains of these antibodies (VHHs and VNARs) can beproduced recombinantly and can recognize antigen in the absence of the remainder of the antibody heavy chain. In common antibodies, the antigen binding region consists of the variable domains of the heavy and light chains (VH and VL). Heavy-chain antibodies can bind antigens despite having only VH domains. In an embodiment, the heavy chain antibody is an antibody derived from cartilaginous fishes (immunoglobulin new antigen receptor (IgNAR)) or camelid ungulates. Non-limiting examples of camelids include dromedaries, camels, llamas and alpacas.

[0291] It is intended that the term “antibody” encompass any Ig class or any Ig subclass (e.g., the IgGl, IgG2, IgG3, and IgG4 subclasses of IgG) obtained from any source (e.g., humans and non-human primates, and in rodents, lagomorphs, caprines, bovines, equines, ovines, etc ).

[0292] The term “Ig class” or "immunoglobulin class”, as used herein, refers to the five classes of immunoglobulin that have been identified in humans and higher mammals, IgG, IgM, IgA, IgD, and IgE. The term “Ig subclass” refers to the two subclasses of IgM (H and L), three subclasses of IgA (IgAl, IgA2, and secretory IgA), and four subclasses of IgG (IgGl, IgG2, IgG3, and IgG4) that have been identified in humans and higher mammals. The antibodies can exist in monomeric or polymeric form; for example, IgM antibodies exist in pentameric f-rm, and IgA antibodies exist in monomeric, dimeric or multimeric form.

[0293] The term “IgG subclass” refers to the four subclasses of immunoglobulin class IgG -IgGl, IgG2, IgG3, and IgG4 that have “been identified in humans and higher mammals by the heavy chains of the immunoglobulins, VI - y4, respectively. The term "single-chain immunoglobulin" or "single-chain antibody" (used interchangeably herein) refers to a protein having a two-polypeptide chain structure consisting of a heavy and a light chain, said chains being stabilized, for example, by interchain peptide linkers, which has the ability to specifically bind antigen. The term "domain" refers to a globular region of a heavy or light chain polypeptide comprising peptide loops (e.g., comprising 3 or 4 peptide loops) stabilized, for example, by pleated sheet and / or intrachain disulfide bond. Domains are further referred to herein as "constant" or "variable", based on the relative lack of sequence variation within the domains of various class members in the case of "constant" domain, or the significant variation within the domains of various class members in the case of a "variable" domain. Antibody or polypeptide "domains" are often referred to interchangeably in the antibody or polypeptide "regions". The "constant" domains of an antibody light chain are referred to interchangeably as "light chain constant regions", "light chain constant domains", "CL" regions or "CL" domains.” The"constant" domains of an antibody heavy chain are referred to interchangeably as "heavy chain constant regions", "heavy chain constant domains", "CH" regions or "CH" domains.” The "variable” domains of an antibody light chain are referred to interchangeably as "light chain variable regions", "light chain variable domains", " VL" regions or "VL" domains.” The "variable” domains of an antibody heavy chain are referred to interchangeably as "heavy chain constant regions", "heavy chain constant domains", "VH" regions or "VH" domains.

[0294] The term "region" can also refer to a part or portion of an antibody chain or antibody chain domain (e.g., a part or portion of a heavy or light chain or a part or portion of a constant or variable domain, as defined herein), as well as more discrete parts or portions of said chains or domains. For example, light and heavy chains or light and heavy chain variable domains include "complementarity determining regions" or "CDRs" interspersed among "framework regions" or "FRs", as defined herein.

[0295] The term "conformation" refers to the tertiary structure of a protein or polypeptide (e.g., an antibody, antibody chain, domain or region thereof). For example, the phrase "light (or heavy) chain conformation" refers to the tertiary structure of a light (or heavy) chain variable region, and the phrase "antibody conformation" or "antibody fragment conformation" refers to the tertiary structure of an antibody or fragment thereof.

[0296] The term “antibody-like protein scaffolds” or “engineered protein scaffolds” broadly encompasses proteinaceous non-immunoglobulin specific-binding agents, typically obtained by combinatorial engineering (such as site-directed random mutagenesis in combination with phage display or other molecular selection techniques). Usually, such scaffolds are derived from robust and small soluble monomeric proteins (such as Kunitz inhibitors or lipocalins) or from a stably folded extra-membrane domain of a cell surface receptor (such as protein A, fibronectin or the ankyrin repeat).

[0297] Such scaffolds have been extensively reviewed in Binz et al. (Engineering novel binding proteins from non-immunoglobulin domains. Nat Biotechnol 2005, 23:1257-1268), Gebauer and Skerra (Engineered protein scaffolds as next-generation antibody therapeutics. Curr Opin Chem Biol. 2009, 13:245-55), Gill and Damle (Biopharmaceutical drug discovery using novel protein scaffolds. Curr Opin Biotechnol 2006, 17:653-658), Skerra (Engineered protein scaffolds for molecular recognition. J Mol Recognit 2000, 13:167-187), and Skerra (Alternative non-antibody scaffolds for molecular recognition. Curr Opin Biotechnol 2007, 18:295-304), andinclude without limitation affibodies, based on the Z-domain of staphylococcal protein A, a three-helix bundle of 58 residues providing an interface on two of its alpha-helices (Nygren, Alternative binding proteins: Affibody binding proteins developed from a small three-helix bundle scaffold. FEBS J 2008, 275:2668-2676); engineered Kunitz domains based on small polypeptides of 58 residues) and robust, disulphide-crosslinked serine protease inhibitor, typically of human origin (e.g., LAC1-D1), which can be engineered for different protease specificities (Nixon and Wood, Engineered protein inhibitors of proteases. Curr Opin Drug Discov Dev 2006, 9:261-268); monobodies or adnectins based on the 10th extracellular domain of human fibronectin III (10Fn3), which adopts an Ig-like beta-sandwich fold (94 residues) with 2-3 exposed loops but lacks the central disulphide bridge (Koide and Koide, Monobodies: antibody mimics based on the scaffold of the fibronectin type III domain. Methods Mol Biol 2007, 352:95-109); anticalins derived from the lipocalins, a diverse family of eight-stranded beta-barrel proteins (ca. 180 residues) that naturally form binding sites for small ligands by means of four structurally variable loops at the open end, which are abundant in humans, insects, and many other organisms (Skerra, Alternative binding proteins: Anticalins — harnessing the structural plasticity of the lipocalin ligand pocket to engineer novel binding activities. FEBS J 2008, 275:2677-2683); DARPins, designed ankyrin repeat domains (166 residues), which provide a rigid interface arising from typically three repeated beta-turns (Stumpp et al., DARPins: a new generation of protein therapeutics. Drug Discov Today 2008, 13:695-701); avimers (multimerized LDLR-A module) (Silverman et al., Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains. Nat Biotechnol 2005, 23:1556-1561); and cysteine-rich knottin peptides (Kolmar” Alternative binding proteins: biological activity and therapeutic potential of cystine-knot miniproteins. FEBS J 2008, 275:2684-2690).

[0298] "Specific binding" of an antibody means that the antibody exhibits appreciable affinity for a particular antigen or epitope and, generally, does not exhibit significant cross reactivity. "Appreciable" binding includes binding with an affinity of at least 25 pM. Antibodies with affinities greater than 1 x 107M'1(or a dissociation coefficient of IpM or less or a dissociation coefficient of Inm or less) typically bind with correspondingly greater specificity. Values intermediate of those set forth herein are also intended to be within the scope of the present disclosure and antibodies of the disclosure bind with a range of affinities, for example, lOOnM or less, 75nM or less, 50nM or less, 25nM or less, for example lOnM or less, 5nM or less, InM orless, or in embodiments 500pM or less, lOOpM or less, 50pM or less or 25pM or less. An antibody that "does not exhibit significant cross reactivity" is one that will not appreciably bind to an entity other than its target (e.g., a different epitope or a different molecule). For example, an antibody that specifically binds to a target molecule will appreciably bind the target molecule but will not significantly react with non-target molecules or peptides. An antibody specific for a particular epitope will, for example, not significantly cross react with remote epitopes on the same protein or peptide. Specific binding can be determined according to any art-recognized means for determining such binding. Preferably, specific binding is determined according to Scatchard analysis and / or competitive binding assays.

[0299] As used herein, the term “affinity” refers to the strength of the binding of a single antigen-combining site with an antigenic determinant. Affinity depends on the closeness of stereochemical fit between antibody combining sites and antigen determinants, on the size of the area of contact between them, on the distribution of charged and hydrophobic groups, etc. Antibody affinity can be measured by equilibrium dialysis or by the kinetic BIACORE™ method. The dissociation constant, Kd, and the association constant, Ka, are quantitative measures of affinity.

[0300] As used herein, the term “monoclonal antibody” refers to an antibody derived from a clonal population of antibody-producing cells (e.g., B lymphocytes or B cells) which is homogeneous in structure and antigen specificity. The term "polyclonal antibody" refers to a plurality of antibodies originating from different clonal populations of antibody-producing cells which are heterogeneous in their structure and epitope specificity, but which recognize a common antigen. Monoclonal and polyclonal antibodies may exist within bodily fluids, as crude preparations, or may be purified, as described herein. "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refineantibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0301] As used herein, a “blocking” antibody or an antibody "antagonist" is one which inhibits or reduces biological activity of the antigen(s) it binds. In an embodiment, the blocking antibodies or antagonist antibodies or portions thereof described herein completely inhibit the biological activity of the antigen(s).

[0302] Antibodies may act as agonists or antagonists of the recognized polypeptides. For example, the present disclosure includes antibodies which disrupt receptor / ligand interactions either partially or fully. The disclosure features both receptor-specific antibodies and ligandspecific antibodies. The disclosure also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine / threonine) of the receptor or of one of its down-stream substrates by immunoprecipitation followed by western blot analysis. In specific embodiments, antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.

[0303] The disclosure also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex. Likewise, encompassed by the disclosure are neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor. Further included in the disclosure are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor. The antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides disclosed herein. The antibodyagonists and antagonists can be made using methods known in the art. See, e.g., PCT publication WO 96 / 40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92 (6): 1981-1988 (1998); Chen et al., Cancer Res. 58( 16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4) : 1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. Ill (Pt2):237-247 (1998); Pitard et al., J. Immunol. Methods 205 (2): 177-190 (1997); Liautard et al., Cytokine 9(4):233-241 (1997); Carlson et al., J. Biol. Chem.272(17): 11295-11301 (1997); Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure 6(9): 1153-1167 (1998); Bartunek et al., Cytokine 8(1): 14-20 (1996).

[0304] The antibodies as defined for the present disclosure include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. For example, but not by way of limitation, the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.Programmable Nuclease

[0305] As used herein, a programmable nuclease refers to a nuclease capable of forming a complex with said guide molecule and wherein the site of nuclease activity on a genomic loci is dictated by the guide molecule. A programmable nuclease may comprise a CRISPR-Cas system or a component thereof (e g., a Cas nuclease) or an OMEGA system or a component thereof (e.g., an IscB nuclease, an IsrB nucelase, an IshB nuclease, a TnpB nuclease, a Fanzor, etc.) (see e.g., Altae-Tran, H.; et al. The Widespread IS200 / IS605 Transposon Family Encodes Diverse Programmable RNA-Guided Endonucleases. Science, 2021, 374:57-65; Karvelis et al., Nature, 599: 692-696 (2021); Hirano et al., Nature, 610:575-581 (2022); and Saito et al., Nature, 620:660-668 (2023); Jiang et al., Science Advances, 9(39), DOI: 10.1126 / sciadv.adk0171 (2023)). The same programmable nuclease may be used with the entire set of guide molecules, or the set of molecules may comprise one or more guides capable of complexing with different types of programmable nucleases.CRISPR-Cas Systems

[0306] The programmable nuclease may be a Cas nuclease. CRISPR-Cas systems can generally fall into two classes based on their architectures of their effector molecules, which are each further subdivided by type and subtype. The two classes are Class 1 and Class 2. Class 1 CRISPR-Cas systems have effector modules composed of multiple Cas proteins, some of which form crRNA-binding complexes, while Class 2 CRISPR-Cas systems include a single, multidomain crRNA-binding protein.

[0307] In some embodiments, the CRISPR-Cas system that can be used to modify a polynucleotide as described herein can be a Class 1 CRISPR-Cas system. Class 1 CRISPR-Cas systems are divided into types I, III, and IV. Makarova et al. 2020. Nat. Rev. 18: 67-83., particularly as described in Figure 1. Type I CRISPR-Cas systems are divided into 9 subtypes (I-A, I-B, I-C, I-D, I-E, I-Fl, I-F2, 1-F3, and IG). Makarova etal., 2020. Class 1, Type I CRISPR-Cas systems can contain a Cas3 protein that can have helicase activity. Type III CRISPR-Cas systems are divided into 6 subtypes (III-A, III-B, III-C, III-D, III-E, and III-F). Type III CRISPR-Cas systems can contain a Cas 10 that can include an RNA recognition motif called Palm and a cyclase domain that can cleave polynucleotides. Makarova et al., 2020. Type IV CRISPR-Cas systems are divided into 3 subtypes. (IV-A, IV-B, and IV-C). Makarova et al., 2020. Class 1 systems also include CRISPR-Cas variants, including Type I-A, I-B, I-E, I-F and I-U variants, which can include variants carried by transposons and plasmids, including versions of subtype I-F encoded by a large family of Tn7-like transposon and smaller groups of Tn7-like transposons that encode similarly degraded subtype I-B systems. Peters et al., PNAS 114 (35) (2017); DOI: 10.1073 / pnas.1709035114; see also, Makarova et al. 2018. The CRISPR Journal, v. 1 , n5, Figure 5.

[0308] The compositions, systems, and methods described in greater detail elsewhere herein can be designed and adapted for use with Class 2 CRISPR-Cas systems. Thus, in some embodiments, the CRISPR-Cas system is a Class 2 CRISPR-Cas system. Class 2 systems are distinguished from Class 1 systems in that they have a single, large, multi-domain effector protein. In certain example embodiments, the Class 2 system can be a Type II, Type V, or Type VI system, which are described in Makarova et al. “Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants” Nature Reviews Microbiology, 18:67-81 (Feb 2020), incorporated herein by reference. Each type of Class 2 system is further divided into subtypes.See Markova et al. 2020, particularly at Figure. 2. Class 2, Type II systems can be divided into 4 subtypes: II-A, II-B, II-C1, and II-C2. Class 2, Type V systems can be divided into 17 subtypes: V-A, V-Bl, V-B2, V-C, V-D, V-E, V-Fl, V-F1(V-U3), V-F2, V-F3, V-G, V-H, V-I, V-K (V-U5), V-Ul, V-U2, and V-U4. Class 2, Type VI systems can be divided into 5 subtypes: VI-A, VI-Bl, VI-B2, VI-C, and VI-D.OMEGA nucleases

[0309] The programmable nuclease may be an OMEGA nuclease. The OMEGA nuclease may be an IscB nuclease, an IsrB nuclease, a TnpB nuclease, or a Fanzor nuclease.IscB nuclease

[0310] In one embodiment, IscB nucleases may comprise a split RuvC nuclease domain comprising RuvC-1, RuvC-II, and RuvC-III subdomains. Some IscB proteins may further comprise a HNH endonuclease domain. In one example embodiment, the RuvC endonuclease domain is split by the insertion of a bridge helix, a HNH domain, or both. However, unlike Cas9, IscB nucleases do not contain a Rec domain. In addition, IscB nucleases may further comprise a conserved N-terminal domain (also referred to herein as a PLMP domain), which is not present in Cas9 proteins. IscB proteins may also further comprise a conserved C-terminal domain. In one example embodiment, an IscB nuclease comprises, moving from the N- to C-terminus, a PLMP domain, a RuvC-I subdomain, a bridge helix, a RuvC-II subdomain, a HNH domain, a RuvC-III subdomain, and a C terminal domain. In one embodiment, IscB nucleic acid-guided nucleases may comprise CRISPR-associated IscB nucleases. In one embodiment, the IscB nucleases are CRISPR-associated proteins, e.g., the loci of the nucleases are associated with an CRISPR array. In one embodiment the IscBs may be referred to as Cas IscBs. The Cas IscB nucleic acid-guided nuclease may comprise one or more domains, e.g., one or more of a X domain (e.g., at N-terminus), a RuvC domain, a Bridge Helix domain, and a Y domain (e.g., at C-terminus). See International Application Publication No. WO 2022 / 087494 Al incorporated herein by reference in its entirety.IsrB nuclease

[0311] IsrBs are homologs of IscB nucleases. IsrB nucleases comprise the PLMP and RuvC domains but do not comprise a HNH domain. In one embodiment, the IsrB nuclease comprises a PLMP domain and a split RuvC but lacks the HNH domain present between the RuvC-II and III subdomains in IscB nucleases. In one embodiment, the IsrB is an OMEAG RNA guided nickase.In one embodiment, the OMEAG RNA guided IsrB nicks a DNA target. In one embodiment, the DNA target is a dsDNA and the nicks occurs on the non-target strand of the dsDNA target. In one embodiment, the IsrB nicks the dsDNA in a guide and TAM specific manner. Accordingly, applications where a nickase is utilized can be used with the IsrB nucleases detailed herein in a manner functionally similar to an IscB that has been inactivated at the HNH domain.IshB nuclease

[0312] As noted above IshBs are IscB homologs and may be referred to herein as an Insertion sequence HNH-like OrfB (IshB) nuclease. IshB nucleases are generally smaller than IsrB or IscB nucleases and contain only the PLMP and HNH domain, but no RuvC domain. In one embodiment, the IshB, or IscB homolog, comprises a PLMP domain and an HNH domain, but does not comprise a RuvC domain.

[0313] Some IshB nucleases may be part of the IS605 OrfB family of transposases. In an embodiment, the IshB nuclease is from Actinoplanes lobatus and has the Genbank accession number MBB4752409. In an embodiment, the RefSeq database accession number for the nuclease with accession number MBB4752409 is WP_188124268 and the INSDC number is GGN95087.TnpB nuclease

[0314] In one aspect, embodiments disclosed herein are directed to compositions comprising a TnpB and an OMEGA RNA capable of forming a complex with the TnpB and directing sitespecific binding of the TnpB to a target sequence on a target polynucleotide.

[0315] TnpB nucleases may comprise a Ruv-C-like domain. Exemplary TnpB sequences are shown in FIG. 1, Table 1A, Table IB, Table 1C and Table 5 of International Patent Publication Application No. WO 2022 / 159892 Al, herein incorporated by reference in its entirety. The RuvC domain may be a split RuvC domain comprising RuvC-I, RuvC-II, and RuvC-III subdomains. The TnpB may further comprise one or more of a HTH domain, a bridge helix domain and a zinc finger domain. TnpB nucleases do not comprise an HNH domain. In one example embodiment, TnpB proteins comprise, starting at the N-terminus a HTH domain, a RuvC-I sub-domain, a bridge helix domain, a RuvC-II sub-domain, a zinger finger domain, and a RuvC-III sub-domain. In one example embodiment, the RuvC-III sub-domain forms the C-terminus of the TnpB nuclease.

[0316] In one embodiment, the TnpB nuclease is from Epsilonproteobacteria bacterium, or Actinoplanes lobatus strain DSM 43150, Actinomadura celluolosilytica strain DSM 45823, Actinomadura namibiensis strain DSM 44197, Alicyclobacillus macrosprangiidus strain DSM 17980, Lipingzhangella halophila strain DSM 102030, or Ktedonobacter recemifer. In one embodiment, the TnpB nuclease is from Ktedonobacter racemifer, or comprises a conserved RNA region with similarity to the 5’ 1TR of K racemifer TnpB loci.Fanzor nuclease

[0317] In one aspect, embodiments disclosed herein are directed to compositions comprising an engineered Fanzor and / or OMEGA RNA capable of forming a complex with the Fanzor and directing site-specific binding of the Fanzor to a target sequence on a target polypeptide.

[0318] Fanzor nucleases may comprise a Ruv-C-like domain. Exemplary Fanzor sequences are shown or encoded by those in Table 1, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, Table 12, Table 13, Table 14, and FIG. 20 of International Patent Application Publication No. WO 2023 / 114872 A3, herein incorporated by reference in its entirety. In some embodiments, the Fanzor nuclease is a nuclease as shown and described in relation with FIGS. 10C-10E, FIG. 35, and FIG. 56A-56D of International Patent Application Publication No. WO 2023 / 114872 A3. The RuvC domain may be a split RuvC domain comprising a RuvC-I, RuvC-II, and RuvC-III subdomains. The Fanzor may further comprise one or more of a HTH domain, a bridge helix domain, a REC domain, a zinc finger domain, or any combination thereof. Fanzor nucleases do not comprise an HNH domain. In one example embodiment, Fanzor proteins comprise, starting at the N-terminus a HTH domain, a RuvC-I sub-domain, a bridge helix domain, a RuvC-II subdomain, a zinger finger domain, and a RuvC-III sub-domain. In one example embodiment, the RuvC-III sub-domain forms the C-terminus of the Fanzor nuclease.Engineered Cells and Organisms Expressing said Engineered AAV capsids

[0319] Described herein are engineered cells that can include one or more of the engineered AAV capsid polynucleotides, polypeptides, vectors, and / or vector systems. In an embodiment, one or more of the engineered AAV capsid polynucleotides can be expressed in the engineered cells. In an embodiment, the engineered cells can be capable of producing engineered AAV capsid proteins and / or engineered AAV capsid particles that are described elsewhere herein. Also described herein are modified or engineered organisms that can include one or more engineered cells described herein. The engineered cells can be engineered to express a cargo molecule (e.g.,a cargo polynucleotide) dependently or independently of an engineered AAV capsid polynucleotide as described elsewhere herein, e.g., packaged within an engineered AAV capsid as described herein.

[0320] A wide variety of animals, plants, algae, fungi, yeast, etc. and animal, plant, algae, fungus, yeast cell or tissue systems may be engineered to express one or more nucleic acid constructs of the engineered AAV capsid system described herein using various transformation methods mentioned elsewhere herein. This can produce organisms that can produce engineered AAV capsid particles, such as for production purposes, engineered AAV capsid design and / or generation, and / or model organisms. In an embodiment, the polynucleotide(s) encoding one or more components of the engineered AAV capsid system described herein can be stably or transiently incorporated into one or more cells of a plant, animal, algae, fungus, and / or yeast or tissue system. In an embodiment, one or more of engineered AAV capsid system polynucleotides are genomically incorporated into one or more cells of a plant, animal, algae, fungus, and / or yeast or tissue system. Further embodiments of the modified organisms and systems are described elsewhere herein. In an embodiment, one or more components of the engineered AAV capsid system described herein are expressed in one or more cells of the plant, animal, algae, fungus, yeast, or tissue systems.Engineered Cells

[0321] Described herein are various embodiments of engineered cells that can include one or more of the engineered AAV capsid system polynucleotides, polypeptides, vectors, and / or vector systems described elsewhere herein. In an embodiment, the cells can express one or more of the engineered AAV capsid polynucleotides and can produce one or more engineered AAV capsid particles, which are described in greater detail herein. Such cells are also referred to herein as “producer cells”. It will be appreciated that these engineered cells are different from “modified cells” described elsewhere herein in that the modified cells are not necessarily producer cells unless they include one or more of the engineered AAV capsid polynucleotides, engineered AAV capsid vectors or other vectors described herein that render the cells capable of producing an engineered AAV capsid particle. Modified cells can be recipient cells of an engineered AAV capsid particles and can, in an embodiment, be modified by the engineered AAV capsid particle(s) and / or a cargo polynucleotide delivered to the recipient cell. Modified cells are discussed in greater detail elsewhere herein. The term modification can be used in connection withmodification of a cell that is not dependent on being a recipient cell. For example, isolated cells can be modified prior to receiving an engineered AAV capsid molecule.

[0322] In an embodiment, the disclosure provides a non-human eukaryotic organism; for example, a multicellular eukaryotic organism, including a eukaryotic host cell containing one or more components of an engineered delivery system described herein according to any of the described embodiments. In other embodiments, the disclosure provides a eukaryotic organism; preferably a multicellular eukaryotic organism, comprising a eukaryotic host cell containing one or more components of an engineered delivery system described herein according to any of the described embodiments. In an embodiment, the organism is a host of AAV.

[0323] In an embodiment, the plants, algae, fungi, yeast, etc., cells or parts obtained are transgenic plants, comprising an exogenous DNA sequence incorporated into the genome of all or part of the cells.

[0324] The engineered cell can be a prokaryotic cell. The prokaryotic cell can be bacterial cell. The prokaryotic cell can be an archaea cell. The bacterial cell can be any suitable bacterial cell. Suitable bacterial cells can be from the genus Escherichia, Bacillus, Lactobacillus, Rhodococcus, Rodhobacter, Synechococcus, Synechoystis, Pseudomonas, Psedoaltermonas, Stenotrophamonas, and Streptomyces Suitable bacterial cells include, but are not limited to Escherichia coli cells, Caulobacter crescentus cells, Rodhobacter sphaeroides cells, Psedoaltermonas haloplanktis cells. Suitable strains of bacterial include, but are not limited to BL21(DE3), DL21(DE3)-pLysS, BL21 Star-pLysS, BL21-SI, BL21-AI, Tuner, Tuner pLysS, Origami, Origami B pLysS, Rosetta, Rosetta pLysS, Rosetta-gami-pLysS, BL21 CodonPlus, AD494, BL2trxB, HMS174, NovaBlue(DE3), BLR, C41(DE3), C43(DE3), Lemo21(DE3), Shuffle T7, ArcticExpress and ArticExpress (DE3).

[0325] The engineered cell can be a eukaryotic cell. The eukaryotic cells may be those of or derived from a particular organism, such as a plant or a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. In an embodiment the engineered cell can be a cell line. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huhl, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panel, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calul, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55,Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLaB, HeLa T4, COS, COS-1, COS-6, C0S-M6A, BS-C-1 monkey kidney epithelial, BALB / 3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1 / 2, C6 / 36, Cal-27, CHO, CHO-7, CHO-1R, CHO-K1, CHO-K2, CHO-T, CHO Dhfr - / -, COR-L23, COR-L23 / CPR, COR-L23 / 5010, COR-L23 / R23, COS-7, COV-434, CML Tl, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6 / AR1, EMT6 / AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepalclc7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB-435, MDCK II, MDCK II, MOR / 0.2R, MONOMAC 6, MTD-1A, My End, NCI-H69 / CPR, NCI-H69 / LX10, NCI-H69 / LX20, NCI-H69 / LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT 2, RenCa, RIN-5F, RMA / RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)).

[0326] In an embodiment, the engineered cell is a muscle cell (e.g. cardiac muscle, skeletal muscle, and / or smooth muscle), bone cell , blood cell, immune cell (including but not limited to B cells, macrophages, T-cells, CAR-T cells, and the like), kidney cells, bladder cells, lung cells, heart cells, liver cells, brain cells, neurons, skin cells, stomach cells, neuronal support cells, intestinal cells, epithelial cells, endothelial cells, stem or other progenitor cells, adrenal gland cells, cartilage cells, and combinations thereof.

[0327] In an embodiment, the engineered cell can be a fungus cell. As used herein, a "fungal cell" refers to any type of eukaryotic cell within the kingdom of fungi. Phyla within the kingdom of fungi include Ascomycota, Basidiomycota, Blastocladiomycota, Chytridiomycota, Glomeromycota, Microsporidia, and Neocallimastigomycota. Fungal cells may include yeasts, molds, and filamentous fungi. In an embodiment, the fungal cell is a yeast cell.

[0328] As used herein, the term “yeast cell”" refers to any fungal cell within the phyla Ascomycota and Basidiomycota. Yeast cells may include budding yeast cells, fission yeast cells, and mold cells. Without being limited to these organisms, many types of yeast used in laboratoryand industrial settings are part of the phylum Ascomycota. In an embodiment, the yeast cell is an S. cerevisiae, Kluyveromyces marxianus, or Issatchenkia orientalis cell. Other yeast cells may include without limitation Candida spp. (e.g., Candida albicans), Yarrowia spp. (e.g., Yarrowia lipolytica), Pichia spp. (e.g., Pichia pastoris), Kluyveromyces spp. (e.g., Kluyveromyces lactis and Kluyveromyces marxianus), Neurospora spp. (e.g., Neurospora crassa), Fusarium spp. (e.g., Fusarium oxysporum), and Issatchenkia spp. (e.g., Issatchenkia orientalis, a.k.a. Pichia kudriavzevii and Candida acidothermophilum). In an embodiment, the fungal cell is a filamentous fungal cell. As used herein, the term “filamentous fungal cell” refers to any type of fungal cell that grows in filaments, i.e., hyphae or mycelia. Examples of filamentous fungal cells may include without limitation Aspergillus spp. (e.g., Aspergillus niger), Trichoderma spp. (e.g., Trichoderma reesei), Rhizopus spp. (e.g., Rhizopus oryzae), and Mortierella spp. (e.g., Mortierella isabellina).

[0329] In an embodiment, the fungal cell is an industrial strain. As used herein, "industrial strain" refers to any strain of fungal cell used in or isolated from an industrial process, e.g., production of a product on a commercial or industrial scale. Industrial strain may refer to a fungal species that is typically used in an industrial process, or it may refer to an isolate of a fungal species that may be also used for non-industrial purposes (e.g., laboratory research). Examples of industrial processes may include fermentation (e.g., in production of food or beverage products), distillation, biofuel production, production of a compound, and production of a polypeptide. Examples of industrial strains can include, without limitation, JAY270 and ATCC4124.

[0330] In an embodiment, the fungal cell is a polyploid cell. As used herein, a "polyploid" cell may refer to any cell whose genome is present in more than one copy. A polyploid cell may refer to a type of cell that is naturally found in a polyploid state, or it may refer to a cell that has been induced to exist in a polyploid state (e.g., through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). A polyploid cell may refer to a cell whose entire genome is polyploid, or it may refer to a cell that is polyploid in a particular genomic locus of interest.

[0331] In an embodiment, the fungal cell is a diploid cell. As used herein, a "diploid" cell may refer to any cell whose genome is present in two copies. A diploid cell may refer to a type of cell that is naturally found in a diploid state, or it may refer to a cell that has been induced to exist in a diploid state (e.g., through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, the S. cerevisiae strainS228C may be maintained in a haploid or diploid state. A diploid cell may refer to a cell whose entire genome is diploid, or it may refer to a cell that is diploid in a particular genomic locus of interest. In an embodiment, the fungal cell is a haploid cell. As used herein, a "haploid" cell may refer to any cell whose genome is present in one copy. A haploid cell may refer to a type of cell that is naturally found in a haploid state, or it may refer to a cell that has been induced to exist in a haploid state (e.g., through specific regulation, alteration, inactivation, activation, or modification of meiosis, cytokinesis, or DNA replication). For example, the S. cerevisiae strain S228C may be maintained in a haploid or diploid state. A haploid cell may refer to a cell whose entire genome is haploid, or it may refer to a cell that is haploid in a particular genomic locus of interest.

[0332] In an embodiment, the engineered cell is a cell obtained from a subject. In an embodiment, the subject is a healthy or non-diseased subject. In an embodiment, the subject is a subject with a desired physiological and / or biological characteristic such that when an engineered AAV capsid particle is produced it can package one or more cargo polynucleotides that can be related to the desired physiological and / or biological characteristic and / or capable of modifying the desired physiological and / or biological characteristic. Thus, the cargo polynucleotides of the produced engineered AAV capsid particle can be capable of transferring the desired characteristic to a recipient cell. In an embodiment, the cargo polynucleotides are capable of modifying a polynucleotide of the engineered cell such that the engineered cell has a desired physiological and / or biological characteristic.

[0333] In an embodiment, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences.

[0334] The engineered cells can be used to produce engineered viral (e.g., AAV) capsid polynucleotides, vectors, and / or particles. In an embodiment, the engineered viral (e.g., AAV) capsid polynucleotides, vectors, and / or particles are produced, harvested, and / or delivered to a subject in need thereof. In an embodiment, the engineered cells are delivered to a subject. Other uses for the engineered cells are described elsewhere herein. In an embodiment, the engineered cells can be included in formulations and / or kits described elsewhere herein.

[0335] The engineered cells can be stored short-term or long-term for use at a later time. Suitable storage methods are generally known in the art. Further, methods of restoring the storedcells for use (such as thawing, reconstitution, and otherwise stimulating metabolism in the engineered cell after storage) at a later time are also generally known in the art.Formulations

[0336] The compositions, polynucleotides, polypeptides, particles, cells, vector systems and combinations thereof described herein can be contained in a formulation, such as a pharmaceutical formulation. In an embodiment, the formulations can be delivered to a subject in need thereof. In an embodiment, component(s) of the engineered AAV capsid system, engineered cells, engineered AAV capsid particles, and / or combinations thereof described herein can be included in a formulation that can be delivered to a subject or a cell. In an embodiment, the formulation is a pharmaceutical formulation. One or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be provided to a subject in need thereof or a cell alone or as an active ingredient, such as in a pharmaceutical formulation. As such, also described herein are pharmaceutical formulations containing an amount of one or more of the polypeptides, polynucleotides, vectors, cells, or combinations thereof described herein. In an embodiment, the pharmaceutical formulation can contain an effective amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. The pharmaceutical formulations described herein can be administered to a subject in need thereof or a cell.

[0337] In an embodiment, the amount of the one or more of the polypeptides, polynucleotides, vectors, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein contained in the pharmaceutical formulation can range from about 1 pg / kg to about 10 mg / kg based upon the body weight of the subject in need thereof or average body weight of the specific patient population to which the pharmaceutical formulation can be administered. The amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein in the pharmaceutical formulation can range from about 1 pg to about 10 g, from about 10 nL to about 10 ml. In embodiments where the pharmaceutical formulation contains one or more cells, the amount can range from about 1 cell to 1 x 102, 1 x 103, 1 x 104, 1 x 10?, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x 1010or more cells. In embodiments where the pharmaceutical formulation contains one or more cells, the amount can range from about 1 cell to 1 x 102, 1 x 103, 1 x 104, 1 x 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x 1010or more cells per nL, pL, mL, or L.

[0338] In an embodiment, were engineered AAV capsid particles are included in the formulation, the formulation can contain 1 to 1 x 101, 1 x 102, 1 x 103, 1 x 104, 1 x IO5, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x IO10, 1 x 1011, 1 x 1012, 1 x 1013, 1 x 1014, 1 x 1015, 1 x 1016, 1 x 1017, 1 x 1018, 1 x 1019, or 1 x IO20transducing units (TU) / mL of the engineered AAV capsid particles. In an embodiment, the formulation can be 0.1 to 100 mL in volume and can contain 1 to 1 x 101, 1 x 102, 1 x 103, 1 x 104, 1 x 10s, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x IO10, 1 x 1011, 1 x 1012, 1 x 1013, 1 x 1014, 1 x 1015, 1 x 1016, 1 x 1017, 1 x 1018, 1 x 1019, or 1 x IO20transducing units (TU) / mL of the engineered AAV capsid particles.Pharmaceutically Acceptable Carriers and Auxiliary Ingredients and Agents

[0339] In an embodiment, the pharmaceutical formulation containing an amount of one or more of the polypeptides, polynucleotides, vectors, cells, virus particles, nanoparticles, other delivery particles, and combinations thereof described herein can further include a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxy methylcellulose, and polyvinyl pyrrolidone, which do not deleteriously react with the active composition.

[0340] The pharmaceutical formulations can be sterilized, and if desired, mixed with auxiliary agents, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and / or aromatic substances, and the like which do not deleteriously react with the active composition.

[0341] In addition to an amount of one or more of the polypeptides, polynucleotides, vectors, cells, engineered AAV capsid particles, nanoparticles, other delivery particles, and combinations thereof described herein, the pharmaceutical formulation can also include an effective amount of an auxiliary active agent, including but not limited to, polynucleotides, amino acids, peptides, polypeptides, antibodies, aptamers, ribozymes, hormones, immunomodulators, antipyretics, anxiolytics, antipsychotics, analgesics, antispasmodics, anti-inflammatories, anti-histamines, anti-infectives, chemotherapeutics, and combinations thereof.

[0342] Suitable hormones include, but are not limited to, amino-acid derived hormones (e.g., melatonin and thyroxine), small peptide hormones and protein hormones (e.g., thyrotropinreleasing hormone, vasopressin, insulin, growth hormone, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone), eicosanoids (e.g., arachidonic acid, lipoxins, and prostaglandins), and steroid hormones (e.g., estradiol, testosterone, tetrahydro testosterone Cortisol). Suitable immunomodulators include, but are not limited to, prednisone, azathioprine, 6-MP, cyclosporine, tacrolimus, methotrexate, interleukins (e.g., IL-2, IL-7, and IL-12) , cytokines (e.g., interferons (e.g., IFN-a, IFN-P, IFN-s, IFN-K, IFN-co, and IFN-y), granulocyte colony-stimulating factor, and imiquimod), chemokines (e.g., CCL3, CCL26 and CXCL7), cytosine phosphate-guanosine, oligodeoxynucleotides, glucans, antibodies, and aptamers).

[0343] Suitable antipyretics include, but are not limited to, non-steroidal anti-inflammatories (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), aspirin and related salicylates (e.g., choline salicylate, magnesium salicylate, and sodium salicylate), paracetamol / acetaminophen, metamizole, nabumetone, phenazone, and quinine.

[0344] Suitable anxiolytics include, but are not limited to, benzodiazepines (e g., alprazolam, bromazepam, chlordiazepoxide, clonazepam, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam, triazolam, and tofisopam), serotonergic antidepressants (e.g., selective serotonin reuptake inhibitors, tricyclic antidepressants, and monoamine oxidase inhibitors), mebicar, fabomotizole, selank, bromantane, emoxypine, azapirones, barbiturates, hydroxyzine, pregabalin, validol, and beta blockers.

[0345] Suitable antipsychotics include, but are not limited to, benperidol, bromoperidol, droperidol, haloperidol, moperone, pipamperone, timiperone, fluspirilene, penfluridol, pimozide, acepromazine, chlorpromazine, cyamemazine, dixyrazine, fluphenazine, levomepromazine, mesoridazine, perazine, pericyazine, perphenazine, pipotiazine, prochlorperazine, promazine, promethazine, prothipendyl, thioproperazine, thioridazine, trifluoperazine, triflupromazine, chlorprothixene, clopenthixol, flupentixol, thiothixene, zuclopenthixol, clotiapine, loxapine, prothipendyl, carpipramine, clocapramine, molindone, mosapramine, sulpiride, veralipride, amisulpride, amoxapine, aripiprazole, asenapine, clozapine, blonanserin, iloperidone, lurasidone, melperone, nemonapride, olanzapine, paliperidone, perospirone, quetiapine, remoxipride, risperidone, sertindole, trimipramine, ziprasidone, zotepine, alstonie, bifeprunox, bitopertin, brexpiprazole, cannabidiol, cariprazine, pimavanserin, pomaglumetad methionil, vabicaserin, xanomeline, and zicronapine.

[0346] Suitable analgesics include, but are not limited to, paracetamol / acetaminophen, nonsteroidal anti-inflammatories (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), opioids (e.g., morphine, codeine, oxycodone, hydrocodone, dihydromorphine, pethidine, buprenorphine), tramadol, norepinephrine, flupirtine, nefopam, orphenadrine, pregabalin, gabapentin, cyclobenzaprine, scopolamine, methadone, ketobemidone, piritramide, and aspirin and related salicylates (e.g., choline salicylate, magnesium salicylate, and sodium salicylate).

[0347] Suitable antispasmodics include, but are not limited to, mebeverine, papaverine, cyclobenzaprine, carisoprodol, orphenadrine, tizanidine, metaxalone, methocarbamol, chlorzoxazone, baclofen, dantrolene, baclofen, tizanidine, and dantrolene. Suitable antiinflammatories include, but are not limited to, prednisone, non-steroidal anti-inflammatories (e.g., ibuprofen, naproxen, ketoprofen, and nimesulide), COX-2 inhibitors (e.g., rofecoxib, celecoxib, and etoricoxib), and immune selective anti-inflammatory derivatives (e.g., submandibular gland peptide-T and its derivatives)

[0348] Suitable anti -histamines include, but are not limited to, Hl-receptor antagonists (e.g., acrivastine, azelastine, bilastine, brompheniramine, buclizine, bromodiphenhydramine, carbinoxamine, cetirizine, chlorpromazine, cyclizine, chlorpheniramine, clemastine, cyproheptadine, desloratadine, dexbrompheniramine, dexchlorpheniramine, dimenhydrinate, dimetindene, diphenhydramine, doxylamine, ebastine, embramine, fexofenadine, hydroxyzine, levocetirizine, loratadine, meclizine, mirtazapine, olopatadine, orphenadrine, phenindamine, pheniramine, phenyltoloxamine, promethazine, pyrilamine, quetiapine, rupatadine, tripelennamine, and triprolidine), H2-receptor antagonists (e.g., cimetidine, famotidine, lafutidine, nizatidine, ranitidine, and roxatidine), tritoqualine, catechin, cromoglicate, nedocromil, and p2-adrenergic agonists.

[0349] Suitable anti-infectives include, but are not limited to, amebicides (e.g., nitazoxanide, paromomycin, metronidazole, tinidazole, chloroquine, miltefosine, amphotericin b, and iodoquinol), aminoglycosides (e.g., paromomycin, tobramycin, gentamicin, amikacin, kanamycin, and neomycin), anthelmintics (e.g., pyrantel, mebendazole, ivermectin, praziquantel, albendazole, thiabendazole, oxamniquine), antifungals (e.g., azole antifungals (e.g., itraconazole, fluconazole, parconazole, ketoconazole, clotrimazole, miconazole, and voriconazole), echinocandins (e.g., caspofungin, anidulafungin, and micafungin), griseofulvin, terbinafine,flucytosine, and polyenes (e.g., nystatin, and amphotericin b), antimalarial agents (e.g., pyrimethamine / sulfadoxine, artemether / lumefantrine, atovaquone / proguanil, quinine, hydroxychloroquine, mefloquine, chloroquine, doxycycline, pyrimethamine, and halofantrine), antituberculosis agents (e.g., aminosalicylates (e.g., aminosalicylic acid), isoniazid / rifampin, isoniazid / pyrazinamide / rifampin, bedaquiline, isoniazid, ethambutol, rifampin, rifabutin, rifapentine, capreomycin, and cycloserine), antivirals (e.g., amantadine, rimantadine, abacavir / lamivudine, emtricitabine / tenofovir, cobicistat / elvitegravir / emtricitabine / tenofovir, efavirenz / emtricitabine / tenofovir, abacavir / lamivudine / zidovudine, lamivudine / zidovudine, emtricitabine / tenofovir, emtricitabine / lopinavir / ritonavir / tenofovir, interferon alfa-2v / ribavirin, peginterferon alfa-2b, maraviroc, raltegravir, dolutegravir, enfuvirtide, foscarnet, fomivirsen, oseltamivir, zanamivir, nevirapine, efavirenz, etravirine, rilpivirine, delavirdine, nevirapine, entecavir, lamivudine, adefovir, sofosbuvir, didanosine, tenofovir, abacavir, zidovudine, stavudine, emtricitabine, zalcitabine, telbivudine, simeprevir, boceprevir, telaprevir, lopinavir / ritonavir, boceprevir, darunavir, ritonavir, tipranavir, atazanavir, nelfinavir, amprenavir, indinavir, saquinavir, ribavirin, valacyclovir, acyclovir, famciclovir, ganciclovir, and valganciclovir), carbapenems (e.g., doripenem, meropenem, ertapenem, and cilastatin / imipenem), cephalosporins (e.g., cefadroxil, cephradine, cefazolin, cephalexin, cefepime, cefazoline, loracarbef, cefotetan, cefuroxime, cefprozil, loracarbef, cefoxitin, cefaclor, ceftibuten, ceftriaxone, cefotaxime, cefpodoxime, cefdinir, cefixime, cefditoren, ceftizoxime, and ceftazidime), glycopeptide antibiotics (e.g., vancomycin, dalbavancin, oritavancin, and telavancin), glycylcyclines (e.g., tigecycline), leprostatics (e.g., clofazimine and thalidomide), lincomycin and derivatives thereof (e.g., clindamycin and lincomycin ), macrolides and derivatives thereof (e.g., telithromycin, fidaxomicin, erythromycin, azithromycin, clarithromycin, dirithromycin, and troleandomycin), linezolid, sulfamethoxazole / trimethoprim, rifaximin, chloramphenicol, Fosfomycin, metronidazole, aztreonam, bacitracin, penicillin (amoxicillin, ampicillin, bacampicillin, carbenicillin, piperacillin, ticarcillin, amoxicillin / clavulanate, ampicillin / sulbactam, piperacillin / tazobactam, clavulanate / ticarcillin, penicillin, procaine penicillin, oxacillin, dicloxacillin, and nafcillin), quinolones (e.g., lomefloxacin, norfloxacin, ofloxacin, gatifloxacin, moxifloxacin, ciprofloxacin, levofloxacin, gemifloxacin, moxifloxacin, cinoxacin, nalidixic acid, enoxacin, grepafloxacin, gatifloxacin, trovafl oxaci n, and sparfloxacin), sulfonamides (e.g., sulfamethoxazole / trimethoprim, sulfasalazine, and sulfisoxazole),tetracyclines (e ., doxycycline, dem ecl ocy cline, minocycline, doxycycline / salicylic acid, doxycycline / omega-3 polyunsaturated fatty acids, and tetracycline), and urinary anti-infectives (e.g., nitrofurantoin, methenamine, Fosfomycin, cinoxacin, nalidixic acid, trimethoprim, and methylene blue).

[0350] Suitable chemotherapeutics include, but are not limited to, paclitaxel, brentuximab vedotin, doxorubicin, 5-FU (fluorouracil), everolimus, pemetrexed, melphalan, pamidronate, anastrozole, exemestane, nelarabine, ofatumumab, bevacizumab, belinostat, tositumomab, carmustine, bleomycin, bosutinib, busulfan, alemtuzumab, irinotecan, vandetanib, bicalutamide, lomustine, daunorubicin, clofarabine, cabozantinib, dactinomycin, ramucirumab, cytarabine, Cytoxan, cyclophosphamide, decitabine, dexamethasone, docetaxel, hydroxyurea, dacarbazine, leuprolide, epirubicin, oxaliplatin, asparaginase, estramustine, cetuximab, vismodegib, asparaginase Erwinia chrysanthemi, amifostine, etoposide, flutamide, toremifene, fulvestrant, letrozole, degarelix, pralatrexate, methotrexate, floxuridine, obinutuzumab, gemcitabine, afatinib, imatinib mesylate, carmustine, eribulin, trastuzumab, altretamine, topotecan, ponatinib, idarubicin, ifosfamide, ibrutinib, axitinib, interferon alfa-2a, gefitinib, romidepsin, ixabepilone, ruxolitinib, cabazitaxel, ado-trastuzumab emtansine, carfilzomib, chlorambucil, sargramostim, cladribine, mitotane, vincristine, procarbazine, megestrol, trametinib, mesna, strontium-89 chloride, mechlorethamine, mitomycin, busulfan, gemtuzumab ozogamicin, vinorelbine, filgrastim, pegfilgrastim, sorafenib, nilutamide, pentostatin, tamoxifen, mitoxantrone, pegaspargase, denileukin diftitox, alitretinoin, carboplatin, pertuzumab, cisplatin, pomalidomide, prednisone, aldesleukin, mercaptopurine, zoledronic acid, lenalidomide, rituximab, octreotide, dasatinib, regorafenib, histrelin, sunitinib, siltuximab, omacetaxine, thioguanine (tioguanine), dabrafenib, erlotinib, bexarotene, temozolomide, thiotepa, thalidomide, Bacillus Calmette-Guerin (BCG), temsirolimus, bendamustine hydrochloride, triptorelin, arsenic trioxide, lapatinib, valrubicin, panitumumab, vinblastine, bortezomib, tretinoin, azacitidine, pazopanib, teniposide, leucovorin, crizotinib, capecitabine, enzalutamide, ipilimumab, goserelin, vorinostat, idelalisib, ceritinib, abiraterone, epothilone, tafluposide, azathioprine, doxifluridine, vindesine, and all-trans retinoic acid.

[0351] In embodiments where there is an auxiliary active agent contained in the pharmaceutical formulation in addition to the one or more of the polypeptides, polynucleotides, CRISPR-Cas complexes, vectors, cells, virus particles, nanoparticles, other delivery particles, andcombinations thereof described herein, amount, such as an effective amount, of the auxiliary active agent will vary depending on the auxiliary active agent. In an embodiment, the amount of the auxiliary active agent ranges from 0.001 micrograms to about 1 milligram. In other embodiments, the amount of the auxiliary active agent ranges from about 0.01 IU to about 1000 IU. In further embodiments, the amount of the auxiliary active agent ranges from 0.001 mL to about 1 mL. In yet other embodiments, the amount of the auxiliary active agent ranges from about 1 % w / w to about 50% w / w of the total pharmaceutical formulation. In additional embodiments, the amount of the auxiliary active agent ranges from about 1 % v / v to about 50% v / v of the total pharmaceutical formulation. In still other embodiments, the amount of the auxiliary active agent ranges from about 1 % w / v to about 50% w / v of the total pharmaceutical formulation.Dosage Forms

[0352] In an embodiment, the pharmaceutical formulations described herein may be in a dosage form. The dosage forms can be adapted for administration by any appropriate route. Appropriate routes include, but are not limited to, rectal, epidural, intracranial, intraocular, inhaled, intranasal, topical (including buccal, sublingual, or transdermal), vaginal, intraurethral, parenteral, intracranial, subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, intraosseous, intracardiac, intraarticular, intracavemous, intrathecal, intravitreal, intracerebral, gingival, subgingival, intracerebroventricular, and intradermal. Such formulations may be prepared by any method known in the art.

[0353] For delivery' to a system or tissue, the AAV formulations described herein may be administered intra-parenchymally, intrathecally, intracerebroventricularly, intracisternally, intravenously, into the carotid artery, systemically or a combination of these. In some aspects, the AAV formulation is administered by intrathecally in equal portions to the cisterna magna and the lumbar spine.

[0354] In some aspects, an AAV pharmaceutical formulation can be administered as a single bolus injection of about 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml, 1 ml, 2 ml, 3 ml, 4 ml, or 5 ml. In other aspects, an AAV pharmaceutical formulation is delivered as an infusion at a rate of 0.001 ml / min to 1 ml / min, (e.g., 0.01 ml / min).

[0355] In certain aspects, more than one administration (e.g., two, three, four, five, six, seven, eight, nine, 10, etc., or more administrations) may be employed to achieve the desired level of gene expression over a period of various intervals, e g., hourly, daily, weekly, monthly, yearly,etc. Dosing can be single dosage or cumulative (serial dosing), and can be readily determined by one skilled in the art. For instance, treatment of a disease or disorder may comprise a one-time administration of an effective dose of a pharmaceutical composition virus vector disclosed herein. Alternatively, treatment of a disease or disorder may comprise multiple administrations of an effective dose of a virus vector carried out over a range of time periods, such as, e g., once daily, twice daily, trice daily, once every few days, or once weekly. In certain aspects, rAAV particles may be administered to multiple locations, for example, 1, 2, 3, 4, or 5 locations simultaneously or staggered over time.

[0356] In an embodiment, the more than one administration may include immunosuppression or immunomodulatory agents (e.g., steroids, anti-B cell antibodies, rapamycin or other mTOR inhibitors). In an embodiment, the immuno-suppression or immunomodulatory agent is an inhibitor, see e.g., WO2021067598A1, hereby incorporated by reference. In an embodiment, the immuno-suppression or immunomodulatory agent is an antibody, see e.g., EP3909602A1, hereby incorporated by reference. In an embodiment, the immuno-suppression or immunomodulatory agent is a protease or glycosidase, see e.g., W02020016318A1, hereby incorporated by reference. In an embodiment, the immuno-suppression or immunomodulatory agent is a steroid, see e.g., WO2021163322, hereby incorporated by reference.

[0357] The timing of administration can vary from individual to individual, depending upon such factors as the severity of an individual's symptoms. For example, an effective dose of a virus vector disclosed herein can be administered to an individual once every six months for an indefinite period, or until the individual no longer requires therapy. A person of ordinary skill in the art will recognize that the condition of the individual can be monitored throughout the course of treatment and that the effective amount of a virus vector disclosed herein that is administered can be adjusted accordingly.

[0358] The pharmaceutical composition is typically in liquid form. Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, magnesium chloride, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, a surfactant, such as pluronic acid (PF68) 0.001% may be used.

[0359] For injection, the active ingredient will be in the form of an aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art arewell able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included, as required.

[0360] Methods of delivering an AAV preparation to a specific region of a system or tissue by stereotactic injection can be found in U.S. Patent No. 10,898,585, the content of which is incorporated by reference herein in its entirety.

[0361] Dosage forms adapted for parenteral administration and / or adapted for any type of injection (e.g., intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, intraosseous, epidural, intracardiac, intraarticular, intracavernous, gingival, subgingival, intrathecal, intravitreal, intracerebral, and intracerebroventricular) can include aqueous and / or non-aqueous sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats, solutes that render the composition isotonic with the blood of the subject, and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents. The dosage forms adapted for parenteral administration can be presented in a single- unit dose or multi-unit dose containers, including but not limited to sealed ampoules or vials. The doses can be lyophilized and resuspended in a sterile carrier to reconstitute the dose prior to administration. Extemporaneous injection solutions and suspensions can be prepared, in an embodiment, from sterile powders, granules, and tablets. See e.g., Glascock, J. J., et al. Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice. J. Vis. Exp. (56), e2968 and Foley CP, et al. Intra-arterial delivery of AAV vectors to the mouse brain after mannitol mediated blood brain barrier disruption. J Control Release. 2014 Dec 28;196:71-78.

[0362] The dosage form can also be prepared to prolong or sustain the release of any ingredient. In an embodiment, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be the ingredient whose release is delayed. In other embodiments, the release of an optionally included auxiliary ingredient is delayed. Suitable methods for delaying the release of an ingredient include, but are not limited to, coating or embedding the ingredients in material in polymers, wax, gels, and the like. Delayed release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets,” eds. Liberman et. al. (New York, Marcel Dekker, Inc., 1989), “Remington - The science and practice of pharmacy”, 20th ed., Lippincott Williams & Wilkins, Baltimore, MD,2000, and “Pharmaceutical dosage forms and drug delivery systems”, 6th Edition, Ansel et al., (Media, PA: Williams and Wilkins, 1995).

[0363] Dosage forms adapted for nasal or inhalation administration include aerosols, solutions, suspension drops, gels, or dry powders. In an embodiment, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein is contained in a dosage form adapted for inhalation is in a particle-size-reduced form that is obtained or obtainable by micronization. In an embodiment, the particle size of the size reduced (e.g., micronized) compound or salt or solvate thereof, is defined by a D50 value of about 0.5 to about 10 microns as measured by an appropriate method known in the art. Dosage forms adapted for administration by inhalation also include particle dusts or mists. Suitable dosage forms wherein the carrier or excipient is a liquid for administration as a nasal spray or drops include aqueous or oil solutions / suspensions of an active ingredient (e.g., the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein and / or auxiliary active agent), which may be generated by various types of metered dose pressurized aerosols, nebulizers, or insufflators.

[0364] In an embodiment, the dosage forms can be aerosol formulations suitable for administration by inhalation. In some of these embodiments, the aerosol formulation can contain a solution or fine suspension of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein and a pharmaceutically acceptable aqueous or non-aqueous solvent. Aerosol formulations can be presented in single or multi-dose quantities in sterile form in a sealed container. For some of these embodiments, the sealed container is a single dose or multi-dose nasal, or an aerosol dispenser fitted with a metering valve (e.g., metered dose inhaler), which is intended for disposal once the contents of the container have been exhausted.

[0365] Where the aerosol dosage form is contained in an aerosol dispenser, the dispenser contains a suitable propellant under pressure, such as compressed air, carbon dioxide, or an organic propellant, including but not limited to a hydrofluorocarbon. The aerosol formulation dosage forms in other embodiments are contained in a pump-atomizer. The pressurized aerosol formulation can also contain a solution or a suspension of one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein. In further embodiments, the aerosol formulation can also contain co-solvents and / or modifiers incorporated to improve, for example, the stability and / or taste and / or fine particle mass characteristics (amountand / or profile) of the formulation. Administration of the aerosol formulation can be once daily or several times daily, for example 2, 3, 4, or 8 times daily, in which 1, 2, or 3 doses are delivered each time.

[0366] For some dosage forms suitable and / or adapted for inhaled administration, the pharmaceutical formulation is a dry powder inhalable formulation. In addition to the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein, an auxiliary active ingredient, and / or pharmaceutically acceptable salt thereof, such a dosage form can contain a powder base such as lactose, glucose, trehalose, mannitol, and / or starch. In some of these embodiments, the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein is in a particle-size reduced form. In further embodiments, a performance modifier, such as L-leucine or another amino acid, cellobiose octaacetate, and / or metals salts of stearic acid, such as magnesium or calcium stearate.

[0367] In an embodiment, the aerosol dosage forms can be arranged so that each metered dose of aerosol contains a predetermined amount of an active ingredient, such as the one or more of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein.

[0368] Dosage forms adapted for ocular administration can include aqueous and / or nonaqueous sterile solutions that can optionally be adapted for injection, and which can optionally contain antioxidants, buffers, bacteriostats, solutes that render the composition isotonic with the eye or fluid contained therein or around the eye of the subject, and aqueous and nonaqueous sterile suspensions, which can include suspending agents and thickening agents.

[0369] For some embodiments, the dosage form contains a predetermined amount of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein per unit dose. In an embodiment, the predetermined amount of the such unit doses may therefore be administered once or more than once a day. Such pharmaceutical formulations may be prepared by any of the methods well known in the art.Effective Amounts

[0370] In an embodiment, the amount of the primary active agent and / or optional secondary agent can be an effective amount, least effective amount, and / or therapeutically effective amount. As used herein, “effective amount”, “effective concentration”, and / or the like refers to the amount, concentration, etc. of the primary and / or optional secondary agent included in theIllpharmaceutical formulation that achieve one or more therapeutic effects or desired effect. As used herein, “least effective”, “least effective concentration”, and / or the like amount refers to the lowest amount, concentration, etc. of the primary and / or optional secondary agent that achieves the one or more therapeutic or other desired effects. As used herein, “therapeutically effective amount”, “therapeutically effective concentration” and / or the like refers to the amount, concentration, etc. of the primary and / or optional secondary agent included in the pharmaceutical formulation that achieves one or more therapeutic effects. In an embodiment, the one or more therapeutic effects comprise evading AAV neutralizing antibodies and transducing a system or tissue.

[0371] The effective dose of engineered AAV particles will depend on multiple factors including the target tissue or system, the transgene being delivered, the route of administration, the degree of antibody evasion required, and individual patient characteristics such as body weight and pre-existing antibody titers. Doses of 5* IO10vg (viral genomes) per mouse can provide robust transduction in both CNS and muscle tissues while demonstrating antibody evasion.

[0372] For human applications, doses can be scaled based on body weight, with typical ranges being approximately 1 x 1012to R IO14vg / kg for systemic administration, though lower or higher doses may be appropriate depending on the specific application. The methods described herein, including the neutralizing antibody assays and in vivo transduction assays, can be used to determine optimal dosing in preclinical models prior to clinical translation.

[0373] In an embodiment, the engineered AAV capsid exhibits enhanced antibody evasion and lower doses may be sufficient to achieve therapeutic efficacy compared to non-evading capsids, for example, in patients with pre-existing neutralizing antibodies. Conversely, the enhanced antibody evasion may enable higher doses to be administered safely, as reduced antibody binding may decrease the risk of complement activation and adverse immune responses. Dose optimization studies using the screening methods described herein can identify the therapeutic window for each specific engineered AAV capsid and transgene combination.

[0374] In an embodiment, the amount or effective amount, particularly where an infective particle is being delivered (e.g., a virus particle having the primary or secondary agent as a cargo), the effective amount of virus particles can be expressed as a titer (plaque forming units per unit of volume) or as a MOI (multiplicity of infection). In an embodiment, the effective amount can be about IxlO1particles per pL, nL, pL, mL, or L to 1X1O20 / particles per pL, nL, pL, mb, or Lor more, such as about IxlO1, IxlO2, IxlO3, IxlO4, IxlO3, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, IxlO13, IxlO14, IxlO15, IxlO16, IxlO17, IxlO18, IxlO19, to / or about IxlO20particles per pL, nL, pL, mL, or L. In an embodiment, the effective titer can be about 1X1O1transforming units per pL, nL, pL, mL, or L to 1X1O20 / transforming units per pL, nL, pL, mL, or L or more, such as about IxlO1, IxlO2, IxlO3, IxlO4, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, IxO13, IxlO14, IxlO13, IxlO16, IxlO17, IxlO18, IxlO19, to / or about IxlO20transforming units per pL, nL, pL, mL, or L or any numerical value or subrange within these ranges. In an embodiment, the MOI of the pharmaceutical formulation can range from about 0.1 to 10 or more, such as 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10 or more or any numerical value or subrange within these ranges.

[0375] In an embodiment, the amount or effective amount, particularly where an infective particle is being delivered (e.g., a virus particle having the primary or secondary agent as a cargo), the effective amount of virus particles can be expressed as vector genomes per kilogram bodyweight (vg / kg). In an embodiment, the effective amount can be about IxlO1vg / kg or more, such as about IxlO2, IxlO3, IxlO4, IxlO5, IxlO6, IxlO7, IxlO8, IxlO9, IxlO10, IxlO11, IxlO12, IxlO13, IxlO14, IxlO13, IxlO16, IxlO17, IxlO18, IxlO19, to / or about IxlO20such that the effective amount can be anything in between, for example, 0.1 to 10 (O.lxlO12to 10xl012). In an embodiment, the pharmaceutical composition is delivered at a dosage between 0.1 x 1012vg / kg to 1 x 1014vg / kg. In an embodiment, the dosage is between 0.1 x 1012vg / kg to 100 x 1012vg / kg. In an embodiment, the dosage is between 1 x 1012vg / kg to 10 x 1012vg / kg. In an embodiment, the dosage is 5 x 1012vg / kg.

[0376] In an embodiment, the effective amount, least effective amount, and / or therapeutically effective amount can be an effective concentration, least effective concentration, and / or therapeutically effective concentration, which can each be any non-zero amount ranging from about 0 to 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570,580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 pM, nM, pM, mM, or M or be any numerical value or subrange within any of these ranges.

[0377] In an embodiment, the primary and / or the optional secondary active agent present in the pharmaceutical formulation can be any non-zero amount ranging from about 0 to 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, 0.5, 0.51, 0.52, 0.53, 0.54, 0.55, 0.56, 0.57, 0.58, 0.59, 0.6, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.7, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, 0.8, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.9, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 % w / w, v / v, or w / v of the pharmaceutical formulation or be any numerical value or subrange within any of these ranges.

[0378] In an embodiment, the amount or effective amount of the one or more of the active agent(s) described herein contained in the pharmaceutical formulation can range from about 1 pg / kg to about 10 mg / kg based upon the body weight of the subject in need thereof or average body weight of the specific patient population to which the pharmaceutical formulation can be administered.

[0379] In an embodiment where there is a secondary agent contained in the pharmaceutical formulation, the effective amount of the secondary active agent will vary depending on the secondary agent, the primary agent, the administration route, subject age, disease, stage of disease, among other things, which will be one of ordinary skill in the art.

[0380] When optionally present in the pharmaceutical formulation, the secondary active agent can be included in the pharmaceutical formulation or can exist as a stand-alone compound or pharmaceutical formulation that can be administered contemporaneously or sequentially with the compound, derivative thereof, or pharmaceutical formulation thereof.

[0381] In an embodiment, the effective amount of the secondary active agent, when optionally present, is any non-zero amount ranging from about 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 % w / w, v / v, or w / v of the total active agents present in the pharmaceutical formulation or any numerical value or subrange within these ranges. In additional embodiments, the effective amount of the secondary active agent is any non-zero amount ranging from about 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 % w / w, v / v, or w / v of the total pharmaceutical formulation or any numerical value or subrange within these ranges.Kits

[0382] Also described herein are kits that contain one or more of the one or more of the compositions, polypeptides, polynucleotides, vectors, cells, or other components described herein and combinations thereof and pharmaceutical formulations described herein. In an embodiment, one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be presented as a combination kit. As used herein, the terms "combination kit" or "kit of parts" refers to the compounds, or formulations and additional components that are used to package, screen, test, sell, market, deliver, and / or administer the combination of elements or a single element, such as the active ingredient, contained therein. Such additional components include but are not limited to, packaging, syringes, blister packages, bottles, and the like. The combination kit can contain one or more of the components (e.g., one or more of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof) or formulation thereof can be provided in a single formulation (e.g., a liquid, lyophilized powder, etc.), or in separate formulations. The separate components or formulations can be contained in a single package or in separate packages within the kit. The kit can also include instructions in a tangible medium of expression that can contain information and / or directions regarding the content of thecomponents and / or formulations contained therein, safety information regarding the content of the components(s) and / or formulation(s) contained therein, information regarding the amounts, dosages, indications for use, screening methods, component design recommendations and / or information, recommended treatment regimen(s) for the components(s) and / or formulations contained therein. As used herein, “tangible medium of expression” refers to a medium that is physically tangible or accessible and is not a mere abstract thought or an unrecorded spoken word. “Tangible medium of expression” includes, but is not limited to, words on a cellulosic or plastic material, or data stored in a suitable computer readable memory form. The data can be stored on a unit device, such as a flash memory drive or CD-ROM or on a server that can be accessed by a user via, e.g., a web interface.

[0383] In one embodiment, the disclosure provides a kit comprising one or more of the components described herein. In an embodiment, the kit comprises a vector system and instructions for using the kit. In an embodiment, the vector system includes a regulatory element operably linked to one or more engineered polynucleotides, such as those containing one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype, and optionally a selective target binding modification, as described elsewhere herein and, optionally, a cargo molecule, which can optionally be operably linked to a regulatory element. The one or more engineered polynucleotides such as those containing one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and optionally a selective target binding modification, as described elsewhere herein and, can be included on the same or different vectors as the cargo molecule in embodiments containing a cargo molecule within the kit.

[0384] In an embodiment, the kit comprises a vector system and instructions for using the kit. In an embodiment, the vector system comprises (a) a first regulatory element operably linked toa direct repeat sequence and one or more insertion sites for inserting one or more guide sequences up- or downstream (whichever applicable) of the direct repeat sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a Cas9 CRISPR complex to a target sequence in a eukaryotic cell, wherein the Cas9 CRISPR complex comprises a Cas9 enzyme complexed with the guide sequence that is hybridized to the target sequence; and / or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Cas9 enzyme comprising a nuclear localization sequence. Where applicable, a tracr sequence may also be provided. In an embodiment, the kit comprises components (a) and (b) located on the same or different vectors of the system. In an embodiment, component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell. In an embodiment, the Cas9 enzyme comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In an embodiment, the CRISPR enzyme is a type V or VI CRISPR system enzyme. In an embodiment, the CRISPR enzyme is a Cas9 enzyme. In an embodiment, the Cas9 enzyme is derived from Francisella tularensis 1, Francisella tularensis subsp. novicida, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW201 l_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6, Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonas macacae Cas9 (e.g., modified to have or be associated with at least one DD), and may include further alteration or mutation of the Cas9, and can be a chimeric Cas9. In an embodiment, the DD-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell. In an embodiment, the DD-CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence. In an embodiment, the DD-CRISPR enzyme lacks or substantially DNA strand cleavage activity (e g., no more than 5% nuclease activity as compared with a wild-type enzyme or enzyme not having the mutation or alteration that decreases nuclease activity). In an embodiment, the first regulatory element is a polymerase III promoter. In an embodiment, the second regulatory element is a polymerase II promoter. In an embodiment, the guide sequence isat least 16, 17, 18, 19, 20, 25 nucleotides, or between 16-30, or between 16-25, or between 16-20 nucleotides in length.Methods of Use for Delivery of Cargo to a System or Tissue

[0385] The compositions including an engineered AAV capsid polypeptide comprising one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and optionally one or more of the cell-selective target binding modifications, engineered AAV capsid system polynucleotides, polypeptides, vector(s), engineered cells, engineered AAV capsid particles can be used generally to package and / or deliver one or more cargos to neurons, glial cells, and endothelial cells of the system or tissue. In an embodiment, delivery is done in cell-selective manner based upon one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype and selectivity of the optional target binding modification. In an embodiment this is conferred by the tropism of the engineered AAV capsid, which can be influenced at least in part by the one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype and inclusion of the optional one or target binding modifications described elsewhere herein. In an embodiment, compositions including one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype and optionally one or more ofthe system or tissue target binding modifications, including engineered AAV capsid particles where the capsids incorporate the one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype and optionally the target binding modification, can be administered to a subject or a cell, tissue, and / or organ and facilitate the transfer and / or integration of the cargo to the recipient cell. In other embodiments, engineered cells capable of producing compositions, such as polypeptides and other particles (e.g., engineered AAV capsids and viral particles), containing one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype and optionally one or more of the target binding modifications can be generated from the polynucleotides, vectors, and vector systems etc., described herein. This includes without limitation, the engineered AAV capsid system molecules (e.g., polynucleotides, vectors, and vector systems, etc.). In an embodiment, the polynucleotides, vectors, and vector systems etc., described herein capable of generating the compositions, such as polypeptides and other particles (e.g., engineered AAV capsids and viral particles), containing one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype and optionally one or more of the target binding modifications can be delivered to a cell or tissue, in vivo, ex vivo, or in vitro. In an embodiment, when delivered to a subject, the composition can transform a subject’s cell in vivo or ex vivo to produce an engineered cell that can be capable of making a composition described herein that contains one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551N, D551K, N552D, D554N, D556K,D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype and optionally one or more of the cell-selective target binding modifications described herein, including but not limited to the engineered AAV capsid particles, which can be released from the engineered cell and deliver cargo molecule(s) to a recipient cell in vivo or produce personalized engineered compositions (e.g., AAV capsid particles) for reintroduction into the subject from which the recipient cell was obtained.

[0386] In an embodiment, an engineered cell can be delivered to a subject, where it can release produced compositions of the present disclosure (including but not limited to engineered AAV capsid particles) such that they can then deliver a cargo (e.g., a cargo polynucleotide(s)) to a recipient cell. These general processes can be used in a variety of ways to treat and / or prevent disease or a symptom thereof in a subject, generate model cells, generate modified organisms, provide cell selection and screening assays, in bioproduction, and in other various applications.

[0387] In an embodiment, the compositions, such as polypeptides and other particles (e.g., engineered AAV capsids and viral particles), containing one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, andN716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and optionally one or more of the target binding modifications, can be delivered to neural cells of the system or tissue. In another example embodiment, the compositions containing one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R550Q, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, andN716D of an AAV9 VP1 polypeptide or an analogous position of a VP1 polypeptide of another AAV serotype, and optionally one or more target binding modifications, can be delivered to glial cells of the system or tissue.

[0388] In an embodiment, the engineered AAV capsid polynucleotides, vectors, and systems thereof can be used to generate engineered AAV capsid variant libraries that can be mined for variants with a desired cell-selectivity. The description provided herein as supported by thevarious Examples can demonstrate that one having a desired cell-selectivity in mind could utilize the present disclosure as described herein to obtain a capsid with the desired cell-selectivity. Modified Tropism

[0389] The adeno-associated virus (AAV) has more than one hundred naturally occurring variants, and this library has been further expanded by various techniques for modifying the viral capsid. These AAV capsid variants have unique antigenic profdes and demonstrate distinct cellular tropisms driven by differences in receptor binding. Additionally, AAV capsids can be chemically modified to alter tropism, produced as hybrid vectors combining the properties of multiple serotypes, and carry peptide insertions that introduce novel receptor-binding activity. Furthermore, directed evolution of shuffled genome libraries can identify engineered variants with unique properties, and rational modification of the viral capsid can alter tropism, reduce blockage by neutralizing antibodies, or enhance transduction efficiency, (reviewed by Castle, M. J., Turunen, H. T., Vandenberghe, L. H. & Wolfe, J. H. Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids. Methods Mol. Biol. 1382, 133-149 (2016)).Regulation of transgene expression by microRNAs

[0390] MicroRNAs (miRNAs) are indeed small, non-coding RNA molecules that play a crucial role in regulating gene expression at the post-transcriptional level. They bind to complementary sequences on messenger RNAs (mRNAs), typically resulting in gene silencing either by degrading the mRNA or by inhibiting its translation into proteins.

[0391] In an embodiment, AAV transgene expression in the various regions of the brain can be further fine-tuned by the incorporation of specific miRNA binding sites into the transgene's 5' or 3'-UTR. Thus, AAV transgene expression can be suppressed in those cells where the microRNA is expressed and the corresponding miRNA binding site is placed in the 5' or 3'-UTR.A summary of miRNA gene expression in specific cell types of the CNS can be found in the Table belo, reproduced from Zolboot, N., Du, J. X., Zampa, F. & Lippi, G. MicroRNAs instruct and maintain cell type diversity in the nervous system. Front. Mol. Neurosci. 14, 646072 (2021).

[0392] In one embodiment, the transgene's 5' or 3'-UTR may contain one or more miR-122 binding sites to suppress spurious transgene expression and potential toxicity in the liver (see, for example, Qiao, C. et al. Liver-specific microRNA- 122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver. Gene Ther. 18, 403-410 (2011)). Inone embodiment, the transgene's 5' or 3'-UTR may contain one or more miR-183 binding sites to suppress spurious transgene expression and potential toxicity in the liver (see, for example, Hordeaux, J.; Buza, E. L.; Jeffrey, B.; Song, C.; Jahan, T.; Yuan, Y.; Zhu, Y.; Bell, P.; Li, M.; Chichester, J. A.; Calcedo, R.; Wilson, J. M. MicroRNA-Mediated Inhibition of Transgene Expression Reduces Dorsal Root Ganglion Toxicity by AAV Vectors in Primates. Science Translational Medicine, 2020, 12).Methods of Treatment

[0393] Provided herein are methods for treating a disease or disorder, the method comprising administering to a subject in need thereof, a composition as disclosed herein to cells of the targeted system or tissue. In an aspect, the compositions used in methods disclosed herein arecapable of increasing transduction of cells of the system or tissue, allowing for delivery of cargo and therapeutics directly to such cell types. In an embodiment, a method is disclosed wherein the cargo is one or more polypeptides.Diseases or Disorders

[0394] In an embodiment, a method is disclosed wherein the disease or disorder is a cancer, neurological disorder, or infection.

[0395] In an embodiment, methods of treatment comprise administering a composition as detailed herein to a subject in need thereof. In an example embodiment, the cancer is a neuroepithelial cancer. In an embodiment, the cancer is a neuroepithelial tumor, for example, Astrocytic tumors, e.g., Diffuse Astrocytoma (fibrillary, protoplasmic, gemistocytic, mixed), Anaplastic (malignant) astrocytoma, Glioblastoma (giant cell, gliosarcoma variants), Pilocytic astrocytoma, Pleomorphic xanthoastrocytoma, or Subependymal giant cell astrocytoma; Oligodendroglial tumors, e.g., Oligodendroglioma, Anaplastic (malignant) Oligodendroglioma, Ependymal tumors, Ependymoma (cellular, papillary, clear cell, tanycytic), Anaplastic (malignant) ependymoma, ependymoma, Subependymoma; Mixed tumors, e.g., Oligoastrocytomaor Anaplastic (malignant) oligoastrocytoma; Choroid plexus tumors, e.g., Choroid Plexus papilloma or Choroid Plexus carcinoma; Neuronal and mixed neuronal -glial tumors, e.g., Gangliocytoma, Gangloglioma, Dysembryoplastic neuroepithelial tumor (DNET), Dysplastic gangliocytoma of the cerebellum (Lhermitte-Duclos), Desmoplastic infantile astrocytoma / ganglioglioma, Central neurocytoma, Anaplastic ganglioglioma, Cerebellar liponeurocytoma, Paraganglioma of the filum terminale; Pineal tumors, e.g., Pineocytoma, Pineoblastoma, Pineal parenchymal tumor of intermediate differentiation; Embryonal tumors, e.g., Medulloblastoma (desmoplastic, large cell, melanotic, medullomyoblastoma), Medulloepithelioma, Supratentorial primitive neuroectodermal tumors, PNETs such as Neuroblastoma, Ganglioneuroblastoma, Ependymoblastoma, or Atypical teratoid / rhabdoid tumor; Neuroblastic tumors, e.g., Olfactory (esthesioneuroblastoma), Olfactory neuroepithelioma, Neuroblastomas of the adrenal gland and sympathetic nervous system; Glial tumors of uncertain etiolo...

Claims

CLAIMSWhat is claimed is:

1. An engineered AAV capsid polypeptide comprising one or more amino acid substitutions selected from D327N, N328K, N329D, K332Q, N452D, N452K, G455Q, G455T, G455K, G455N, K462E, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551N, D551K, N552D, D554N, D556K, D556N, K557Q, K557E, D657N, N663D, K664Q, K664E, D665N, N668K, and N716D of an AAV9 VP1 polypeptide or an analogous position of a VP 1 polypeptide of another AAV serotype.

2. The engineered AAV capsid polypeptide of claim 1, wherein the amino acid substitutions comprise N425K / D, E500D, P504T, A510K, R550Q, D551N / K, D554N, and K557Q.

3. The engineered AAV capsid polypeptide of claim 2, wherein the amino acid substitutions further comprise 1 to 12 substitutions selected from the group consisting of D327N, N328K, N329D, K332Q, K462E / Q, R533Q, D657N, D657N, N663D, K664Q, N665N, and N668K.

4. The engineered AAV capsid polypeptide of claim 3, wherein the amino acid substitutions further comprise D327N, K332Q, and D657N.

5. The engineered AAV capsid polypeptide of claim 3, wherein additional amino acid substitutions comprise N328K and N329D.

6. The engineered AAV capsid polypeptide of claim 3, wherein additional amino acid substitutions comprise K462E / Q, and N668K.

7. The engineered AAV capsid polypeptide of claim 3, wherein an additional amino acid substitution comprises N668K.

8. The engineered AAV capsid polypeptide of claim 3, wherein an additional amino acid substitution comprises N663D.

9. The engineered AAV capsid polypeptide of claim 2, wherein the amino acid substitutions comprise D327N, N328K, N329D, K332Q, N452D, E500D, A502S, P504T, A510K, R550Q, D551N, D554N, K557Q, D657N, N663D, and D665N.

10. The engineered AAV capsid polypeptide of claim 2, wherein the amino acid substitutions comprise D327N, N328K, N329D, K332Q, N452D, E500D, A502S, P504T, A510K, R550Q, D551N, D554N, K557Q and D657N.

11. The engineered AAV capsid polypeptide of claim 2, wherein the amino acid substitutions comprise D327N, K332Q, N452D, E500D, A502S, P504T, A510K, R550Q, D551N, D554N, K557Q, D657N, N663D, and N668K.

12. The engineered AAV capsid polypeptide of claim 2, wherein the amino acid substitutions comprise N452K, K462E, E500D, A502S, P504T, A510K, R550Q, D551N, D554N, K557Q, D657N, andN663D.

13. The engineered AAV capsid polypeptide of claim 2, wherein the amino acid substitutions comprise N452K, K462Q, E500D, A502S, P504T, A510K, R533Q, R55OQ, D551K, D554N, K557Q, K664Q, andN668K.

14. The engineered AAV capsid polypeptide of claim 1, wherein the amino acid substitutions comprise G455Q / T / K / N, D551N, N552D, K557E / Q, K664E / Q, D665N, andN668K.

15. The engineered AAV capsid polypeptide of claim 14, wherein amino acid substitutions further comprise 1 to 5 substitutions selected from the group consisting of D554N, D556K / N, N663D, and N716D.

16. The engineered AAV capsid polypeptide of claim 14, wherein an additional amino acid substitution comprises D556K / N.

17. The engineered AAV capsid polypeptide of claim 14, wherein an additional amino acid substitution comprises D554N.

18. The engineered AAV capsid polypeptide of claim 14, wherein an additional amino acid substitution comprises N716D.

19. The engineered AAV capsid polypeptide of claim 14, wherein the amino acid substitutions comprise G455Q, D551N, N552D, D556K, K557E, K664E, D665N, N668K, andN716D.

20. The engineered AAV capsid polypeptide of claim 14, wherein the amino acid substitutions comprise G455T, D551N, N552D, D556K, K557E, K664E, D665N, N668K, and N716D.

21. The engineered AAV capsid polypeptide of claim 14, wherein the amino acid substitutions comprise G455K, D551N, N552D, D556K, K557E, K664E, D665N, N668K, andN716D.

22. The engineered AAV capsid polypeptide of claim 14, wherein the amino acid substitutions comprise G455N, D551N, N552D, D554N, D556N, K557E, N663D, K664Q, D665N, and N668K.

23. The engineered AAV capsid polypeptide of any one of the preceding claims, wherein the engineered AAV capsid polypeptide is selected from SEQ ID NO: 26-34 and 1641-1648.

24. The engineered AAV capsid polypeptide of any one of the preceding claims further comprising a targeting moiety.

25. The engineered AAV capsid polypeptide of claim 24, wherein the targeting moiety is an insertion or substitution in loop IV, loop VIII, or both.

26. The engineered AAV capsid polypeptide claims 24 or 25, wherein the targeting moiety is a central nervous system (CNS) targeting moiety.

27. The engineered AAV capsid polypeptide of claim 26, wherein the CNS targeting moiety binds a transferrin receptor (TfR.1), a CD59, a CA4, an ALPL, a Ly6a, a Ly6c, or a Car4 protein.

28. The engineered AAV capsid polypeptide of claim 27, wherein the targeting moiety is a TfR.1 binding targeting moiety.

29. The engineered AAV capsid polypeptide of claim 28, wherein the TfRl binding targeting moiety is selected from SEQ ID NO: 40-167 or 269-1606.

30. The engineered AAV capsid polypeptide of claim 28, wherein the engineered AAV capsid polypeptide is selected from SEQ ID NO: 7-21, 40-167, and 1649-1656.

31. The engineered AAV capsid polypeptide of claim 27, wherein the targeting moiety is a CD59 targeting moiety.

32. The engineered AAV capsid polypeptide of claim 27, wherein the targeting moiety is a CA4 targeting moiety.

33. The engineered AAV capsid polypeptide of claim 32, wherein the CA4 targeting moiety is selected from SEQ ID NO: 168-169.

34. The engineered AAV capsid polypeptide of claim 24, wherein the targeting moiety is a muscle-specific targeting moiety.

35. The engineered AAV capsid polypeptide of claim 34, wherein the muscle-specific targeting moiety comprises a RGD motif.

36. The engineered AAV capsid polypeptide of claim 35, wherein the RGD motif is selected from SEQ ID NO: 170-191, 1685-1699, 1705, 1708-1709.

37. The engineered AAV capsid polypeptide of claim 34, wherein the engineered AAV capsid polypeptide is selected from SEQ ID NO: 35-39 and 1657-1684.

38. The engineered AAV capsid polypeptide of claim 34, wherein the muscle-specific targeting moiety is selected from SEQ ID NO: 1700-1704 and 1706-1707.

39. The engineered AAV capsid polypeptide of any one of the preceding claims, wherein the AAV capsid polypeptide is an AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV rh.74, or AAV rh.10.

40. An engineered AAV particle comprising the engineered AAV capsid polypeptide of any one of the preceding claims and further comprising a recombinant AAV genome encoding a transgene.

41. The engineered AAV particle of claim 40, wherein the transgene encodes a therapeutic polypeptide, an antibody or fragment thereof, a shRNA, miRNA, a CRISPR-Cas system, Transcription Activator-like Effector (TALE)- or Zinc Finger Protein (ZFP)-based transcriptional activator; repressor; or epigenomic silencer, a RNA encoding a partial gene fragment designed for trans-splicing into an endogenous RNA, one or more transfer RNAs, or a component thereof, or an OMEGA system or any component thereof.

42. The engineered AAV of claims 40 or 41, wherein the transgene is operably linked to a regulatory sequence that promotes expression in a CNS or muscle.

43. A pharmaceutical composition comprising the recombinant engineered AAV particle of any one of claims 40 to 42 and an acceptable carrier.

44. A method of delivering a polypeptide or polynucleotide to the CNS of a subject comprising administering an engineered AAV particle of claim 40, wherein the engineered AAV particle comprises a CNS targeting moiety and wherein the engineered AAV particle exhibits increased evasion of AAV-neutralizing antibodies relative to a reference AAV particle.

45. The method of claim 44, wherein the CNS targeting moiety binds a TfRl receptor and wherein the engineered AAV particle exhibits increased transduction of the CNS relative to a reference AAV9.

46. The method of claim 41, wherein the engineered AAV particle is administered systemically.

47. The method of any one of claims 44 to 46, wherein the subject suffers from a CNS disease or disorder.

48. The method of any one of claims 44 to 47, wherein the subject has AAV-neutralizing antibodies.

49. An AAV library comprising a population of engineered recombinant AAV particles wherein each member of the population of engineered recombinant AAV particles comprises any one or more of the engineered adeno associated virus (AAV) capsid polypeptides of claims 1-39.

50. A method of screening an AAV capsid library for a recombinant AAV particle that has reduced binding affinity for a subject’s or a group of subjects’ one or more antibodies, said method comprising:a) receiving a sample from a subject or group of subjects, wherein the sample comprises one or more antibodies;b) assaying the AAV capsid library of claim 49 with the sample for reduced binding affinity of the one or more antibodies for one or more AAV particles in the AAV capsid library relative to an AAV particle with a reference capsidc) selecting one or more recombinant AAV particles that have reduced binding affinity for the one or more antibodies to the AAV particle; andd) selecting for maintained or enhanced receptor-mediated binding or transduction of cells in culture or in an organism.

51. A method of prescreening a subj ect for treatment comprising:a) receiving a sample from a subject or group of subjects, wherein the sample comprises one or more antibodies;b) assaying the AAV capsid library of claim 49 with the sample for reduced binding affinity of the one or more antibodies for one or more AAV particles in the AAV capsid library relative to an AAV particle with a reference capsid; andc) selecting one or more recombinant AAV particles which have reduced binding affinity for the one or more antibodies to the AAV particle.

52. The method of any one of claims 50-51 further comprising determining one or more neutralizing antibodies of the subject.

53. A cultured host cell comprising an expression vector encoding the engineered AAV capsid polypeptide of any one of claims 1-39.

Images