Novel AAV capsids binding to human CD59

Abstract

Applicants engineered peptide-modified AAV9 capsids for system-wide enhanced organ transduction by directly engineering a binding interaction with the human GPI-linked glycoprotein CD59. Adeno-associated vimses (AAVs) are used by numerous approved and investigational gene therapies. However, native AAVs have limited tissue tropisms and have comparatively low extrahepatic delivery efficiencies. While capsid engineering has largely focused on targeting specific organs, treating multisystem disorders requires efficient gene delivery to many organs. Here Applicants describe novel engineered capsids that bind CD59.

Description

NOVEL AAV CAPSIDS BINDING TO HUMAN CD59CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 631,415, filed April 08, 2024. The content of the above-identified application is hereby incorporated herein by reference in its entirety.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-6015WP ST26. xml"; Size is 6,851,023 bytes, created on April 1, 2025) is herein incorporated by reference in its entirety.TECHNICAL FIELD

[0004] The subject matter disclosed herein relates generally to enhancing transduction of an engineered AAV capsid into cells of the central nervous system (CNS) through interaction with CD59 expressed on the surface of the cells. In an example described herein, at least one of the capsid’s proteins is modified to include an n-mer motif. 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 engineered AAV capsids and a method of delivering the cargo to the CNS by either administering the AAV capsids directly to a host organism in vivo or transducing cells with the AAV capsids ex vivo and transplanting the cells back into the host organism.BACKGROUND

[0005] Gene therapies for multisystem disorders require efficient gene delivery to many organs. However, natural adeno-associated viruses (AAVs) have limited extrahepatic tissue tropisms, and capsid engineering has largely focused on targeting specific organs such as the central nervous system (CNS), liver, retina, or muscle. To target multiple organs, Applicants usedrecently described receptor-targeting platform to target peptide-modified AAV9 capsids to a human receptor that is broadly expressed across diverse cell types, including the CNS and muscle. These receptor-targeting capsids exhibited enhanced binding and transduction of cells in transgenic mice that expressed the human receptor. Two of the capsids, BI306 (sequence 1.1) and BI309 (sequence 2.1), efficiently transduced the CNS to an extent comparable to AAV-PHP.eB, a mouse CNS-targeting capsid, when intravenously administered to mice expressing the human receptor in the brain microvasculature (Fig. 20A). This enhanced tropism was absent in transgenic mice treated with wild type AAV9 or in mice not expressing the human receptor. When BI306 was intravenously administered to transgenic mice ubiquitously expressing the human receptor, it achieved substantially enhanced CNS and muscle transduction compared to wild type mice or transgenic mice injected with AAV9 (Fig. 20B). BI306 was also dramatically de-targeted from the liver compared to AAV9, even in animals where the human receptor was ubiquitously expressed (Fig. 20B). Notably, Applicants demonstrated that BI306 can more efficiently transduce cells that express human or macaque, but not mouse, orthologs of the targeted receptor (Fig. 20C). The findings show that BI306 has the potential to be a cross-species receptor-targeting AAV that can mediate enhanced in vivo gene delivery in a receptor-dependent manner. Based on the broad expression of the receptor in humans, BI306 and similar capsids may enable enhanced gene delivery to multiple organs after systemic administration.SUMMARY

[0006] In some aspects, the techniques described herein relate to an engineered adeno- associated virus (AAV) capsid polypeptide including a CD59 targeting moiety defined by a n-mer of the formula X1-X2-X3-X4-X5-X6-X7, inserted at any position between 450-461 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype, and wherein XI, X2, X3, X4, X5, X6, and X7 each represent an amino acid inserted into the capsid polypeptide.

[0007] In an embodiment, the engineered adeno-associated virus (AAV) capsid polypeptide further includes first removing one or more amino acids at any position between 450-461 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype. In an embodiment, amino acids at position 451-460 are removed. In an embodiment, the engineered adeno-associated virus (AAV) capsid polypeptide further includes removing the aminoacid at position 449 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype and inserting a new amino acid. In an embodiment, the new amino acid is arginine. In an embodiment, the n-mer is of the formula having an amino acid sequence of EFNNGSD (SEQ ID NO: 89) or GAASLMP (SEQ ID NO: 109).

[0008] In an embodiment, the AAV9 capsid polypeptide includes a K449R mutation, or at an analogous position of a capsid polypeptide of another AAV serotype. In an embodiment, the targeting moiety includes any one of the amino acid sequences of SEQ ID NO: 89-5983. In an embodiment, the targeting moiety is selected from any one of the amino acid sequences listed in Table A, or any combination thereof. In an embodiment, the capsid polypeptide includes a VP1, VP2, or VP3 polypeptide, or a combination thereof. In an embodiment, the other AAV serotype includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rhlO.

[0009] In some aspects, the techniques described herein relate to an engineered AAV particle including the engineered AAV capsid polypeptide, and further including a recombinant AAV genome configured to express a transgene.

[0010] In an embodiment, the transgene encodes a therapeutic polypeptide, an antibody or fragment thereof, an siRNA, a CRISPR-Cas system, a Transcription Activator-like Effector (TALE)- or Zinc Finger Protein (ZFP)-based transcriptional activator; repressor; or an epigenomic silencer, 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, the transgene is operably linked to a regulatory sequence that promotes expression in the nervous system.

[0011] In some aspects, the techniques described herein relate to pharmaceutical composition comprising the recombinant engineered AAV particle of any one of any of those described herein and an acceptable carrier.

[0012] In some aspects, the techniques described herein relate to a method of delivering a polypeptide or polynucleotide to the central nervous system (CNS) of a subject comprising administering the pharmaceutical composition of any of those described herein to the subject.

[0013] In some aspects, the techniques described herein relate to a method, wherein the pharmaceutical composition is administered systemically or directly to the CNS.

[0014] In an embodiment, said method including (1) a polynucleotide encoding the engineered AAV capsid polypeptide of any one of those described herein, (2) a polynucleotide encoding a recombinant AAV genome including a transgene operably linked to a regulatory sequence and flanked by AAV ITR sequences, and optionally (3) a polynucleotide encoding adenoviral helper genes, under conditions sufficient for the production of recombinant engineered AAV particles; and recovering the recombinant engineered AAV particles from said culture.

[0015] In some aspects, the techniques described herein relate to a cultured host cell containing a recombinant nucleic acid molecule encoding the engineered AAV capsid polypeptide of any of those described herein.

[0016] In some aspects, the techniques described herein relate to an AAV library including a population of variant engineered recombinant AAV particles including a variant recombinant AAV capsid polypeptide targeting moiety defined by a n-mer of the formula X1-X2-X3-X4-X5- X6-X7, inserted at any position between 450-461 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype, and wherein XI, X2, X3, X4, X5, X6, and X7 represent an amino acid inserted into the capsid polypeptide, wherein the modification has been selected for binding of a CD59 protein and / or increased tropism for the CNS relevant to a reference AAV particle without the modification, and optionally, further includes first removing one or more amino acids at any position between 450-461 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype and optionally further includes removing the amino acid at position 449 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype and inserting a new amino acid.

[0017] In an embodiment, the other AAV serotype includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rh.10.

[0018] In some aspects, the techniques described herein relate to method of screening an AAV library for a recombinant AAV particle that binds a CD59 protein and / or has increased tropism for the CNS, said method comprising assaying the AAV library of any of those described herein for increased binding to a CD59 protein and / or tropism for the CNS relative to an AAV vector with a reference capsid, and selecting those recombinant AAV vectors that have increased binding of the CD59 protein and / or enhanced tropism for the CNS.

[0019] In some aspects, the techniques described herein relate to a method for training a machine learning algorithm including: (a) receiving, by at least one computing device, a plurality of AAV capsid polypeptide sequences including a targeting moiety for binding to a CD59 protein; (b) training, by at least one computing device, with the plurality of AAV capsid polypeptide sequences including a modification for binding to the CD59 protein, a CD59 protein targeting machine learning model; and (c) deploying, by at least one computing device, the CD59 protein targeting machine learning algorithm.

[0020] In an embodiment, the CD59 protein targeting machine learning model is trained to identify one or more sequences from the plurality of sequences having increased binding to the CD59 protein. In an embodiment, the CD59 protein targeting machine learning model is trained to identify one or more sequences from the plurality of sequences that decreases transduction of off-target tissues. In an embodiment, the targeting moiety defined by a n-mer of the formula XI- X2-X3-X4-X5-X6-X7, and wherein XI, X2, X3, X4, X5, X6, and X7 represent an amino acid inserted into at an amino acid position in the capsid polypeptide.

[0021] In an embodiment, the training includes unsupervised learning, supervised learning, semi-supervised learning, reinforcement learning, transfer learning, incremental learning, curriculum learning, learning to learn, or contrastive learning. In an embodiment, the CD59 protein targeting machine learning model includes linear classifiers, logistic classifiers, random forest, artificial neural networks, matrix factorization, support vector machines, K-means clustering, or K-nearest neighbor. In an embodiment, the CD59 protein targeting machine learning model includes Boltzmann machines, Bayesian networks, autoregressive models, variational autoencoders (VAEs), diffusion models, energy-based models, flow-based models, generative adversarial networks (GANs), mixture models, hidden Markov models, or large language models (LLMs). In an embodiment, the CD59 protein targeting machine learning model includes convolutional neural networks (CNNs), recurrent neural networks (RNNs), long short-term memory models (LSTMs), gated recurrent units (GRUs), capsule networks, attention mechanisms, or transformer networks. In an embodiment, the CD59 protein targeting machine learning model is pre-trained, and further trained to predict CD59 protein targeting by a plurality of sequences.

[0022] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of 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 - Receptor-dependent organ targeting of engineered AAVs. (1A) Luciferase signal in brain tissue from mice expressing the target receptor in the brain vasculature injected with receptor-targeting capsids compared to PHP.eB. (IB) Luciferase signal in brain, muscle, and liver tissue from mice expressing the target receptor in both CNS and muscle injected with BI306 (capsid 1.1) or AAV9 compared to wild type mice injected with BI306.

[0025] FIG. 2 - BI306 transduces CHO cells stably expressing the human or macaque orthologs of the target receptor more efficiently than AAV9.

[0026] FIG. 3 - RNA-seq data from the Genotype-Tissue Expression (GTEx) project shows comparable hCD59 expression across all organs, with elevated expression in the lungs, proximal digestive tract, breast, cardiac and skeletal muscle, and fat. Additionally, hCD59 is upregulated in solid tumors (Ouyang et al., 2016, Int J Oncol).

[0027] FIG. 4 - Normalized single cell RNA levels from the Human Protein Atlas show that CD59 is expressed in both neurons and glia in the brain, tubular cells in the kidney, smooth muscle and endothelial cells in the muscle, and endothelial cells in lungs.

[0028] FIG. 5 - Library performance in vivo of hCD59-binding capsids: In a synthetic peptide display library, wild type AAV9 was negatively enriched for in the brains of both wild type C57BL / 6 mice as well as mice bred from (LSL)hCD59 x Tie2Cre mice which express hCD59 in endothelial cells. Both capsid sequences 1.1 (BI306) and 2.1 (BI309) were both enriched in the brains of (LSL)hCD59 x Tie2Cre mice compared to wild type mice.

[0029] FIG. 6 - Novel AAV capsid sequences: (SEQ ID NO: 6005-6007) The 10 amino acid sequence from positions 451-460 of AAV9 VP1 were substituted with a 7mer peptide to confer AAV9 the ability to bind to hCD59 and transduce organs in in mice expressing hCD59. The engineered capsids also have a silent mutation K449R.

[0030] FIG. 7 - Production titer of hCD59-binding capsids: AAVs were produced in 500 mL suspension HEK cell cultures. Capsids 1.1, 1.2, and AAV9 were packaged with a CAG-NLS- mScarlet-P2A-Luc transgene.

[0031] FIG. 8 - Individual characterization of Sequences 1.1 and 2.1 in comparison to AAV9 in vitro-. hCD59 binding capsids packaging CAG-NLS- mScarlet-P2A-Luc were applied at 5e3 vg / cell to CHO cells stably transfected with human CD59, macaque CD59, mouse CD59, or an empty pLenti sequence. Luciferase activity was measured 3 days after virus application. Both capsids tested have enhanced luciferase activity in CHO cells expressing human CD59 compared to the parental AAV9 serotype, demonstrating interaction of the capsids with hCD59. Sequence 1.1 has enhanced luciferase activity in CHO cells expressing macaque CD59 as well, demonstrating cross-species applicability. Additionally, Sequence 1.1 has decreased transduction in control CHO cells compared to AAV9 and Sequence 2.1, suggesting less off-targeting interactions.

[0032] FIG. 9 - Characterization of hCD59-binding AAVs in transiently transfected HEK cells: Relative transduction of Sequences 1.1 and 1.2 compared to AAV9 in HEK293T cells transiently transfected with human, macaque, or marmoset CD59. Both sequences have enhanced transduction in cells over-expressing human CD59, and Sequence 1.1 has enhanced expression in cells over-expressing macaque CD59 as well. A decrease in transduction is see for Sequence 1.1 in untransfected HEK cells, consistent with observations from CHO cell transduction data.

[0033] FIG. 10 - Characterization of hCD59-binding AAVs in hCMEC / D3 cells: Relative transduction of Sequences 1.1 and 1.2 compared to AAV9 in a human brain endothelial cell line, hCMEC / D3 and in hCMEC / D3 AAVR knock-out cells. AAVR is and AAV receptor required for the entry of multiple AAV serotypes into cells. Both sequence 1.1 and 1.2 have enhanced transduction in hCMEC / D3 cells compared to AAV9, indicating that endogenous expression of hCD59 is sufficient for AAV-targeting. Both capsids are AAVR-dependent, a common feature of most AAVs.

[0034] FIG. 11 - Individual characterization of hCD59 binding AAVs in vivo-. hCD59- binding AAVs were injected into mice bred from (LSL)-hCD59 mice crossed with the Cre mouse lines Tie2-Cre, CMV-Cre, CAGGCre-ERT[TM], and HSA79-Cre to express hCD59 in different cell types according to the Cre expression pattern of each parental Cre line. (LSL)-hCD59 x CAGGCre-ERT[TM] mice were treated with tamoxifen 3 weeks prior to AAV injection. The transgene packaged was CAG-NLS-mScarlet-P2A-Luc. NLS is a nuclear localization sequence and P2A- Luc encodes for a self cleaving peptide followed by luciferase. Mice were dosed withlei 1 vg of hCD59-binding AAVs intravenously, and expression of luciferase and mScarlet was evaluated 2-3 weeks following the injection.

[0035] FIG. 12 - Endothelial hCD59 expression in (LSL)hCD59 x Tie2-Cre mice: Immunostaining for hCD59 shows widespread hCD59 expression throughout the brain vasculature in (LSL)hCD59 x Tie2Cre mice. A representative image of the thalamus is shown.

[0036] FIG. 13 - Individual characterization of capsid sequence 1.1 ex vivo in (LSL)hCD59 x Tie2-Cre mice: lei 1 vg of capsid 1.1 or AAV9 was injected into the indicated mouse lines. 14 days post-injection, mice were injected with luciferin, and tissue was collected and imaged ex vivo for luciferase activity. hCD59 expression is expected to be on endothelial cells, including in the brain. Brain and spinal cord luciferase signal is enhanced in (LSL)hCD59 x Tie2- Cre mice compared to 1.1 injected in mice without hCD59 expression or AAV9 in wild type mice. Liver transduction was decreased for mice injected with 1.1 regardless of hCD59 or Cre expression.

[0037] FIG. 14 - Transduction of the CNS in (LSL)hCD59 x Tie2-Cre mice: The AAVs Sequence 1.1, Sequence 2.1, and AAV9 packaging the transgene CAG-NLS-mScarlet-P2A-Luc were injected into adult mice bred from the cross of (LSL)hCD59 x Tie2-Cre mice. These mice express hCD59 on endothelial cells in the brain vasculature. Enhanced mScarlet signal in brains injected with hCD59-binding capsids 1.1 and 2.1 compared to AAV9 demonstrate that capsid interaction with hCD59 enabled crossing of the BBB and transduction of the CNS.

[0038] FIG. 15 - Individual characterization of capsid sequence 1.1 ex vivo in (LSL)hCD59 x HSA-Cre mice: lei 1 vg of capsid 1.1 or AAV9 was injected into the indicated mouse lines. 14 days post-injection, mice were injected with luciferin, and tissue was collected and imaged ex vivo for luciferase activity. hCD59 expression is expected to be in muscle tissue. Skeletal muscle luciferase signal is enhanced in (LSL)hCD59 x HSA- Cre mice compared to AAV9 in wild type mice. Liver transduction was decreased for mice injected with 1.1 regardless of hCD59 or Cre expression.

[0039] FIG. 16 - Individual characterization of capsid sequence 1.1 ex vivo in (LSL)hCD59 x CMV-Cre mice: lei 1 vg of capsid 1.1 or AAV9 was injected into the indicated mouse lines. 14 days post-injection, mice were injected with luciferin, and tissue was collected and imaged ex vivo for luciferase activity. Ubiquitous expression of hCD59 is expected in this line of mice. Both brain and skeletal muscle luciferase signal are enhanced in (LSL)hCD59 x CMV-Cre mice compared to AAV9 in in CMV-Cre crossed or wild type mice. Liver transduction was decreased for mice injected with 1.1 regardless of hCD59 or Cre expression. This indicates that multiple organs are able to be targeted in a single animal with 1.1.

[0040] FIG. 17 - Individual characterization of capsid sequence 1.1 ex vivo in (LSL)hCD59 x CAGG-Cre mice: lei 1 vg of capsid 1.1 or AAV9 was injected into the indicated mouse lines. 14 days post-injection, mice were injected with luciferin, and tissue was collected and imaged ex vivo for luciferase activity. Ubiquitous expression of hCD59 is expected in this line of mice. Consistent with CMV-Cre crossed mice, both brain and skeletal muscle luciferase signal are enhanced in (LSL)hCD59 x CAGG-Cre mice compared to AAV9 in in CAGG-Cre crossed or wild type mice. Liver transduction was decreased for mice injected with 1.1 regardless of hCD59 or Cre expression.

[0041] FIG. 18 - In vivo validation of cross-species affinity of hCD59 binding capsid 1.1: NOD scid gamma (NSG) mice were injected sequentially with two doses of AAV. Mice were first dosed with lei 1 vg of BI30:CAG-humanCD59-miR122BS-WPRE, BI30:CAG-macaqueCD59- miR122BS- WPRE, or BI30:CAG-mouseLY6A-miR122BS-WPRE as a negative control. BI30 is a previously reported AAV capsid (Krolak et al. 2022) that targets the mouse brain vasculature, and miR122BS is a binding site for microRNA-122 to down-regulate transgene mRNA in the liver. Three weeks following the initial injection, mice were dosed with lei 1 vg of Capsid l.LCAG- NLS-mScarlet-P2A-Luc. NLS is a nuclear localization sequence, and P2A-Luc encodes for a self cleaving peptide followed by luciferase. Expression of mScarlet was evaluated three weeks following the second injection.

[0042] FIG. 19 - In vivo validation of cross-species affinity of hCD59 binding capsid 1.1: When Capsid 1.1 packaging an mScarlet transgene is injected using the previously described two- dose strategy into NSG mice expressing either human or macaque CD59 in the brain vasculature, transduction is observed in the endothelial cells and the brain parenchyma. In contrast, Capsid 1.1 does not transduce control mice expressing the receptor Ly6A. This indicates that CD59-binding Capsid 1.1 has cross-species functionality in vivo.

[0043] FIG. 20A-20C - Human receptor-targeted AAVs efficiently transduced the brain and muscle but were de-targeted from the liver in transgenic mice. (20A) Representative images show the luciferase signal in brain tissue from transgenic mice expressing the targeted human receptor in the brain vasculature injected with ssAAV-CAG-NLS-mScarlet-P2A-Luciferase-SV40pA packaged into human receptor-targeting capsids (BI306 or BI309) or AAV- PHP.eB. (20B) Representative images show the luciferase signal in brain, muscle, and liver tissue from wild type mice or transgenic mice ubiquitously expressing the human receptor that were injected with BI306 (1.1) or AAV9. (20C) BI306 (1.1) transduced CHO cells stably expressing the human or macaque orthologs of the targeted human receptor more efficiently than AAV9.

[0044] FIG. 21 - AAV9 capsid residues 539-605 aligned to other previously described capsids. (SEQ ID NO: 6008-6027) The 7-mer insertion site between AAV9 residue 588 and 589 is shown. The black bars above the alignment highlight surrounding residues that were 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).

[0045] FIG. 22 - (SEQ ID NO: 6028-6040) CD59 targeting moiety - example insertions between residues 558 and 559 AAV9 VP1.

[0046] FIG. 23 - (SEQ ID NO: 1, 6041-6051) Example serotype sequence alignment.

[0047] FIG. 24 - (SEQ ID NO: 1, 6052-6054) CD59 binding moiety insertion site in AAV9VP1 capsid.

[0048] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTSDefinitions

[0049] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew e / czZ. (eds ), The Encyclopedia of Molecular Biology, published byBlackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011).

[0050] Titles or subtitles may be used in the specification for the sole convenience of the reader but are not intended to influence the scope of the present disclosure or to limit any aspect of the disclosure to any subsection, subtitle, or paragraph.

[0051] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise. The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0052] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints. For example, “1-5 ng” or a range of “1 ng to 5 ng” is intended to encompass 1 ng, 2 ng, 3 ng, 4 ng, 5 ng, 1-2 ng, 1-3 ng, 1- 4 ng, 1-5 ng, 2-3 ng, 2-4 ng, 2-5 ng, 3-4 ng, 3-5 ng, and 4-5 ng.

[0053] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, +7-5% or less, + / - 1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0054] As used herein, a “biological sample” may contain whole cells and / or live cells and / or cell debris. The biological sample may contain (or be derived from) a “bodily fluid”. The present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat,tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures.

[0055] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, lab animals and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0056] As used herein, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements, and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a nonlimiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one aspect, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another aspect, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another aspect, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0057] As used herein, the terms “treat,” “treatment,” “treating” refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a disorder. The term “treating” includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder. Treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Alternatively, treatment is “effective” if the progression of a disorder is reduced or halted. That is, “treatment” includes not just the improvement of symptoms or markers, but also a cessation of, or at least slowing of, progress or worsening of symptoms compared to what would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or moresymptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (whether partial or total), and / or decreased mortality, whether detectable or undetectable. The term “treatment” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0058] The term “expression vector”, as used herein, refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of a transgene product in a cell or in an in vitro assay. For example, a transgene expression vector, disclosed herein, comprises a promoter operatively linked to a transgene transcription unit comprising a transcription initiation site, a 5' untranslated region (UTR), a transgene nucleotide sequence and a 3’ untranslated region (UTR) comprising one or more post-transcriptional regulatory elements, e.g., a polyadenylation sequence.

[0059] The term "operably linked" refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, the term refers to the functional relationship of a transcriptional regulatory sequence and a transgene to be transcribed. For example, a promoter or enhancer sequence is operably linked to a transgene if it, e g., stimulates or modulates the transgene transcription in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory' sequences that are operably linked to a sequence are contiguous to that sequence or are separated by short spacer sequences, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

[0060] As used herein, “recombinant” refers to nucleic acids or polypeptides that are genetically engineered.

[0061] A "transgene" is a polynucleotide sequence that may encode an RNA (mRNA) that is translated into protein. For example, a transgene may comprise a cDNA sequence. In other embodiments, a transgene may encode on “non-coding” RNA that is not translated into protein (e g. guide RNAs, ribozymes, aptamers, antisense RNAs, piwi-interacting RNAs (piRNAs), short interfering RNAs (siRNAs), microRNAs (miRNAs), shRNAs or recombinant U RNAs). In an embodiment, the transgene nucleotide sequence may comprise one or more introns. In other aspects, the transgene can be polycistronic (e.g., two coding regions separated by internal ribosome entry site (IRES)). In an embodiment, a transgene may encode more than one protein. In oneaspect, a transgene comprises a "protein coding sequence" or a sequence that encodes a particular protein or polypeptide, i .e., a nucleic acid sequence that is capable of being transcribed into mRNA and translated into a polypeptide in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence may be determined by a start codon at the 5' terminus (N-terminus) and a translation stop nonsense codon at the 3' terminus (C- terminus). A coding sequence can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic nucleic acids. A transcription termination sequence will usually be located 3' to the coding sequence. A “serotype” is traditionally defined on the basis of a lack of cross-reactivity between antibodies to one virus as compared to another virus. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). Under the traditional definition, a serotype means that the virus of interest has been tested against serum specific for all existing and characterized serotypes for neutralizing activity and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates are discovered and capsid mutants generated, there may or may not be serological differences with any of the currently existing serotypes. Thus, in cases where the new AAV has no serological difference, this new AAV would be a subgroup or variant of the corresponding serotype. In many cases, serology testing for neutralizing activity has yet to be performed on mutant viruses with capsid sequence modifications to determine if they are of another serotype according to the traditional definition of serotype. Accordingly, for the sake of convenience and to avoid repetition, the term “serotype” broadly refers to both serologically distinct viruses (e.g., AAV) as well as viruses (e.g., AAV) that are not serologically distinct that may be within a subgroup or a variant of a given serotype.

[0062] Transduction” refers to the transfer of a transgene into a recipient host cell by a viral vector. Transduction of a target cell by an rAAV virion of the invention leads to transfer of the transgene contained in that rAAV virion into the transduced cell. “Host cell” or “target cell” refers to the cell into which the DNA delivery takes place, such as the cells of the CNS or HEK293T cells in case of the in vitro transduction assay. AAV vectors are able to transduce both dividing and non-dividing cells. In a cell comprising a gene product of interest, such as for example GFP, the gene product of interest has been introduced / transferred / transduced by rAAV “transduction”of the cell. A cell into which the transgene has been introduced is referred to as a “transduced” cell.

[0063] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment”, “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.OVERVIEW

[0064] Embodiments disclosed herein provide CD59 targeting moieties which promote transduction into the CNS through its interaction with the cell surface CD59. These CD59 targeting moieties may be incorporated into particles, such as viral capsid delivery particles, including AAV9 particles and AAV particles of other serotypes, to confer tropism on the delivery particles and promote transduction of CNS. Exemplary CNS tissues include brain and spinal cord tissue. Exemplary CNS cell types include neurons, ependymal cells, and glial cells, e.g., microglia, astrocytes, oligodendrocytes, and NG2-glia progenitors, pericytes, as well as endothelial cells. Further embodiments disclosed herein provide for a vector system comprising one or more vectors encoding AAV capsids according to embodiments described herein. Accordingly, embodiments disclosed herein provide compositions capable of delivering cargos with enhanced selectivity and efficiency to the CNS vasculature. Embodiments disclosed herein also provide vector systems for the generation and loading of such delivery particles with a cargo. Likewise, embodimentsdisclosed herein provide methods for use of such compositions to target CNS endothelial cells, in vitro and in vivo, with implications for both therapeutic and research purposes.

[0065] Additional feature and advantages of the aforementioned embodiments are further described below.CD59 targeting moieties and Compositions Thereof

[0066] In an embodiment, compositions are provided herein of AAV capsids comprising capsid proteins having a CD59 targeting moiety sequence conferring on the capsid an enhanced tropism for endothelial cells of the CNS. A CD59 targeting moiety with an enhanced tropism for endothelial cells of the CNS promotes, increases, or otherwise improves binding to, and in some cases, transduction of the CNS as compared to a natural or wild-type target moiety. This CD59 targeting moiety may be coupled directly to a cargo to be delivered such as an oligonucleotide or polypeptide. Alternatively, the targeting molecule may be incorporated into a delivery particle, such as an AAV particle (for example, being incorporated into an AAV capsid protein) to confer tropism for endothelial cells of the CNS on the delivery particle. A non-limiting example of delivery particle is a viral capsid particle. In such embodiments, the CD59 targeting moiety may be incorporated into a viral capsid polypeptide such that the CD59 targeting moiety is incorporated into the assembled viral capsid. However, other particle delivery systems where the CD59 targeting moiety may be incorporated or attached, for example on exosomes, liposomes, lipid nanoparticles, virus-like particles, ribonucleoproteins, nanobodies, antibodies, or antibody fragments are also envisioned and encompassed as alternative embodiments herein.

[0067] In embodiments, provided herein is a composition comprising a CD59 targeting moiety effective to increase transduction of CNS via binding to a CD59, optionally further comprising a cargo coupled to or otherwise associated with the CD59 targeting moiety. A CD59 targeting moiety with an increased transduction promotes, enhances, or otherwise improves binding to, and in some cases, transduction of the CNS as compared to a natural or wild-type target moiety. In an embodiment, the CD59 targeting moiety binds to a CD59 polypeptide. In an embodiment, the n- mer is an amino acid sequence of length n. The length of the n-mer may be any necessary length to transduce the CNS. In an embodiment, the n-mer is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids in length. In an embodiment, the n-mer motif has a length of at least 7 amino acids. In an embodiment, a composition comprising a targeting moiety effective to increase transduction ofCNS tissues comprises a n-mer motif, the n-mer motif comprising or consisting of an amino acid sequence of any of those described herein.

[0068] In an embodiment, the n-mer can be used to increase transduction in target cells i.e., CNS cells and tissues. 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 CD59 targeting moiety for example inclusion of one or more CD59 targeting moieties in a composition can result in an increase in transduction and or transduction efficiency by 10%, 20%, 30%, 40%, 50%, 60% 70% 80% 90% a 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 endothelial cells, in one embodiment increase in endothelial cells of the CNS, for example, the central nervous system vasculature. In an embodiment, the transduction and / or transduction efficiency is increased or enhanced in cells of the CNS. In an embodiment, the transduction and / or transduction efficiency is increased or enhanced in endothelial cells. In an embodiment, the composition comprising a n- mer is selective to a target cell as compared to other cell types and / or other virus particles. As used herein, ‘selective’ and ‘cell-selective’ refers to preferential targeting for cells as compared to other cell types. Preferably, the CD59 targeting moiety is selective for a desired target (e.g., cell, organ, system e.g., CD59 tissues) or set of targets by at least 2: 1, 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 (e.g., CNS). In an embodiment, the composition comprising a CD59 targeting moiety described herein can have an increased uptake, delivery rate, transduction rate, efficiency, amount, or a combination thereof in a target cell (e.g., endothelial cells across the CNS e.g., the brain) as compared to other cells types (e.g. hepatocytes) and / or other virus particles (e.g., AAVs not containing the CD59 targeting moiety) and other compositions that do not contain the cell- selective n-mer motif of the present invention.

[0069] In an embodiment, the n-mer motif is selected from the group consisting of any of those described herein. In an embodiment, the n-mer is selected from any of the amino acid sequences in Table A, or any combination thereof. In an embodiment, the n-mer motif is selected from a peptide having an amino acid sequence of one of SEQ ID NO: 89-5983. In an embodiment, theCD59 targeting moiety is part of (e g., inserted between consecutive amino acids of) a viral capsid protein, including an AAV capsid protein.Table A. Sequences of n-mers, AAVs, and n-mer family motifs of the disclosure.

[0070] The sequences previously disclosed in Table A filed in US provisional 63 / 631,415 are included as SEQ ID NO 1 to 5983 in the accompanying Sequence Listing of this application.

[0071] In an embodiment, the n-mer is selected from the group consisting of GS[AVLMP][RQSLHNT]M[LQRHMNVI]P (SEQ ID NO: 11),GS[AVLM][RY]L[AMNLQSV]P (SEQ ID NO: 12), G[SG][APVLGM][RYHM]L[MLQSI]P (SEQ ID NO: 13), GS[PRVWLAF][SMLADH][MS][SQLWRFMAH][PR] (SEQ ID NO: 14), GS[PVR][GALSQ]I[ARQMLVWHS]P (SEQ ID NO: 15),GS[PVRWY][SMAVKH]L[QLMHVA]P (SEQ ID NO: 16),GS[PVRYLI][ALIV][MAVI][LARSH]P (SEQ ID NO: 17), GS[LRPAF][LKQSH]L[SAMNQ]P(SEQ ID NO: 18), GS[LAMQG][GRSKL]I[LSTRQIG]P (SEQ ID NO: 19),GS[LPVKMA][GA]L[RYASMG]P (SEQ ID NO: 20),GS[LAPVQEK][SALQTDMRE]F[RSAGQVLT]P (SEQ ID NO: 21),GS[LPVAMI][GA][HWV][SALRGMW]P (SEQ ID NO: 22), GS[LSTQKM]GF[GAELRM]P(SEQ ID NO: 23), GS[LMPRAV]G[VY][RLSWMQ]P (SEQ ID NO: 24), GS[LRQPMTA]GM[SARGLKH]P (SEQ ID NO: 25),GS [RLP VF Y] [ AD VGN] [LQH] [LRF YA]P (SEQ ID NO: 26), GA[PLVRA][LSRANKQMT]V[SQMAGTN]P (SEQ ID NO: 27), GA[PTRKSEM][GSDRVEQ]F[SARGQLT]P (SEQ ID NO: 28),GA[PLTI][ATQHILY]L[AMEIN]P (SEQ ID NO: 29),GA[PLRQ][GSARNDL][YWM][GQMASEL]P (SEQ ID NO: 30), GA[PLRFVY][RLMGHQF] [AT] [LMRQWNSH]P (SEQ ID NO: 31), GA[PALMVH]SL[GAQLFH]P (SEQ ID NO: 32), [GR]A[LHV][GLQH]A[RYW]P (SEQ IDNO: 33), GA[LVAPQ][GTSAQEMH]M[GASVQMDK]P (SEQ ID NO: 34),GAR[SDLQEGA]L[SMAVLRN]P (SEQ ID NO: 35), GAR[GLHA][LM][TLMAE]P (SEQ ID NO: 36), GG[RLTAVHQ][GNDAQSH]F[MALRGS]P (SEQ ID NO: 37),GA[NAPYSQI][RQLM]VLP (SEQ ID NO: 38), GA[AS]RV[SVLTAIE]P (SEQ ID NO: 39), GA[LQSTHN]GV[GRLAMVT]P (SEQ ID NO: 40),G[TGVS][LVARQ][GRTN][MIFY][WLRQFY]P (SEQ ID NO: 41), GAR[GQAMHS]V[LGT]P(SEQ ID NO: 42), GA[AGQS][TASMKD]F[GLVSR]P (SEQ ID NO: 43), GA[QLHPW][ASGDNM]V[RK]P (SEQ ID NO: 44), GA[MPHLV][LSMI]V[RKAG]P (SEQ ID NO: 45), GA[ASVPQTM][GRSLTAN]I[SEGVTMAL]P (SEQ ID NO: 46), GA[RLKPQAVM][AGTQDLVHSKE]I[QSGLRKAV]P (SEQ ID NO: 47),GA[LQHDTVME][GQ]W[QGSENKM]P (SEQ ID NO: 48),GA[PLAQRVS][GASQDLNHE][HW][AGSLRNWHVM]P (SEQ ID NO: 49), GA[SNRLV][RKLNAQ]M[LQSA]P (SEQ ID NO: 50), GA[VMPRYAI][GDSEM]H[RMQS]P(SEQ ID NO: 51), GA[SIPGYMFA][KRTQLHN]L[MQIKHDL]P (SEQ ID NO: 52),GA[VEQSLGD][RK]L[VSGLIMA]P (SEQ ID NO: 53),GA[RISQM][SLMTDN]M[LGHSVW]P (SEQ ID NO: 54),GA[AQSVKH][LMNQETKA]L[GVKSIQL]P (SEQ ID NO: 55),GA[APRHI][GVLTS]M[RGL]P (SEQ ID NO: 56), GA[QKAY][NHFLDG]L[VLSGMT]P (SEQ ID NO: 57), GA[APLV][LATKQS]L[STAG]P (SEQ ID NO: 58), GA[SIEPLV][SGAQT][MHI]RP (SEQ ID NO: 59), GA[NVSLTQKM][GN]L[LVGMIQ]P (SEQ ID NO: 60), GA[VPQRSKM][GLNHAD]L[SLADG]P (SEQ ID NO: 61),GA[SPLVIT][SAGTQH]LRP (SEQ ID NO: 62), GA[PLVAIS][RGMSHK]M[SWNFGE]A (SEQ ID NO: 63), GA[PRS][GRSLAKMQ]V[RWSLYNMH]A (SEQ ID NO: 64),GA[VPLIFW][KNDQAH]L[VWLSFMQAY]A (SEQ ID NO: 65),GA[LPKWVHAQ][QANMLDE]L[GLSRY]A (SEQ ID NO: 66), GA[PRAYMV]SL[LGS]A (SEQ ID NO: 67), GA[APSLQ][RQNMSKH]L[GMLWYA]A (SEQ ID NO: 68), GA[ALPVM]RL[LASTVE]A (SEQ ID NO: 69), GA[PLARTIS][GRST][IVF][GSWRMN]A (SEQ ID NO: 70), GA[PLAVSK][GSQAN]F[SGRLAYTWM]A (SEQ ID NO: 71), GA[PVRLASQHT][GASNL]M[GMLYHF]A (SEQ ID NO: 72),GA[LPVAS][RGHL][VM][WSGLY][ASP] (SEQ ID NO: 73), GA[PVIKT]GL[DHTENMYA]A (SEQ ID NO: 74), GA[LVQPARTMS][RSAKNLH]I[GSLATEDM]A (SEQ ID NO: 75), GA[SRTQV]GL[GLS]A (SEQ ID NO: 76), GA[PSVALYH][LAGQHM]L[SAEGHNW]A (SEQ ID NO: 77), GAR[AQNE]L[GMS]A (SEQ ID NO: 78), [GR]A[AHP][RM][IL][LNC][AN] (SEQ ID NO: 79), GA[RAPVQSH][GRTASKEQ][FHY][GASLWD]A (SEQ ID NO: 80), GS[PVT]G[FI][RYSWHGN]A (SEQ ID NO: 81), [GK][SG][PRLSVM][GM][MIFQ][WHN]A (SEQ ID NO: 82), E[FHT]NN[TSLKGA][PMTSGA][HDALWNTY] (SEQ ID NO: 83), E[FHT]NH[RK][APSQDGCTV][LIVYFQKR] (SEQ ID NO: 84),E[GF][FWY]M[GMAS][MQSG][DASQ] (SEQ ID NO: 85),G[WF][PVSL][GLSA]G[RGSWNHM][PAG] (SEQ ID NO: 86),GW[ASPQVKHRLE][GSMAHL][STN][GRSH][YWFG] (SEQ ID NO: 87), and GW[PSV][GH][SAT][GRNMLQ][GFW] (SEQ ID NO: 88) wherein one amino acid in each bracket is individually selected to form the n-mer X1-X2-X3-X4-X5-X6-X7.CD59 BindingGlycosylphosphatidylinositol Anchors

[0072] The role of the glycosylphosphatidylinositol (GPI) is to anchor proteins to a cellsurface. GPI anchors are glycolipid posttranslational modifications to proteins and are found throughout eukaryotes. GPI anchors fundamentally consists of phosphatidylinositol, glycans (including one glucosamine and three mannoses), and a terminal phosphoethanolamine. The GPI anchors are assembled in the endoplasmic reticulum and then the terminal phosphoethanolamine is amide-bonded to a carboxyl-terminus of a protein. The GPI anchor backbone may include a phosphoethanolamine and / or glycan side branches depending on the cell type and protein. The lipid portion may include a l-alkyl-2-acyl phosphatidylinositol, diacyl phosphatidylinositol, or inositol-phosphoceramide.

[0073] All GPI-anchored proteins include a trigger sequence that signals for the addition of a GPI anchor and is removed after the GPI anchor has been added. The now anchored GPI is further modified and eventually brought to the cell surface for display. Common features associated with GPI-anchored proteins include: association with membrane microdomains; exist on the cell surface as transient homodimers; undergo specific endocytosis; and transduce signals for proliferation or cell motility. See e.g., Kinoshita, T. Glycosylphosphatidylinositol (GPI) Anchors: Biochemistry and Cell Biology: Introduction to a Thematic Review Series. Journal of Lipid Research, 2016, 57, 4-5.CD 59

[0074] One particular GPI-anchored protein is CD59, which is a membrane attack complexinhibiting protein. It is broadly expressed in human tissue.

[0075] CD59, located on chromosome 11 (i.e., 11 pl3), is a protein that in humans is encoded by the CD59 gene (Gene Identifier).

[0076] In an embodiment, the CD59 targeting moiety binds to the CNS. In an embodiment, the CD59 targeting moiety binds to one or more of the CD59. As used herein, the term “CD59”refers to any one of the alternatively spliced variants, orthologues, paralogues. In an embodiment, the term “a CD59” refers to any one of the alternatively spliced variants, orthologues, paralogues. Engineered Viral Capsids

[0077] Described herein are various embodiments of engineered viral capsids, such as adeno- associated virus (AAV) capsids, that can be engineered to confer cell-selective tropism, such as CNS tissue- and cell-specific tropism, to an engineered viral particle. Engineered viral capsids can be adenoviral or AAV capsids. The engineered capsids can be included in an engineered virus particle (e.g., an engineered adenoviral or AAV virus particle), and can confer cell-selective tropism to the engineered viral particle. The engineered viral capsids described herein can include one or more engineered viral capsid proteins described herein. The engineered viral capsids described herein can include one or more engineered viral capsid proteins described herein that can contain one or more CD59 targeting moiety sequence as described elsewhere herein.

[0078] The engineered viral capsids can be variants of a wild-type viral capsid. For example, in an embodiment, the engineered AAV capsids can be variants of wild-type AAV capsids. In an embodiment, the wild-type AAV capsids can be composed of VP1, VP2, VP3 capsid proteins or a combination thereof. In other words, the engineered AAV capsids can include one or more variants of a wild-type VP1, wild-type VP2, and / or wild-type VP3 capsid proteins.

[0079] In an embodiment, the serotype of the reference wild-type AAV capsid can be AAV9, AAV9 K449R (or K449R AAV9), 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 A586Rmutant, 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, 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 and variants thereof. See e.g., US20210380969, the content of which is incorporated by reference herein in its entirety. In an embodiment, the serotype of the wild-type AAV capsid can be AAV9. The engineered AAV capsids can have a different tropism than that of the reference wild-type AAV capsid.

[0080] In an embodiment, the CD59 targeting moieties comprises of modifying the AAV capsid polypeptide. In an embodiment, an engineered adeno-associated virus (AAV) capsid polypeptide comprising a CD59 targeting moiety defined by a n-mer of the formula X1-X2-X3-X4- X5-X6-X7, inserted at any position between 450-461 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype, and wherein Xi, X2, X3, X4, X5, Xe, and X7 represent an amino acid inserted at an amino acid position in the capsid polypeptide.

[0081] The core of each wild-type AAV viral protein contains an eight-stranded beta-barrel motif (betaB to betal) and an alpha-helix (alphaA) that are conserved in autonomous parvovirus capsids (see e.g., DiMattia et al. 2012. J. Virol. 86(12):6947-6958). Structural variable regions (VRs), also referred to as “loops”, occur in the surface loops that connect the beta-strands, which cluster to produce local variations in the capsid surface. AAVs have 12 variable regions (also referred to as hypervariable regions) (see e.g., Weitzman and Linden. 2011. “Adeno-Associated Virus Biology.” In Snyder, R.O., Moullier, P. (eds.) Totowa, NJ: Humana Press). In an embodiment, Xi, X2, X3, X4, X5, Xe, and X7 modify amino acids in one or more of the 12 variable regions in the wild-type AVV capsid proteins. In an embodiment, the Xi, X2, X3, X4, X5, Xe, and X7modify 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 embodiment, the CD59 targeting moiety is inserted or substituted in loop IV and / or loop VIII.

[0082] In an embodiment, the CD59 targeting moiety comprises of amino acids 586-588 and 589-592 of a 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 AAV9, AAV9 K449R (or K449R AAV9), 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.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, AAVA8RA586R 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, AAVCBr-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, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9 and variants thereof. In an embodiment, X1-X2-X3 comprises of any sequential amino acids 449-459 and X4-X5-X6-X7 comprises of any amino 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.

[0083] In an embodiment, the CD59 targeting moiety is incorporated into a viral protein, such as a capsid protein, including but not limited to adenoviral or AAV proteins. In an embodiment, the n-mer is located between two amino acids of the viral protein such that the CD59 targetingmoiety is external (i.e., is presented on the surface of) to a viral capsid. 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 consecutive amino acids in a variable amino acid region in a viral capsid protein.

[0084] In an embodiment, the n-mer can be inserted between two consecutive amino acids in a variable amino acid region in an AAV capsid protein. 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, the one or more n-mers can be 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 embodiment, the CD59 targeting moiety is inserted or substituted in loop IV and / or loop VIII. In an embodiment, the n-mer or 7-mer is a CD59 targeting moiety.

[0085] 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 acid 590 and 591). 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. 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. 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. In an embodiment, the engineered capsid is a modified AAV6 capsid and can have a n-mer inserted at or a neighbor of amino acid 585 and optionally Y705-731, T492V, K531E. In an embodiment, the engineered capsid is a modified AAV8 capsid and can have a n-mer inserted after or a neighbor of amino acid 585 and 590. In an embodiment, the engineered capsid is a modified AAV9 capsid and can have a n-mer inserted in between amino acid 588 and 589. (Buning, H.; Srivastava, A. Capsid Modifications 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 7-mer motif inserted between amino acids 588 and 589 of an AAV9 viral protein. SEQ ID NO: 1 is a 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 consecutiveamino acids within amino acids 451-460 of a capsid protein of AAV9 viral protein. SEQ ID NO: l is a reference AAV9 capsid sequence for at least referencing the insertion sites discussed above.

[0086] It will be appreciated that n-mers can be inserted in analogous positions in AAV viral proteins of other wild-type serotypes or engineered capsid variants, such as but not limited to, AAV9, AAV9 K449R (or K449R AAV9), 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.50, 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.40, 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. 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, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9 and variants thereof. See e.g., US20210380969, the content of which is incorporated by reference herein in its entirety. In an embodiment as previously discussed, the n-mer(s) can be inserted between any two contiguous amino acids within the AAV viral protein and in an embodiment the insertion is made in a variable region.

[0087] In an embodiment, the first 1, 2, 3, or 4 amino acids of a CD59 targeting moiety can replace 1, 2, 3, or 4 amino acids of a polypeptide into which it is inserted and preceding theinsertion site. Using an AAV as another non-limiting 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 insert can replace amino acids 586, 587, and 588 such that the amino acid immediately preceding the CD59 targeting moiety 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 embodiment, the AAV capsid protein is selected from SEQ ID NO: 1.Table B. Example AAV VP1 amino acid sequences

[0088] In an embodiment, the n-mer comprises EFNNGSD (SEQ ID NO: 89) or GAASLMP (SEQ ID NO: 109). In an embodiment, the CD59 targeting moiety comprises of the amino acid sequence of one of SEQ ID Nos: 89-5983.

[0089] In an embodiment, in addition to the n-mer motif(s), the CD59 targeting moiety can include a polypeptide, a polynucleotide, a lipid, a polymer, a sugar, or a combination thereof.

[0090] 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 poly adenylation signal can be an SV40 polyadenylation signal.

[0091] 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. 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 the transgene and regulatory sequences are flanked by AAV ITR sequences.

[0092] 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. The host cell system may also include a construct that expresses a recombinant AAV 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 the transgene and regulatory sequences are flanked by AAV ITR sequences. 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 polyadenylation tail. In an embodiment, the AAV capsid encoding polynucleotide can be operably coupled to a promoter. In an embodiment, the regulatory sequence that regulates the expression of the transgene is a promoter and can be a tissue- or cell type-specific promoter. In an embodiment, the tissue-specific promoter is specific for muscle (e.g., cardiac, skeletal, and / or smooth muscle), neurons or othernervous system cells (e.g., astrocytes, glial cells, Schwann cells, ependymal cells, pericyte, oligodendrocyte, oligodendrocyte progenitor), specific neuronal 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 a constitutive promoter. 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 regulates the expression of the transgene is a promoter and can be a cell state regulating promotor or drug inducible promotor.

[0093] 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 operatively-linked transgene in neurons as compared to expression in non-neuronal cells. Nonlimiting 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 HUMNFL, L04147); a synapsin promoter (see, e.g., GenBank HUMSYNIB, M553O1); a thy-1 promoter (see, e.g., Chen et al„ (1987) Cell, 51 :7-19; Llewellyn et al. (2010) Nat. Med., 16(10): 1161-1 166); a serotonin receptor promoter (see, e.g., GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Oh et al., (2009) Gene Then, 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) EMBO J., 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 an embodiment, 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 tissue-specific expression element for oligodendrocytes include the myelin basic protein (MBP) promoter. In an embodiment, 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 cellspecific 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).

[0094] In other aspects, a promoter can be an inducible promoter (i.e., a promoter whose activity is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein). In an embodiment, a promoter may be a temporally restricted promoter that drives expression depending on the temporal context in which the promoter is found. 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 etal. Proc. Natl. Aced. 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 havetargets in mammalian cells, the possibility of interference with cellular 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. and N. 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 in eukaryotic inducible expression systems using different strategies to control activation and repression of expression. Activation of expression is mediated by a chimeric transactivator 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 entireties). 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 a promoter which is 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 non-coding 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 of 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).

[0095] Additional promotors 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. PCT (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, John, Andrew DELGADO, Elizabeth JENQUIN, Jana, Rose WANG, Eric, Tzy-Shi. GENE THERAPY VECTORS. World Patent, incorporated herein by reference in their entirety.Further Capsid Modifications

[0096] In an embodiment, the viral capsid protein may comprise one or more mutations relative to wild type. In an embodiment, the one or more mutations comprise a K449R substitution in a capsid polypeptide of AAV 9 20507, or a substitution in an analogous position of a capsid polypeptide from AAV9, AAV9 K449R (or K449R AAV9), 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, 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, 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 5R1, 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. l l, 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.48R1, 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. 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, AAVF17 / HSC17, AAVF2 / HSC2, AAVF3 / HSC3, AAVF4 / HSC4, AAVF5 / HSC5, AAVF6 / HSC6, AAVF7 / HSC7, AAVF8 / HSC8, and / or AAVF9 / HSC9 and variants thereof. In an embodiment, the K449R substituted AAV capsid is selected from SEQ ID NO: 2.

[0097] In an embodiment, the viral capsid protein may comprise additional targeting motifs that are in addition to the CD59 targeting moiety of the present disclosure. Without being bound by theory the additional targeting moieties can be antibodies or fragments thereof. In an embodiment, the additional targeting moiety can be any molecule or composition capable of recognizing, binding, attaching to, or otherwise interacting with a binding partner that can be present on the surface of a target cell. Binding partners include, but are not limited to, nucleic acids, proteins, peptides, sugars, fats, or any combination thereof or any other molecule or molecules that are present on the surface of a target cell. In an embodiment, the binding partner is unique to a cell type or cell state or a to a group of related cell types or cell states. In an embodiment, the binding partner is a receptor, channel, or other complex present on the surface of a target cell. These additional targeting moieties can be used to target, e.g., specific cell types or cell states within those the set of target cells targeted by the CD59 targeting moiety. As used herein, “cell state” is used to describe transient elements of a cell’s identity. Cell state can be thought of as the transient characteristic profile or phenotype of a cell. Cell states arise transiently during time-dependent processes, either in a temporal progression that is unidirectional (e.g., during differentiation, or following an environmental stimulus) or in a state vacillation that is not necessarily unidirectional and in which the cell may return to the origin state. Vacillating processescan be oscillatory (e.g., cell-cycle or circadian rhythm) or can transition between states with no predefined order (e.g., due to stochastic, or environmentally controlled, molecular events). These time-dependent processes may occur transiently within a stable cell type (as in a transient environmental response), or may lead to a new, distinct type (as in differentiation). See e.g., Wagner et al., 2016. Nat Biotechnol. 34(11): 1145-1160.

[0098] In an embodiment, the additional targeting moiety is or includes a peptide or a polypeptide. In an embodiment, the additional targeting moiety is or includes an antibody or fragment thereof. Exemplary antibodies and fragments thereof are described in greater detail elsewhere herein, see e.g., discussion on exemplary cargos. In an embodiment, the additional targeting moiety is or includes an aptamer. In an embodiment, the additional targeting moiety is or includes a small molecule. In an embodiment, the additional targeting moiety is or includes a nucleic acid (e.g., DNA or RNA). In an embodiment, the additional targeting moiety is or includes a receptor. In an embodiment, the additional targeting moiety is or includes a receptor ligand. In an embodiment, the additional targeting moiety is or includes a carbohydrate (e.g., a sugar). In an embodiment, the additional targeting moiety is or includes a lipid. In an embodiment, the additional targeting moiety is an engineered protein scaffold. In an embodiment, the additional targeting moiety is an affibody. In an embodiment, the additional targeting moiety is an antibody mimetic. In an embodiment, the additional targeting moiety is an engineered binding protein, such as a designed ankyrin repeat proteins (DARPins) (see e.g., Pluckthun et al., Annu. Rev. Pharmacol. Toxicol. (2015) 55(1): 489-511), avimers (Silverman et al., Nat. Biotechnol. (2005) 23 (12): 1556— 1561 and Jeong et al. Nat. Biotechnol. (2005) 23(12): 1493-1494), or affibodies (see e.g., Nord et al., Nat. Biotechnol. (1997) 15(8):772-777). In an embodiment, the additional targeting moiety is a receptor ligand or binding protein. In an embodiment, the additional targeting moiety is attached or otherwise coupled to the capsid surface. In an embodiment, the additional targeting moiety is encoded by a vector that produces a capsid of the present invention described herein.

[0099] In an embodiment, the capsid polypeptide can be covalently modified by the covalent coupling of at least one compound comprising a lactam moiety (e.g., P-lactam) to at least one amino group of an amino acid residue of the capsid of the AAV vectors (see, for example, the published International PCT application No. PCT / EP2021 / 080832 and U.S. Patent No. US 11382988, the contents of which are incorporated by reference herein in their entireties). In other embodiments, a ligand, e.g., a 7-mer covalently linked to a primary amino group of a capsidpolypeptide via a CSNH- bond, (see, e.g., U.S. Patent No. 11,648,319, the content of which is incorporated by reference herein in its entirety).

[0100] In an embodiment, the CD59 targeting moiety can be bound to an AAV capsid polypeptide through a specific protein: protein binding pair that forms a covalent, e.g., isopeptide bond. For example, the Spy Tag / Spy Catcher bioconjugation technology can be used to append a targeting ligand, e.g., the CD59 targeting moiety, to the surface of a capsid protein, where it can specifically bind a CD59, expressed on the cell of interest (see, for example, the published U.S. Patent Application No. 2020 / 0140492, the content of which is incorporated by reference herein in its entirety). According to this embodiment, the CD59 targeting moiety can be fused in frame to the 13 amino acid SpyTag peptide. Advantages of this approach include binding of the SpyTag - CD59 targeting moiety to a fully assembled AAV capsid and the relative ease of testing different 7-mer CD59 targeting moieties using the same AAV preparation. The SpyCatcher moiety can be bound to the AAV capsid polypeptide either covalently or non-covalently, for example, using a protein binding domain-specific for one or more epitopes on the surface of the capsid polypeptide.Engineered Vectors and Vector Systems

[0101] Also provided herein are vectors and vector systems that can encode one or more of the engineered polypeptides described herein that includes one or more of the CD59 targeting moieties of the present invention, including but not limited to engineered viral polynucleotides (e.g., polynucleotides encoding engineered AAV capsid proteins). In a preferred embodiment, provided herein is a vector system comprising one or more vectors encoding a CD59 targeting moiety effective to increase transduction of CNS, optionally further comprising a vector encoding a recombinant viral genome comprising a transgene. In preferred embodiment, the CD59 targeting moiety encoded in the vector system binds to CD59. As used in this context, engineered viral capsid polynucleotides refers to any one or more of the polynucleotides described herein encoding an engineered viral capsid as described elsewhere herein and / or polynucleotide(s) encoding one or more engineered viral capsid proteins described elsewhere herein. Further, where the vector includes an engineered viral capsid polynucleotide described herein, the vector can also be referred to and considered an engineered vector or system thereof although not specifically noted as such. In embodiments, the vector can contain one or more polynucleotides encoding one or more elements of an engineered viral capsid described herein. The vectors and systems thereof can be useful in producing bacterial, fungal, yeast, plant cells, animal cells, and transgenic animals thatcan express one or more components of the engineered viral capsid, particle, or other compositions described herein. Within the scope of this disclosure are vectors containing one or more of the polynucleotide sequences described herein. One or more of the polynucleotides that are part of the engineered viral capsid and system thereof described herein can be included in a vector or vector system.

[0102] In an embodiment, a vector used in the production of the rAAVs disclosed herein comprises a rep gene and 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 as well. 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.

[0103] 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. In an embodiment, the vector includes one or more minimal splice regulatory elements. 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 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 herein. It some embodiments, the vector does not include one or more minimal splice regulatory elements, modified splice regulatory agent, splice acceptor, and / or splice donor.

[0104] 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. Otheruses 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 which 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 so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements.

[0105] 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 type of vector 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 carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication 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 genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing 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.

[0106] 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 on the basis of 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 definedelsewhere herein. 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 expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell).

[0107] In an embodiment, the vector can be a bicistronic vector. 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, 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.Cell-based Vector Amplification and Expression

[0108] Vectors can be designed for expression of one or more elements of the engineered viral (e.g., AAV) capsid system or other compositions containing a CD59 targeting moiety of the present disclosure described herein (e.g., nucleic acid transcripts, proteins, enzymes, and combinations thereof) in a suitable host cell.

[0109] In an embodiment, the suitable host cell is a prokaryotic cell. 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 a eukaryotic cell. In an embodiment, the suitable host cell is a suitable bacterial cell. Suitable bacterial cells include, but are not limited to, bacterial cells from the bacteria of the species Escherichia coli. Many suitable strains of E. coli are known in the art for expression of vectors. These include, but are not limited to Pirl, Stbl2, Stbl3, Stbl4, TOP10, XL1 Blue, and XL10 Gold. In an embodiment, the host cell is a suitable insect cell. Suitable insect cells include those from Spodopter a frugiperda. Suitable strains of S. frugiperda cells include, but are not limited to, Sf9 and Sf21. 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 embryofibroblasts (CEFs). Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).

[0110] 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 as any 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 in Yeast Protocols, 2nd edition, Xiao, W ., ed. (Humana Press, New York, 2007) andBuckholz, 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.

[0111] In an embodiment, the vector is a baculovirus vector or expression vector and can be suitable for expression of 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 (EX-CELL 405).

[0112] In an embodiment, the vector is a mammalian expression vector. In an embodiment, the mammalian expression vector is capable of expressing one or more polynucleotides and / orpolypeptides in a mammalian cell. Examples of mammalian expression vectors include, but are not limited to, pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, etal., 1987. EMBO J. 6: 187-195). The mammalian expression vector can include one or more suitable regulatory elements capable of controlling expression of the 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 detail on suitable regulatory elements is described elsewhere herein.

[0113] 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: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0114] In an embodiment, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid 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. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific 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). With regards to these prokaryotic and eukaryotic vectors, mention is made of U.S. Patent 6,750,059, the contents of which are incorporated by reference herein in their entirety. Other embodiments can utilize viral vectors, with regards to which mention is made of U.S. Patent application 13 / 092,085, the contents of which are incorporated by reference herein in their entirety. Tissue-specific regulatory elements are known in the art and in this regard, mention is made of U.S. Patent 7,776,321, the contents of which are incorporated by reference herein in their entirety. In an embodiment, a regulatoryelement can be operably linked to a transgene in a recombinant genome packaged by the engineered AAV capsid system so as 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.

[0115] 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.

[0116] In an embodiment, the vector can be a fusion vector or fusion expression vector. In an embodiment, fusion vectors add a number of amino acids to a protein encoded therein, such as to 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 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 other fusion moiety subsequent to 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 l id (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).

[0117] In an embodiment, one or more vectors driving expression of one or more elements of an engineered viral (e.g., AAV) capsid system or other composition containing a CD59 targeting moiety described herein are introduced into a host cell such that expression of the elements of the engineered delivery system described herein direct formation of an engineered viral (e.g., AAV) capsid system or other composition containing a CD59 targeting moiety described herein (including but not limited to an engineered gene transfer agent particle, which is described in greater detail elsewhere herein). For example, different elements of the engineered viral (e.g., AAV) capsid system or other composition containing a CD59 targeting moiety described herein can each be operably linked to separate regulatory elements on separate vectors. RNA(s) of different elements of the engineered delivery system described herein can be delivered to an animal or mammal or cell thereof to produce an animal or mammal or cell thereof that constitutively or inducibly or conditionally expresses different elements of the engineered viral (e.g., AAV) capsid system or other composition containing a CD59 targeting moiety described herein that incorporates one or more elements of the engineered viral (e.g., AAV) capsid system or other composition containing a CD59 targeting moiety described herein or contains one or more cells that incorporates and / or expresses one or more elements of the engineered viral (e.g., AAV) capsid system or other composition containing a CD59 targeting moiety described herein.

[0118] 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 proteins or other composition containing a CD59 targeting moiety described herein, embedded within one or more intron sequences (e.g., each in a different intron, two or more in at least one intron, or all in a single intron). In an embodiment, the engineered polynucleotides of the present invention (including but not limited to engineered viral polynucleotides) can be operably linked to and expressed from the same promoter.Vector Features

[0119] 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 there from, or 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 additional features included can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc.Regulatory Elements

[0120] 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). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter can direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e g., liver, brain), or particular cell types (e.g., lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, 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 III promoters), 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, thedihydrofolate reductase promoter, the -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).

[0121] In an embodiment, the regulatory sequence can be a regulatory sequence 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 herein in their entirety. 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 polynucleotide sequences is less than 4.4Kb.

[0122] 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, -actin, RSV, and PGK. Suitable constitutive promoters for bacterial cells, yeast cells, and fungal cells are generally known in the art, such as a T-7 promoter for bacterial expression and an alcohol dehydrogenase promoter for expression in yeast.

[0123] In an embodiment, the regulatory element can be a regulated 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 as previously discussed elsewhere herein. 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 certain environmental conditions, and / or during a specific state of development. Suitable tissue specific promoters can include, but are not limited to, liver specific promoters (e.g., APOA2, 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)), centralnervous system cell promoters (SYN1, GFAP, EMA, NES, MOBP, MBP, TH, FOXA2 (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, FOXA2, 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.

[0124] 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 any suitable 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.

[0125] 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, nucleus, ribosome, endoplasmic reticulum, Golgi apparatus, chloroplast, mitochondria, vacuole, lysosome, cytoskeleton, plasma membrane, cell wall, peroxisome, centrioles, etc.CpG content

[0126] 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 deletedand / or substituted. In one aspect, deletion, or reduction in the number of CpG islands can reduce the immunogenicity of the rAAV vector. This results from a reduction or complete inhibition in TLR-9 binding to the rAAV vector DNA sequence, which occurs 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 decreased methylation and therefore decreased transgene silencing. In an embodiment, it is the minimal functional ITR in which one or more CpG islands 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.

[0127] In an embodiment, at least 1 CpG motif is deleted and / or substituted, e g., at least 4 or more or 8 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 is deleted, substituted with a different nucleotide, or any combination of deletions and substitutions.

[0128] In an embodiment, 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 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

[0129] 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 oversize 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. Oversize 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, reassembly of these truncated genomes in the target cell nucleus has been proposed as a mechanism for oversize AAV vector transduction. Independent of transduction mechanism and in vivo efficacy, the heterogeneity in oversize AAV genome sizes is a major limitation for their application in human gene therapy.

[0130] 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 by the dual AAV vectors and inverted terminal repeat (1TR)- mediated head-to-tail concatemerization of the two halves, trans-splicing results in the production of a mature mRNA and full-size protein. Trans-splicing has been successfully used to express large genes in muscle and retina.

[0131] 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.

[0132] A third dual AAV strategy (hybrid) is based on adding a highly recombinogenic region from an exogenous gene (i.e., 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 in order 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.Selectable Markers and Tags

[0133] One or more of the engineered polynucleotides of the present disclosure (e.g., an engineered viral (e.g., AAV) capsid polynucleotide) can be operably linked, fused to, or otherwise modified to include a polynucleotide that encodes or is a selectable marker or tag, which can be a polynucleotide or polypeptide. In an embodiment, the polypeptide encoding a polypeptide selectable marker can be incorporated in the engineered polynucleotide of the present disclosure (e.g., an engineered viral (e.g., AAV) capsid polynucleotide) such that the selectable marker polypeptide, when translated, is inserted between two amino acids between the N- and C- terminus of an engineered polypeptide (e.g., the engineered AAV capsid polypeptide) or at theN- and / or C- terminus of the engineered polypeptide (e.g., an engineered AAV capsid polypeptide). In anembodiment, the selectable marker or tag is a polynucleotide barcode or unique molecular identifier (UMI).

[0134] It will be appreciated that the polynucleotide encoding such selectable markers or tags can be incorporated into a polynucleotide encoding one or more components of the engineered AAV capsid system described herein in an appropriate manner to allow expression of the selectable marker or tag. Such techniques and methods are described elsewhere herein and will be instantly appreciated by one of ordinary skill in the art in view of this disclosure. Many such selectable markers and tags are generally known in the art and are intended to be within the scope of this disclosure.

[0135] Suitable selectable markers and tags include, but are not limited to, affinity tags, such as chitin binding protein (CBP), maltose binding protein (MBP), glutathione-S-transferase (GST), poly(His) tag; solubilization tags such as thioredoxin (TRX) and poly(NANP), MBP, and GST; chromatography tags such as those consisting of polyanionic amino acids, such as FLAG-tag; epitope tags such as V5-tag, Myc-tag, HA-tag and NE-tag; protein tags that can allow specific enzymatic modification (such as biotinylation by biotin ligase) or chemical modification (such as reaction with FlAsH-EDT2 for fluorescence imaging), DNA and / or RNA segments that contain restriction enzyme or other enzyme cleavage sites; DNA segments that encode products that provide resistance against otherwise toxic compounds including antibiotics, such as, spectinomycin, ampicillin, kanamycin, tetracycline, Basta, neomycin phosphotransferase II (NEO), hygromycin phosphotransferase (HPT) and the like; DNA and / or RNA segments that encode products that are otherwise lacking in the recipient cell (e.g., tRNA genes, auxotrophic markers); DNA and / or RNA segments that encode products which can be readily identified (e.g., phenotypic markers such as P-galactosidase, GUS; fluorescent proteins such as green fluorescent protein (GFP), cyan (CFP), yellow (YFP), red (REP), luciferase, and cell surface proteins); polynucleotides that can generate one or more new primer sites for PCR (e.g., the juxtaposition of two DNA sequences not previously juxtaposed), DNA sequences not acted upon or acted upon by a restriction endonuclease or other DNA modifying enzyme, chemical, etc.; epitope tags (e.g. GFP, FLAG- and His-tags), and, DNA sequences that make a molecular barcode or unique molecular identifier (UMI), DNA sequences required for a specific modification (e.g., methylation) that allows its identification. Other suitable markers will be appreciated by those of skill in the art.

[0136] Selectable markers and tags can be operably linked to one or more components of the engineered AAV capsid system or other compositions and / or systems described herein via suitable linker, such as a glycine or glycine serine linkers as short as GS or GG up to (GGGGG)a (SEQ ID NO: 6002) or (GGGGSfi (SEQ ID NO: 6003). Other suitable linkers are described elsewhere herein.

[0137] The vector or vector system can include one or more polynucleotides encoding one or more CD59 targeting moieties. In an embodiment, the CD59 targeting moiety encoding polynucleotides can be included in the vector or vector system, such as a viral vector system, such that they are expressed within and / or on the virus particle(s) produced such that the virus particles can be targeted to specific cells, tissues, organs, etc. In an embodiment, the CD59 targeting moiety encoding polynucleotides can be included in the vector or vector system such that the engineered polynucleotide(s) of the present disclosure (e.g., an engineered viral (e.g., AAV) capsid polynucleotide(s)) and / or products expressed therefrom include the CD59 targeting moiety and can be targeted to specific cells, tissues, organs, etc. In an embodiment, such as non-viral carriers, the CD59 targeting moiety can be attached to the carrier (e.g., polymer, lipid, inorganic molecule etc.) and can be capable of targeting the carrier and any attached or associated engineered polynucleotide(s) of the present disclosure, the engineered polypeptides, or other compositions of the present disclosure described herein, to specific cells, tissues, organs, etc. In an embodiment, the specific cells are CNS cells.Cell-free Vector and Polynucleotide Expression

[0138] In an embodiment, the polynucleotide(s) encoding a CD59 targeting moiety 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 from a vector or suitable polynucleotide in a cell-free in vitro system. 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 in vitro translation systems replicate the processes of RNA and protein synthesis, respectively, outside of the cellular environment. Vectors and suitable polynucleotides for in vitro transcription can include T7, SP6, T3, promoter regulatory sequences that can be recognized and acted upon by an appropriate polymerase to transcribe the polynucleotide or vector.

[0139] In vitro translation can be stand-alone (e.g., translation of a purified polyribonucleotide) or linked / 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 that are needed for translation of exogenous RNA (e.g., 70S or 80S ribosomes, tRNAs, aminoacyl-tRNA, synthetases, initiation, elongation factors, termination factors, etc.). 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 co-factors (Mg2+, K+, etc.). As previously mentioned, in vitro translation can be based on RNA or DNA 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 utilize a DNA template as a starting material (e.g., E coli-based systems). In these systems transcription and translation are coupled and DNA is first transcribed into RNA, which is subsequently translated. Suitable standard and coupled cell-free translation systems are generally known in the art and are commercially available.Codon Optimization of Vector Polynucleotides

[0140] As described elsewhere herein, the polynucleotide encoding a CD59 targeting moiety of the present disclosure and / or other polynucleotides described herein, or the transgene contained within the recombinant AAV genome can be codon optimized. In an embodiment, polynucleotides of the engineered AAV capsid system described herein can be codon optimized. In an embodiment, one or more polynucleotides contained in a vector (“vector polynucleotides”) described herein that are in addition to an optionally codon optimized polynucleotide encoding a CD59 targeting moiety, including but not limited to, embodiments of the engineered AAV capsid system described herein, can be codon optimized. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g., about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), whichis in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.or.jp / codon / and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available. In an embodiment, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a DNA / RNA-targeting Cas protein corresponds to the most frequently used codon for a particular amino acid. As to codon usage in yeast, reference is made to the online Yeast Genome database available at www.yeastgenome.org / community / codon_usage.shtml, or Codon selection in yeast, Bennetzen and Hall, J Biol Chem. 1982 Mar 25;257(6):3026-31. As to codon usage in plants including algae, reference is made to Codon usage in higher plants, green algae, and cyanobacteria, Campbell and Gowri, Plant Physiol. 1990 Jan; 92(1): 1-11.; as well as Codon usage in plant genes, Murray et al, Nucleic Acids Res. 1989 Jan 25;17(2):477-98; or Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages, Morton BR, J Mol Evol. 1998 Apr;46(4):449-59.

[0141] The vector polynucleotide can be codon optimized for expression in a specific celltype, tissue type, organ type, and / or subject type. In an embodiment, a codon optimized sequence is a sequence optimized for expression in a eukaryote, e.g., humans (i.e., being optimized for expression in a human or human cell), or for another eukaryote, such as another animal (e.g., a mammal or avian) as is described elsewhere herein. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In an embodiment, the polynucleotide is codon optimized for a specific cell type. Such cell types can include, but are not limited to, epithelial cells (including skin cells, cells lining the gastrointestinal tract, cells lining other hollow organs), nerve cells (nerves, brain cells, spinal column cells, nerve support cells (e.g., astrocytes, glial cells, Schwann cells etc.) , muscle cells (e.g., cardiac muscle, smooth muscle cells, and skeletal muscle cells), connective tissue cells (fat and other soft tissue padding cells, bonecells, tendon cells, cartilage cells), blood cells, stem cells and other progenitor cells, immune system cells, germ cells, and combinations thereof. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In an embodiment, the polynucleotide is codon optimized for a specific tissue type. Such tissue types can include, but are not limited to, muscle tissue, connective tissue, nervous tissue, and epithelial tissue. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein. In an embodiment, the polynucleotide is codon optimized for a specific organ. Such organs include, but are not limited to, muscles, skin, intestines, liver, spleen, brain, lungs, stomach, heart, kidneys, gallbladder, pancreas, bladder, thyroid, bone, blood vessels, blood, and combinations thereof. Such codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein.

[0142] In an embodiment, a vector polynucleotide is codon optimized for expression in particular cells, such as prokaryotic or eukaryotic cells. 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 discussed herein, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate.Non-Viral Vectors and Carriers

[0143] In an embodiment, the vector is a non-viral vector or carrier. In an embodiment, non- viral vectors can have the advantage(s) of reduced toxicity and / or immunogenicity and / or increased bio-safety as compared to viral vectors The terms of art “Non-viral vectors and carriers” and as used herein in this context refers to molecules and / or compositions that are not based on one or more component of a virus or virus genome (excluding any nucleotide to be delivered and / or expressed by the non-viral vector) that can be capable of attaching to, incorporating, coupling, and / or otherwise interacting with an engineered capsid polynucleotide (e.g., an engineered AAV capsid polynucleotide) or other composition of the present disclosure described herein and can be capable of ferrying the polynucleotide to a cell and / or expressing the polynucleotide. It will be appreciated that this does not exclude the inclusion of a virus-based polynucleotide that is to be delivered. For example, if a gRNA to be delivered is directed against a virus component and it is inserted or otherwise coupled to an otherwise non-viral vector or carrier, this would not make said vector a “viral vector”. Non-viral vectors and carriers include naked polynucleotides, chemicalbased carriers, polynucleotide (non-viral) based vectors, and particle-based carriers. It will beappreciated that the term “vector” as used in the context of non-viral vectors and carriers refers to polynucleotide vectors and “carriers” used in this context refers to a non-nucleic acid or polynucleotide molecule or composition that be attached to or otherwise interact with a polynucleotide to be delivered, such as an engineered AAV capsid polynucleotide of the present disclosure.Naked Polynucleotides

[0144] In an embodiment one or more engineered AAV capsid polynucleotides or other polynucleotides of the present disclosure described elsewhere herein can be included in a naked polynucleotide. The term of art “naked polynucleotide” as used herein refers to polynucleotides that are not associated with another molecule (e.g., proteins, lipids, and / or other molecules) that can often help protect it from environmental factors and / or degradation. As used herein, associated with includes, but is not limited to, linked to, adhered to, adsorbed to, enclosed in, enclosed in or within, mixed with, and the like. Naked polynucleotides that include one or more of the engineered AAV capsid polynucleotides or other polynucleotides of the present disclosure described herein can be delivered directly to a host cell and optionally expressed therein. The naked polynucleotides can have any suitable two- and three-dimensional configurations. By way of non-limiting examples, naked polynucleotides can be single-stranded molecules, double stranded molecules, circular molecules (e.g., plasmids and artificial chromosomes), molecules that contain portions that are single stranded and portions that are double stranded (e.g., ribozymes), and the like. In an embodiment, the naked polynucleotide contains only the engineered AAV capsid polynucleotide(s) or other polynucleotides of the present disclosure. In an embodiment, the naked polynucleotide can contain other nucleic acids and / or polynucleotides in addition to the engineered AAV capsid polynucleotide(s) or other polynucleotides of the present disclosure described elsewhere herein. The naked polynucleotides can include one or more elements of a transposon system. Transposons and system thereof are described in greater detail elsewhere herein.Non- Viral Polynucleotide Vectors

[0145] In an embodiment, one or more of the engineered AAV capsid polynucleotides or other polynucleotides of the present disclosure can be included in a non-viral polynucleotide vector. Suitable non-viral polynucleotide vectors include, but are not limited to, transposon vectors and vector systems, plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, AR(antibiotic resistance)-free plasmids and miniplasmids, circular covalently closed vectors (e.g.,minicircles, minivectors, miniknots,), linear covalently closed vectors (“dumbbell shaped”), MIDGE (minimalistic immunologically defined gene expression) vectors, MiLV (micro-linear vector) vectors, Ministrings, mini-intronic plasmids, PSK systems (post-segregationally killing systems), ORT (operator repressor titration) plasmids, and the like. See e.g., Hardee et al. 2017. Genes. 8(2):65.

[0146] In an embodiment, the non-viral polynucleotide vector can have a conditional origin of replication. In an embodiment, the non-viral polynucleotide vector can be an ORT plasmid. In an embodiment, the non-viral polynucleotide vector can have a minimalistic immunologically defined gene expression. In an embodiment, the non-viral polynucleotide vector can have one or more post-segregationally killing system genes. In an embodiment, the non-viral polynucleotide vector is AR-free. In an embodiment, the non-viral polynucleotide vector is a minivector. In an embodiment, the non-viral polynucleotide vector includes a nuclear localization signal. In an embodiment, the non-viral polynucleotide vector can include one or more CpG motifs. In an embodiment, the non-viral polynucleotide vectors can include one or more scaffold / matrix attachment regions (S / MARs). See e.g., Mirkovitch et al. 1984. Cell. 39:223-232, Wong et al. 2015. Adv. Genet. 89: 113-152, whose techniques and vectors can be adapted for use in the present disclosure. S / MARs are AT-rich sequences that play a role in the spatial organization of chromosomes through DNA loop base attachment to the nuclear matrix. S / MARs are often found close to regulatory elements such as promoters, enhancers, and origins of DNA replication. Inclusion of one or S / MARs can facilitate a once-per-cell-cycle replication to maintain the non- viral polynucleotide vector as an episome in daughter cells. In an embodiment, the S / MAR sequence is located downstream of an actively transcribed polynucleotide (e.g., one or more engineered AAV capsid polynucleotides or other polynucleotides or molecules of the present disclosure) included in the non-viral polynucleotide vector. In an embodiment, the S / MAR can be a S / MAR from the beta-interferon gene cluster. See e.g., Verghese et al. 2014. Nucleic Acid Res. 42:e53; Xu et al. 2016. Sci. China Life Sci. 59:1024-1033; Jin et al. 2016. 8:702-711; Koirala et al. 2014. Adv. Exp. Med. Biol. 801 :703-709; and Nehlsen et al. 2006. Gene Ther. Mol. Biol. 10:233-244, whose techniques and vectors can be adapted for use in the present disclosure.

[0147] In an embodiment, the non-viral vector is a transposon vector or system thereof. As used herein, “transposon” (also referred to as transposable element) refers to a polynucleotide sequence that is capable of moving form location in a genome to another. There are several classesof transposons. Transposons include retrotransposons and DNA transposons. Retrotransposons require the transcription of the polynucleotide that is moved (or transposed) in order to transpose the polynucleotide to a new genome or polynucleotide. DNA transposons are those that do not require reverse transcription of the polynucleotide that is moved (or transposed) in order to transpose the polynucleotide to a new genome or polynucleotide. In an embodiment, the non-viral polynucleotide vector can be a retrotransposon vector. In an embodiment, the retrotransposon vector includes long terminal repeats. In an embodiment, the retrotransposon vector does not include long terminal repeats. In an embodiment, the non-viral polynucleotide vector can be a DNA transposon vector. DNA transposon vectors can include a polynucleotide sequence encoding a transposase. In an embodiment, the transposon vector is configured as a non-autonomous transposon vector, meaning that the transposition does not occur spontaneously on its own. In some of these embodiments, the transposon vector lacks one or more polynucleotide sequences encoding proteins required for transposition. In an embodiment, the non-autonomous transposon vectors lack one or more Ac elements.

[0148] In an embodiment, a non-viral polynucleotide transposon vector system can include a first polynucleotide vector that contains the engineered AAV capsid polynucleotide(s) or other polynucleotides, or molecules of the present disclosure described herein flanked on the 5’ and 3’ ends by transposon terminal inverted repeats (TIRs) and a second polynucleotide vector that includes a polynucleotide capable of encoding a transposase coupled to a promoter to drive expression of the transposase. When both are expressed in the same cell the transposase can be expressed from the second vector and can transpose the material between the TIRs on the first vector (e.g., the engineered AAV capsid polynucleotide(s) or other polynucleotides or molecules of the present disclosure) and integrate it into one or more positions in the host cell’s genome. In an embodiment the transposon vector or system thereof can be configured as a gene trap. In an embodiment, the TIRs can be configured to flank a strong splice acceptor site followed by a reporter and / or other gene (e.g., one or more of the engineered AAV capsid polynucleotide(s) or other polynucleotides or molecules of the present disclosure) and a strong poly A tail. When transposition occurs while using this vector or system thereof, the transposon can insert into an intron of a gene and the inserted reporter or other gene can provoke a mis-splicing process and as a result it in activates the trapped gene.

[0149] Any suitable transposon system can be used. Suitable transposon and systems thereof can include Sleeping Beauty transposon system (Tcl / mariner superfamily) (see e.g., Ivies et al. 1997. Cell. 91(4): 501-510), piggyBac (piggyBac superfamily) (see e.g., Li et al. 2013 110(25): E2279-E2287 and Yusa et al. 2011. PNAS. 108(4): 1531-1536), Tol2 (superfamily hAT), Frog Prince (Tcl / mariner superfamily) (see e.g., Miskey et al. 2003 Nucleic Acid Res. 31(23):6873- 6881) and variants thereof.Chemical Carriers

[0150] In an embodiment, the engineered AAV capsid polynucleotide(s) or other polynucleotides or other molecules of the present disclosure described herein can be coupled to a chemical carrier. Chemical carriers that can be suitable for delivery of polynucleotides can be broadly classified into the following classes: (i) inorganic particles, (ii) lipid-based, (iii) polymer- based, and (iv) peptide based. They can be categorized as (1) those that can form condensed complexes with a polynucleotide (such as the engineered AAV capsid polynucleotide(s) of the present disclosure), (2) those capable of targeting specific cells, (3) those capable of increasing delivery of the polynucleotide or other molecules (such as the engineered AAV capsid polynucleotide(s) )of the present disclosure to the nucleus or cytosol of a host cell, (4) those capable of disintegrating from DNA / RNA in the cytosol of a host cell, and (5) those capable of sustained or controlled release. It will be appreciated that any one given chemical carrier can include features from multiple categories. The term “particle” as used herein, refers to any suitable sized particles for delivery of the compositions (including particles, polypeptides, polynucleotides, and other compositions described herein) present disclosure described herein. Suitable sizes include macro-, micro-, and nano-sized particles.

[0151] In an embodiment, the non-viral carrier can be an inorganic particle. In an embodiment, the inorganic particle, can be a nanoparticle. The inorganic particles can be configured and optimized by varying size, shape, and / or porosity. In an embodiment, the inorganic particles are optimized to escape from the reticulo endothelial system. In an embodiment, the inorganic particles can be optimized to protect an entrapped molecule from degradation. The suitable inorganic particles that can be used as non-viral carriers in this context can include, but are not limited to, calcium phosphate, silica, metals (e.g., gold, platinum, silver, palladium, rhodium, osmium, iridium, ruthenium, mercury, copper, rhenium, titanium, niobium, tantalum, and combinations thereof), magnetic compounds, particles, and materials, (e.g., supermagnetic iron oxide andmagnetite), quantum dots, fullerenes (e.g., carbon nanoparticles, nanotubes, nanostrings, and the like), and combinations thereof. Other suitable inorganic non-viral carriers are discussed elsewhere herein.

[0152] In an embodiment, the non-viral carrier can be lipid-based. Suitable lipid-based carriers are also described in greater detail herein. In an embodiment, the lipid-based carrier includes a cationic lipid or an amphiphilic lipid that is capable of binding or otherwise interacting with a negative charge on the polynucleotide to be delivered (e.g., such as an engineered AAV capsid polynucleotide of the present disclosure). In an embodiment, chemical non-viral carrier systems can include a polynucleotide (such as the engineered AAV capsid polynucleotide(s)) or other composition or molecule of the present disclosure) and a lipid (such as a cationic lipid). These are also referred to in the art as lipoplexes. Other embodiments of lipoplexes are described elsewhere herein. In an embodiment, the non-viral lipid-based carrier can be a lipid nano emulsion. Lipid nano emulsions can be formed by the dispersion of an immiscible liquid in another stabilized emulsifying agent and can have particles of about 200 nm that are composed of the lipid, water, and surfactant that can contain the polynucleotide to be delivered (e.g., the engineered AAV capsid polynucleotide(s) of the present disclosure). In an embodiment, the lipid-based non-viral carrier can be a solid lipid particle or nanoparticle.

[0153] In an embodiment, the non-viral carrier can be peptide-based. In an embodiment, the peptide-based non-viral carrier can include one or more cationic amino acids. In an embodiment, 35 to 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100 % of the amino acids are cationic. In an embodiment, peptide carriers can be used in conjunction with other types of carriers (e.g., polymer-based carriers and lipid-based carriers to functionalize these carriers). In an embodiment, the functionalization is targeting a host cell. Suitable polymers that can be included in the polymer- based non-viral carrier can include, but are not limited to, polyethylenimine (PEI), chitosan, poly (DL-lactide) (PLA), poly (DL-Lactide-co-glycoside) (PLGA), dendrimers (see e.g., US Pat. Pub. 2017 / 0079916 whose techniques and compositions can be adapted for use with the engineered AAV capsid polynucleotides of the present disclosure), polymethacrylate, and combinations thereof.

[0154] In an embodiment, the non-viral carrier can be configured to release an engineered delivery system polynucleotide that is associated with or attached to the non-viral carrier in response to an external stimulus, such as pH, temperature, osmolarity, concentration of a specificmolecule or composition (e.g., calcium, NaCl, and the like), pressure and the like. In an embodiment, the non-viral carrier can be a particle that is configured includes one or more of the engineered AAV capsid polynucleotides or other compositions of the present disclosure describe herein and an environmental triggering agent response element, and optionally a triggering agent. In an embodiment, the particle can include a polymer that can be selected from the group of polymethacrylates and polyacrylates. In an embodiment, the non-viral particle can include one or more embodiments of the compositions microparticles described in US Pat. Pubs. 20150232883 and 20050123596, whose techniques and compositions can be adapted for use in the present disclosure.

[0155] In an embodiment, the non-viral carrier can be a polymer-based carrier. In an embodiment, the polymer is cationic or is predominantly cationic such that it can interact in a charge-dependent manner with the negatively charged polynucleotide to be delivered (such as the engineered AAV capsid polynucleotide(s) of the present disclosure). Polymer-based systems are described in greater detail elsewhere herein.Viral Vectors

[0156] In an embodiment, the vector is a viral vector. The term of art “viral vector” and as used herein in this context refers to polynucleotide based vectors that contain one or more elements from or based upon one or more elements of a virus that can be capable of expressing and packaging a polynucleotide, such as an engineered AAV capsid polynucleotide, cargo, or other composition or molecule of the present disclosure, into a virus particle and producing said virus particle when used alone or with one or more other viral vectors (such as in a viral vector system). Viral vectors and systems thereof can be used for producing viral particles for delivery of and / or expression and / or generation of one or more compositions of the present disclosure described herein (including, but not limited to, any viral particle and associated cargo). The viral vector can be part of a viral vector system involving multiple vectors. In an embodiment, systems incorporating multiple viral vectors can increase the safety of these systems. Suitable viral vectors can include adenoviral-based vectors, adeno associated vectors, helper-dependent adenoviral (HdAd) vectors, hybrid adenoviral vectors, and the like. Other embodiments of viral vectors and viral particles produce therefrom are described elsewhere herein. In an embodiment, the viral vectors are configured to produce replication incompetent viral particles for improved safety of these systems.Adenoviral vectors. Helper-dependent Adenoviral vectors, and Hybrid Adenoviral Vectors

[0157] In an embodiment, the vector can be an adenoviral vector. In an embodiment, the adenoviral vector can include elements such that the virus particle produced using the vector or system thereof can be serotype 2, 5, or 9. In an embodiment, the polynucleotide to be delivered via the adenoviral particle can be up to about 8 kb. Thus, in an embodiment, an adenoviral vector can include a DNA polynucleotide to be delivered that can range in size from about 0.001 kb to about 8 kb. Adenoviral vectors have been used successfully in several contexts (see e.g., Teramato et al. 2000. Lancet. 355: 1911-1912; Lai et al. 2002. DNA Cell. Biol. 21 :895-913; Flotte et al., 1996. Hum. Gene. Ther. 7: 1145-1159; and Kay et al. 2000. Nat. Genet. 24:257-261. The vector can encode the engineered AAV capsids, said capsids forming adenoviral particles.

[0158] In an embodiment the vector can be a helper-dependent adenoviral vector or system thereof. These are also referred to in the field as “gutless” or “gutted” vectors and are a modified generation of adenoviral vectors (see e.g., Thrasher et al. 2006. Nature. 443:E5-7). In an embodiment of the helper-dependent adenoviral vector system one vector (the helper) can contain all the viral genes required for replication but contains a conditional gene defect in the packaging domain. The second vector of the system can contain only the ends of the viral genome, one or more engineered AAV capsid polynucleotides, and the native packaging recognition signal, which can allow selective packaged release from the cells (see e.g., Cideciyan et al. 2009. N Engl J Med. 361 :725-727). Helper-dependent Adenoviral vector systems have been successful for gene delivery in several contexts (see e.g., Simonelli et al. 2010. J Am Soc Gene Ther. 18:643-650; Cideciyan et al. 2009. N Engl J Med. 361 :725-727; Crane et al. 2012. Gene Ther. 19(4):443-452; Alba et al. 2005. Gene Ther. 12: 18-S27; Croyle et al. 2005. Gene Ther. 12:579-587; Amalfitano et al. 1998. J. Virol. 72:926-933; and Morral et al. 1999. PNAS. 96: 12816-12821). The techniques and vectors described in these publications can be adapted for inclusion and delivery of the engineered AAV capsid polynucleotides described herein. In an embodiment, the polynucleotide to be delivered via the viral particle produced from a helper-dependent adenoviral vector or system thereof can be up to about 38 kb. Thus, in an embodiment, an adenoviral vector can include a DNA polynucleotide to be delivered that can range in size from about 0.001 kb to about 37 kb (see e.g., Rosewell et al. 2011. J. Genet. Syndr. Gene Ther. Suppl. 5:001).

[0159] In an embodiment, the vector is a hybrid-adenoviral vector or system thereof. Hybrid adenoviral vectors are composed of the high transduction efficiency of a gene-deleted adenoviralvector and the long-term genome-integrating potential of adeno-associated, and transposon based- gene transfer. In an embodiment, such hybrid vector systems can result in stable transduction and limited integration site. See e.g., Balague et al. 2000. Blood. 95:820-828; Morral et al. 1998. Hum. Gene Ther. 9:2709-2716; Kubo and Mitani. 2003. J. Virol. 77(5): 2964-2971; Zhang et al. 2013. PloS One. 8(10) e76771; and Cooney et al. 2015. Mol. Ther. 23(4):667-674), whose techniques and vectors described therein can be modified and adapted for use in the engineered AAV capsid system of the present disclosure. In an embodiment, a hybrid-adenoviral vector can include one or more features of a retrovirus and / or an adeno-associated virus. In an embodiment the hybrid- adenoviral vector can include one or more features of a spuma retrovirus or foamy virus (FV). See e.g., Ehrhardt et al. 2007. Mol. Ther. 15:146-156 and Liu et al. 2007. Mol. Ther. 15:1834-1841, whose techniques and vectors described therein can be modified and adapted for use in the engineered AAV capsid system of the present disclosure. Advantages of using one or more features from the FVs in the hybrid-adenoviral vector or system thereof can include the ability of the viral particles produced therefrom to infect a broad range of cells, a large packaging capacity as compared to other retroviruses, and the ability to persist in quiescent (non-dividing) cells. See also e.g., Ehrhardt et al. 2007. Mol. Ther. 156: 146-156 and Shuji et al. 2011. Mol. Ther. 19:76-82, whose techniques and vectors described therein can be modified and adapted for use in the engineered AAV capsid system of the present disclosure.Adeno Associated Vectors

[0160] In an embodiment, the engineered vector or system thereof can be an adeno-associated vector (AAV). See, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); and Muzyczka, J. Clin. Invest. 94: 1351 (1994). Although similar to adenoviral vectors in some of their features, AAVs have some deficiency in their replication and / or pathogenicity and thus can be safer than adenoviral vectors. In an embodiment, the AAV can integrate into a specific site on chromosome 19 of a human cell with no observable side effects. In an embodiment, the capacity of the AAV vector, system thereof, and / or AAV particles can be up to about 4.7 kb. The AAV vector or system thereof can include one or more engineered capsid polynucleotides described herein.

[0161] The AAV vector or system thereof can be operably linked to a regulatory sequence, said regulatory sequence encoding one or more regulatory molecules. In an embodiment the regulatory molecules can be promoters, enhancers, repressors and the like, which are described ingreater detail elsewhere herein. In an embodiment, the AAV vector or system thereof can include one or more polynucleotides that can encode one or more regulatory proteins. In an embodiment, the promoter can be a tissue specific promoter as previously discussed. In an embodiment, the tissue specific promoter can drive expression of an engineered capsid AAV capsid polynucleotide described herein.

[0162] The AAV vector or system thereof can include one or more polynucleotides that can encode one or more capsid proteins, such as the engineered AAV capsid proteins described elsewhere herein. The engineered capsid proteins can be capable of assembling into a protein shell (an engineered capsid) of the AAV virus particle. The engineered capsid can have a cell-, tissue-, and / or organ-specific tropism.

[0163] In an embodiment, the AAV vector or system thereof can include one or more adenovirus helper factors or polynucleotides that can encode one or more adenovirus helper factors. Such adenovirus helper factors can include, but are not limited, El A, E1B, E2A, E4ORF6, and VA RNAs. In an embodiment, a producing host cell line expresses one or more of the adenovirus helper factors.

[0164] The AAV vector or system thereof can be configured to produce AAV particles having a specific serotype. In an embodiment, the serotype can be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9 or any combinations thereof. In an embodiment, the AAV can be AAV1, AAV2, AAV5, AAV9 or any combination thereof. One can select the AAV of the AAV with regard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5, 9 or a hybrid capsid AAV1, AAV2, AAV5, AAV9 or any combination thereof for targeting brain and / or neuronal cells; and one can select AAV4 for targeting cardiac tissue; and one can select AAV8 for delivery to the liver. Thus, in an embodiment, an AAV vector or system thereof capable of producing AAV particles capable of targeting the brain and / or neuronal cells can be configured to generate AAV particles having serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof. In an embodiment, an AAV vector or system thereof capable of producing AAV particles capable of targeting cardiac tissue can be configured to generate an AAV particle having an AAV4 serotype. In an embodiment, an AAV vector or system thereof capable of producing AAV particles capable of targeting the liver can be configured to generate an AAV having an AAV8 serotype. See also Srivastava. 2017. Curr. Opin. Virol. 21 :75-80.

[0165] The nucleotide sequences of the genomes of the AAV serotypes are known in the art. For example, the complete genome of AAV1 is provided in GenBank Accession No. NC 002077; the complete genome of AAV2 is provided in GenBank Accession No. NC 001401 and Srivastava et al., J. Virol., 45: 555-564 (1983); the complete genome of AAV3 is provided in GenBank Accession No. NC 1829; the complete genome of AAV4 is provided in GenBank Accession No. NC 001829: the AAV5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV6 is provided in GenBank Accession No. NC 00 1862; at least portions of AAV 7 and AAV8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively; the AAV9 genome is provided in Gao et al., J. Virol., 78; 6381-6388 (2004); tlie AAV10 genome is provided in Mol. Then, 13(1): 67-76 (2006); the AAV11 genome is provided in Virology, 330(2): 375-383 (2004); AAV PHP.B is described by Deverman et al., Nature Biotech. 34(2), 204-209 and its sequence deposited under GenBank Accession No. KU056473.1. Exemplary reviews of AAV serotypes may be found in Choi et al (2005) Curr Gene Ther 5(3); 299-310 and Wu et al (2006) Molecular Therapy 14(3), 316-327.

[0166] It will be appreciated that while the different serotypes can provide some level of cell, tissue, and / or organ specificity, each serotype still is multi-tropic and thus can result in tissuetoxicity if using that serotype to target a tissue that the serotype is less efficient in transducing. Thus, in addition to achieving some tissue targeting capacity via selecting an AAV of a particular serotype, it will be appreciated that the tropism of the AAV serotype can be modified by an engineered AAV capsid described herein. As described elsewhere herein, variants of wild-type AAV of any serotype can be generated via a method described herein and determined to have a particular cell-specific tropism, which can be the same or different as that of the reference wildtype AAV serotype. In an embodiment, the cell, tissue, and / or specificity of the wild-type serotype can be enhanced (e.g., made more selective or specific for a particular cell type that the serotype is already biased towards). For example, wild-type AAV9 is biased towards muscle and brain in humans (see e.g., Srivastava. 2017. Curr. Opin. Virol. 21 :75-80.) By including an engineered AAV capsid and / or capsid protein variant of wild-type AAV9 as described herein, the bias for e.g., brain can be reduced or eliminated and / or the septicity increased such that the brain specificity appears reduced in comparison, thus enhancing the specificity for the muscle as compared to the wild-type AAV9. As previously mentioned, inclusion of an engineered capsid and / or capsid protein variant of a wild-type AAV serotype can have a different tropism than the wild-type reference AAVserotype. For example, an engineered AAV capsid and / or capsid protein variant of AAV9 can have specificity for a tissue other than muscle or brain in humans.

[0167] In an embodiment, the AAV vector is a hybrid AAV vector or system thereof. Hybrid AAVs are AAVs that include genomes with elements from one serotype that are packaged into a capsid derived from at least one different serotype. For example, if it is the rAAV2 / 5 that is to be produced, and if the production method is based on the helper-free, transient transfection method discussed below, the 1st plasmid and the 3rd plasmid (the adeno helper plasmid) will be the same as discussed for rAAV2 production. However, the 2nd plasmid, the pRepCap will be different. In this plasmid, called pRep2 / Cap5, the Rep gene is still derived from AAV2, while the Cap gene is derived from AAV5. The production scheme is the same as the above-mentioned approach for AAV2 production. The resulting rAAV is called rAAV2 / 5, in which the genome is based on recombinant AAV2, while the capsid is based on AAV5. It is assumed the cell or tissue-tropism displayed by this AAV2 / 5 hybrid virus should be the same as that of AAV5. It will be appreciated that wild-type hybrid AAV particles suffer the same specificity issues as with the non-hybrid wildtype serotypes previously discussed.

[0168] Advantages achieved by the wild-type based hybrid AAV systems can be combined with the increased and customizable cell-specificity that can be achieved with the engineered AAV capsids can be combined by generating a hybrid AAV that can include an engineered AAV capsid described elsewhere herein. It will be appreciated that hybrid AAVs can contain an engineered AAV capsid containing a genome with elements from a different serotype than the reference wildtype serotype that the engineered AAV capsid is a variant of. For example, a hybrid AAV can be produced that includes an engineered AAV capsid that is a variant of an AAV9 serotype that is used to package a genome that contains components (e.g., AAV2 ITRs) from an AAV2 serotype. As with wild-type based hybrid AAVs previously discussed, the tropism of the resulting AAV particle will be that of the engineered AAV capsid.

[0169] A tabulation of certain wild-type AAV serotypes as to these cells can be found in Grimm, D. et al, J. Virol. 82: 5887-5911 (2008) reproduced below as Table C. Further tropism details can be found in Srivastava. 2017. Curr. Opin. Virol. 21 :75-80 as previously discussed.

[0170] In an embodiment, the AAV vector or system thereof is AAV rh.74 or AAV rh.10.

[0171] In an embodiment, the AAV vector or system thereof is configured as a “gutless” vector, similar to that described in connection with a retroviral vector. In an embodiment, the “gutless” AAV vector or system thereof can have the cis-acting viral DNA elements involved in genome amplification and packaging in linkage with the heterologous sequences of interest (e.g., a transgene encoding a therapeutic protein or nucleic acid of interest)).Table D: Example virus sequences - see FIG. 21 for an example alignment of the example virus sequences.Vectors Encoding the Transsene

[0172] 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. Forexample, 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 forms the essence of all AAV vectors.

[0173] In an embodiment, additional modifications may be implemented to further increase the efficacy of the AAV. 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.

[0174] 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 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 encapsulated into the virion shell. Wild-type AAVs package either a single plus-strand or minus-strand DNA. The modified scAAV intermediates are delivered to the nucleus, these plus and minus strands instantaneously anneal to form dsDNA.

[0175] In an embodiment, the cis-elements are optimized for targeted delivery. The ciselements are optimized because the packaging capacity of AAVs is restricted. In an embodiment, small cis-elements replace long promoter sequences for the delivery of large therapeutic transgenes (e.g., 4.4-4.5 kbs).

[0176] 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 the two or more fragments.

[0177] 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 producesthe transgene cassette. In an embodiment, overlapping fragments may be added to the end of the individual AAV vectors to encourage homologous recombination.

[0178] In example approach 3, a hybrid dual-vector incorporates an overlapping region with intron splice sites in the split vector transgenes. Approach 3 uses 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' splicing elements, respectively), and splicing provide the appropriate transgene protein. This strategy may increase the expression of full functional protein.

[0179] In example approach 4, an AAV genome is cross-packaged into the capsids of other parvoviruses thus creating chimeric vectors. In example approach 5, intein-mediated protein transsplicing is used. Intein catalyzes protein splicing thereby causing the ligation of two polypeptides via trans-splicing (this approach is similar to intron-mediated RNA splicing). Multiple AAV vectors are delivered to the same cells. Each of the AAV vectors encode one of the fragments of target proteins, the fragments are flanked by short split inteins. 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

[0180] 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 to the vector(s) described herein. Suitable recombination and / or cloning techniques and / or methods can include, but not limited to, those described in U.S. Application publication No. US 2004-0171156 Al. Other suitable methods and techniques are described elsewhere herein.

[0181] Construction of recombinant AAV vectors is described in a number of 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.

[0182] 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, oneor 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.

[0183] Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of an engineered AAV capsid system described herein 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.Virus Particle Production from Viral VectorsAAV Particle Production

[0184] There are two main strategies for producing AAV particles from AAV vectors and systems thereof, such as those described herein, which depend on how 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 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 skill in the art will appreciate various methods and variations thereof that are both helper and -helper free and as well as the different advantages of each system.

[0185] As used herein, “trans-splicing” means joining a first RNA molecule containing one or more exons (e.g., exogenous exons or exons that are part of a CDS of a trans-splicing molecule) to a second RNA molecule (e.g., a pre-mRNA molecule, e.g., an endogenous pre-mRNA molecule) and replacing a portion of the second RNA molecule with a portion of the first RNA molecule through a spliceosome-mediated mechanism.

[0186] A “nucleic acid trans-splicing molecule” or “trans-splicing molecule” has three main elements: (a) a binding domain that confers specificity by tethering the trans-splicing molecule toits target gene (e.g., pre-mRNA); (b) a splicing domain (e.g., a splicing domain having a 3' or 5' splice site); and (c) a CDS configured to be trans-spliced onto the target nucleic acid, which can replace one or more exons in the target nucleic acid (e.g., one or more mutated exons). A “pre- mRNA trans-splicing molecule” or “RTM” refers to a nucleic acid trans-splicing molecule that targets pre-mRNA. The terms “nucleic acid trans-splicing molecule” and “trans-splicing molecule” refer to both (1) DNA that encodes RNA, wherein the RNA transcript is the effector molecule that physically binds the target pre-mRNA, and (2) the RNA transcript itself.

[0187] Trans-splicing methodology is described in detail, for example, in published U.S. Patent Application No. 2024 / 0091381.

[0188] The engineered AAV vectors and systems thereof described herein can be produced by any of these methods.Vector and Virus Particle Delivery

[0189] A vector (including non-viral carriers) described herein can be introduced into host cells to thereby produce 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).

[0190] AAV capsids prepared from one or more engineered AAV capsid polynucleotides can be used to deliver a recombinant AAV 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 examples, 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.

[0191] 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 ofadministration 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.

[0192] 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.

[0193] 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.

[0194] 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 beselected 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.Engineered Virus Particles Including an Engineered Viral (e.g., AAV) Capsid

[0195] Also described herein are engineered virus particles (also referred to here and elsewhere herein as “engineered viral particles” that can contain an engineered viral capsid (e.g., AAV capsid, referred to as “engineered AAV particles”) as described in detail elsewhere herein. It will be appreciated that the engineered AAV particles can be adenovirus-based particles, helper adenovirus-based particles, AAV-based particles, or hybrid adenovirus-based particles that contain at least one engineered AAV capsid proteins as previously described. An engineered AAV capsid is one that that contains one or more engineered AAV capsid proteins as are described elsewhere herein. In an embodiment, the engineered AAV particles can include 1-60 engineered AAV capsid proteins described herein. In an embodiment, the engineered AAV particles 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 particles can contain 0-59 wild-type AAV capsid proteins. In an embodiment, the engineered AAV particles 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 wild-type AAV capsid proteins. The engineered AAV particles can thus include one or more CD59 targeting moieties as is previously described.

[0196] The engineered AAV particle can include one or more cargo polynucleotides. 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.Example Cargos

[0197] The CD59 targeting moieties can be coupled to or otherwise associated with a cargo. Cargos can include any molecule that is capable of being coupled to or associated with the CD59 targeting moieties described herein. 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, and thelike), 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, antiinflammatories, 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.

[0198] In an embodiment, the cargo is capable of treating or preventing a CNS disease or disorder, details of which are described herein.

[0199] In an embodiment, the cargo is a morpholino, a peptide-linked morpholino, an antisense oligonucleotide, a PMO, a therapeutic transgene, a polynucleotide encoding a therapeutic polypeptide or peptide, a 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.

[0200] In an embodiment, one or more CD59 targeting moieties described herein is directly attached to the cargo. In an embodiment, one or more CD59 targeting moieties described herein is indirectly coupled to the cargo, such as via a linker molecule. In an embodiment, one or more one or more CD59 targeting moieties described herein is coupled to associated with a polypeptide or other particle that is coupled to, attached to, encapsulates, and / or contains a cargo.

[0201] Exemplary particles include, without limitation, viral particles (e.g., viral capsids, which is inclusive of bacteriophage capsids), polysomes, liposomes, nanoparticles, microparticles, exosomes, micelles, and the like. The term “nanoparticle” as used herein includes a nanoscale deposit of a homogenous or heterogeneous material. Nanoparticles may be regular or irregular in shape and may be formed from a plurality of co-deposited particles that form a composite nanoscale particle. Nanoparticles may be generally spherical in shape or have a composite shape formed from a plurality of co-deposited generally spherical particles. Exemplary shapes for the nanoparticles include, but are not limited to, spherical, rod, elliptical, cylindrical, disc, and the like. In an embodiment, the nanoparticles have a substantially spherical shape.Cargo Polynucleotides

[0202] 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 capsid containing a CD59 targeting moiety of the present disclosure. The cargo polynucleotides can be packaged into an engineered viral (e.g., AAV) particle, which can be delivered to, e.g., a cell. In an embodiment, the cargo polynucleotide can be capable of modifying a polynucleotide (e.g., 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) techniques.

[0203] In an embodiment, the cargo molecule is a polynucleotide that is or can encode a vaccine. In an embodiment, the cargo molecule is a polynucleotide encoding an antibody.RNA Interference Agents

[0204] In an embodiment, the one or more polynucleotides may encode one or more RNA interference agents. RNA interference agents are RNA molecules capable of suppressing gene expressions. Examples of RNA interference agentsinclude, but are not limited to, small interfering RNAs (siRNA), microRNAs (miRNA), and short hairpin RNAs (shRNA).

[0205] In an embodiment, the interference RNA may be a siRNAs. Small interfering RNA (siRNA) molecules are capable of inhibiting target gene expression by interfering RNA. siRNAs may be chemically synthesized, or may be obtained by in vitro transcription, or may be synthesized in vivo in 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. Lengthof protuberant region is independent from total length of siRNA molecule. siRNAs may act by post-transcriptional degradation or silencing of 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.

[0206] In an embodiment, the cargo polynucleotide is an RNAi molecule, antisense molecule, and / or a gene silencing oligonucleotide or a polynucleotide that encodes an RNAi molecule, antisense molecule, and / or gene silencing oligonucleotide.

[0207] 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, 42 41, 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.

[0208] 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

[0209] In an embodiment, the cargo molecule can be a polynucleotide or polypeptide 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. 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, morpholines, 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.

[0210] In an embodiment, the cargo molecule is or encodes a gene editing system or component thereof. In an embodiment, the cargo molecule is or 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.

[0211] In an embodiment, the cargo molecule can be a polynucleotide or polypeptide 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 (suchas single stranded RNA viruses). 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 it treats or prevents a neurological disease or disorder described further herein.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] 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

[0217] In an embodiment, the cargo molecule may one or more polypeptides or may be a nucleic acid 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 using the engineered delivery vesicles described herein, including but not limited to, secretory proteins, immunomodulatory proteins, anti-fibrotic proteins, proteins that promotetissue regeneration and / or transplant survival 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

[0218] 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 non-antibody 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.

[0219] In an embodiment, the antibody is a fragment or portion thereof. In an 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 by recombinant DNAtechniques, 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.

[0220] 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).

[0221] 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.

[0222] 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 one or moreserine 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.

[0223] 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 singledomain 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. Nonlimiting examples of camelids include dromedaries, camels, llamas and alpacas.

[0224] 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 ).

[0225] 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.

[0226] 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 “ingle-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.

[0227] 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.

[0228] 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.

[0229] 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).

[0230] 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., LACI-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).

[0231] “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 107M1(or a dissociation coefficient of I pM 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.

[0232] 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.

[0233] 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.

[0234] 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).

[0235] 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.

[0236] 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 antibody agonistsand 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., I. 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).

[0237] 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

[0238] 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 etal., 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

[0239] 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.

[0240] 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, LB, I-C, I-D, LE, I-Fl, I-F2, I-F3, and IG). Makarova et al., 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, IILB, III-C, III-D, IILE, and IILF). 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 LA, LB, LE, LF and LU 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 LB 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.

[0241] 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 an embodiment, 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: ILA, ILB, 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-B1, VI-B2, VI-C, and VI-D.OMEGA nucleases

[0242] 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

[0243] 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 an 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 an 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

[0244] 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 OMEGA RNA guided nickase. In one embodiment, the OMEGA 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 oneembodiment, 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

[0245] 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 noRuvC domain. In one embodiment, the IshB, or IscB homolog, comprises a PLMP domain and an HNH domain, but does not comprise a RuvC domain.

[0246] 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

[0247] 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.

[0248] 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 an 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 an embodiment, the RuvC-III sub-domain forms the C-terminus of the TnpB nuclease.

[0249] 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, AU cyclobacillus macrosprangiidus strain DSM 17980, Lipingzhangella halophila strain DSM 102030, or Ktedonobacter recemifer. In oneembodiment, the TnpB nuclease is from Ktedonobacter racemifer, or comprises a conserved RNA region with similarity to the 5’ ITR of K. racemifer TnpB loci.Fanzor nuclease

[0250] 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.

[0251] 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 an embodiment, Fanzor 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 an embodiment, the RuvC-III sub-domain forms the C-terminus of the Fanzor nuclease.Engineered Cells and Organisms Expressing said Engineered AAV capsids

[0252] 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.

[0253] 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

[0254] 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 with modification 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.

[0255] 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.

[0256] 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.

[0257] 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, Psedoalter monas, Stenotrophamonas, and Streptomyces Suitable bacterial cells include, but are not limited to Escherichia coli cells, Caulobacter crescentus cells, Rodhobacter sphaeroides cells, Psedoalter monas 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, UMS 174, NovaBlue(DE3), BLR, C41(DE3), C43(DE3), Lemo21(DE3), Shuffle T7, ArcticExpress and ArticExpress (DE3).

[0258] 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, BcLl, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB / 3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mousefibroblasts, 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-IR, CHO-K1, CHO-K2, CHO-T, CHO DhfrL23 / 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, MONO-MAC 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-1 A / 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.)).

[0259] 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.

[0260] 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.

[0261] 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 laboratory and 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., Yarrowialipolytica), 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 fdamentous 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).

[0262] 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.

[0263] 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.

[0264] 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 strain S228C 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 cellI l lwhose 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.

[0265] 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 a 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.

[0266] 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.

[0267] 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.

[0268] 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 stored cells 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.

[0269] In some aspects, the techniques described herein relate to a method of manufacturing a recombinant engineered AAV including culturing mammalian cells comprising; (1) a polynucleotide encoding the engineered AAV capsid polypeptide of any one of any of thosedescribed herein; (2) a polynucleotide encoding a recombinant AAV genome including a transgene operably linked to a regulatory sequence and flanked by AAV ITR sequences, and optionally (3) a polynucleotide encoding adenoviral helper genes, under conditions sufficient for the production of recombinant engineered AAV particles; and recovering the recombinant engineered AAV particles from said culture. See e g., Grieger, J. C., Soltys, S. M. & Samulski, R. J. Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector From the Culture Media for GMP FIX and FLT1 Clinical Vector. Mol. Ther. 24, 287-297 (2016), hereby incorporated by reference.Formulations

[0270] 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 used to generate polypeptides and other particles that include one or more CNS-specific CD59 targeting moieties described herein. 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.

[0271] 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 bodyweight 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 105, 1 x 106, 1 x 107, 1 x 108, 1 x 109, 1 x IO10or 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 IO10or more cells per nL, pL, mL, or L.

[0272] 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 105, 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 IO17, 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 105, 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

[0273] 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.

[0274] 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.

[0275] In addition to an amount of one or more of the polypeptides, polynucleotides, vectors, cells, engineered AAV capsid particles, nanoparticles, other delivery particles, and combinationsthereof 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.

[0276] 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-0, 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).

[0277] 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.

[0278] 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.

[0279] 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.

[0280] 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).

[0281] 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)

[0282] 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.

[0283] 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, nelfmavir, 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, trovafloxacin, and sparfloxacin), sulfonamides (e.g., sulfamethoxazole / trimethoprim, sulfasalazine, and sulfisoxazole), tetracyclines (e.g., doxycycline, demeclocycline, 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).

[0284] 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.

[0285] 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, and combinations 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 an embodiment, 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

[0286] 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, intracavernous, intrathecal, intravitreal, intracerebral, gingival, subgingival, intracerebroventricular, and intradermal. Such formulations may be prepared by any method known in the art.

[0287] For delivery to the CNS, the AAV formulations described herein may be administered intra-parenchymally, intrathecally, intracerebroventricularly, intraci sternally, intravenously, into the carotid artery, systemically or a combination of these. In an embodiment, the AAV formulation is administered by intrathecally in equal portions to the cistema magna and the lumbar spine.

[0288] In an embodiment, 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).

[0289] In an embodiment, 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 an embodiment, rAAV particles may be administered to multiple locations, for example, 1, 2, 3, 4, or 5 locations simultaneously or staggered over time.

[0290] In an embodiment, the more than one administration may include immuno-suppression 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.

[0291] 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.

[0292] 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.

[0293] 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 are well 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.

[0294] Methods of delivering an AAV preparation to a specific region of the CNS 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.

[0295] 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 multiunit 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.

[0296] 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 orembedding 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).

[0297] 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.

[0298] 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 nonaqueous 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.

[0299] 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 an embodiment, 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 (amount and / 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.

[0300] 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 an embodiment, 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.

[0301] 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.

[0302] 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.

[0303] 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

[0304] 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 the pharmaceutical 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 transducing the CNS.

[0305] 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, mL, or L or more, such as about IxlO1, IxlO2, IxlO3, IxlO4, IxlO5, 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 1X101transforming 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, IxlO15, 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.

[0306] 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 kilogrambodyweight (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, IxlO1’, 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 I x lO12vg / kg to 10 x 1012vg / kg. In an embodiment, the dosage is 5 x 1012vg / kg.

[0307] 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 O 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.

[0308] 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.

[0309] 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.

[0310] 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.

[0311] 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.

[0312] 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, ...

Claims

CLAIMSWhat is claimed is:

1. An engineered adeno-associated virus (AAV) capsid polypeptide comprising a CD59 targeting moiety defined by a n-mer of the formula X1-X2-X3-X4-X5-X6-X7, inserted at any position between 450-461 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype, and wherein Xi, X2, X3, X4, X5, Xe, and X7 each represent an amino acid inserted into the capsid polypeptide.

2. The engineered AAV capsid polypeptide of claim 1, further comprises first removing one or more amino acids at any position between 450-461 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype.

3. The engineered AAV capsid polypeptide of claim 2, wherein amino acids at position 451- 460 are removed.

4. The engineered AAV capsid polypeptide of any one of the preceding claims, further comprises removing the amino acid at position 449 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype and inserting a new amino acid.

5. The engineered AAV capsid polypeptide of claim 4, wherein the new amino acid is arginine.

6. The engineered AAV capsid polypeptide of any one of the preceding claims, wherein the n-mer is of the formula having an amino acid sequence of EFNNGSD (SEQ ID NO: 89) or GAASLMP (SEQ ID NO: 109).

7. The engineered AAV capsid polypeptide of any of the preceding claims, wherein the AAV9 capsid polypeptide comprises a K449R mutation, or at an analogous position of a capsid polypeptide of a different AAV serotype.

8. The engineered AAV capsid polypeptide of any of the preceding claims, wherein the targeting moiety comprises any one of the amino acid sequences of SEQ ID NO: 89-5983.

9. The engineered AAV capsid polypeptide of any of the preceding claims, wherein the targeting moiety is selected from any one of the amino acid sequences listed in Table A, or any combination thereof.

10. The engineered AAV capsid polypeptide of any of the preceding claims, wherein the capsid polypeptide comprises a VP1, VP2, or VP3 polypeptide, or a combination thereof.

11. The engineered AAV capsid polypeptide of any of the preceding claims, wherein the different AAV serotype comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rhlO.

12. An engineered AAV particle comprising the engineered AAV capsid polypeptide of any one of the preceding claims, and further comprising a recombinant AAV genome configured to express a transgene.

13. The engineered AAV particle of claim 12, wherein the transgene encodes a therapeutic polypeptide, an antibody or fragment thereof, an siRNA, a CRISPR-Cas system, a Transcription Activatordike Effector (TALE)- or Zinc Finger Protein (ZFP)-based transcriptional activator; repressor; or an epigenomic silencer, 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.

14. The engineered AAV particle of claims 12 or 13, wherein the transgene is operably linked to a regulatory sequence that promotes expression in a nervous system.

15. A pharmaceutical composition comprising the recombinant engineered AAV particle of any one of claims 12 to 14 and an acceptable carrier.

16. A method of delivering a polypeptide or polynucleotide to the central nervous system (CNS) of a subject comprising administering the pharmaceutical composition of claim 15 to the subject.

17. The method of claim 16, wherein the pharmaceutical composition is administered systemically or directly to the CNS.

18. A method of manufacturing a recombinant engineered AAV, said method comprising: culturing mammalian cells comprising;(1) a polynucleotide encoding the engineered AAV capsid polypeptide of any one of claims 1 to 11,(2) a polynucleotide encoding a recombinant AAV genome comprising a transgene operably linked to a regulatory sequence and flanked by AAV ITR sequences, and optionally(3) a polynucleotide encoding adenoviral helper genes, under conditions sufficient for production of recombinant engineered AAV particles; and recovering the recombinant engineered AAV particles from said culture.

19. A cultured host cell containing a recombinant nucleic acid molecule encoding the engineered AAV capsid polypeptide of any one of claims 1 to 11.

20. An AAV library comprising a population of variant engineered recombinant AAV particles comprising a variant recombinant AAV capsid polypeptide with a modification comprising a targeting moiety defined by a n-mer of the formula X1-X2-X3-X4-X5-X6-X7, inserted at any position between 450-461 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype, and wherein Xi, X2, X3, X4, X5, Xe, and X7 represent an amino acid inserted into the capsid polypeptide, wherein the modification has been selected for binding of a CD59 protein and / or increased tropism for the CNS relevant to a reference AAV particle without the modification, and optionally, further comprises first removing one or more amino acids at any position between 450-461 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptide of another AAV serotype and optionally further comprises removing the amino acid at position 449 of an AAV9 capsid polypeptide, or in an analogous position of a capsid polypeptide of a different AAV serotype and inserting a new amino acid.

21. The AAV library of claim 20, wherein the different AAV serotype comprises AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rh.10.

22. A method of screening an AAV library for a recombinant AAV particle that binds a CD59 protein and / or has increased tropism for the CNS, said method comprising assaying the AAV library of claim 20 for increased binding to a CD59 protein and / or tropism for the CNS relative to an AAV vector with a reference capsid, and selecting those recombinant AAV vectors that have increased binding of the CD59 protein and / or enhanced tropism for the CNS.

23. A method for training a machine learning algorithm comprising: a. receiving, by at least one computing device, a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein; b. training, by at least one computing device, with the plurality of AAV capsid polypeptide sequences comprising a modification for binding to the CD59 protein, a CD59 protein targeting machine learning model; and c. deploying, by at least one computing device, the CD59 protein targeting machine learning algorithm.

24. The method of claim 23, wherein the CD59 protein targeting machine learning model is trained to identify one or more sequences from the plurality of sequences having increased binding to the CD59 protein.

25. The method of any one of claims 23-24, wherein the CD59 protein targeting machine learning model is trained to identify one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.

26. The method of any one of claims 23-25, wherein the targeting moiety defined by a n-mer of the formula X1-X2-X3-X4-X5-X6-X7, and wherein Xi, X2, X3, X4, X5, Xe, and X7 represent an amino acid inserted into at an amino acid position in the capsid polypeptide.

27. The method of any one of claims 23-26, wherein the training comprises unsupervised learning, supervised learning, semi-supervised learning, reinforcement learning, transfer learning, incremental learning, curriculum learning, learning to learn, or contrastive learning.

28. The method of any one of claims 23-27, wherein the CD59 protein targeting machine learning model comprises linear classifiers, logistic classifiers, random forest, artificial neural networks, matrix factorization, support vector machines, K-means clustering, or K- nearest neighbor.

29. The method of any one of claims 23-28, wherein the CD59 protein targeting machine learning model comprises Boltzmann machines, Bayesian networks, autoregressive models, variational autoencoders (VAEs), diffusion models, energy-based models, flowbased models, generative adversarial networks (GANs), mixture models, hidden Markov models, or large language models (LLMs).

30. The method of any one of claims 23-29, wherein the CD59 protein targeting machine learning model comprises convolutional neural networks (CNNs), recurrent neural networks (RNNs), long short-term memory models (LSTMs), gated recurrent units (GRUs), capsule networks, attention mechanisms, or transformer networks.

31. The method of any one of claims 23-30, the CD59 protein targeting machine learning model is pre-trained, and further trained to predict CD59 protein targeting by a plurality of sequences.

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