[go: up one dir, main page]

WO2025217163A2 - Nouveaux capsides aav se liant à cd59 humain - Google Patents

Nouveaux capsides aav se liant à cd59 humain

Info

Publication number
WO2025217163A2
WO2025217163A2 PCT/US2025/023652 US2025023652W WO2025217163A2 WO 2025217163 A2 WO2025217163 A2 WO 2025217163A2 US 2025023652 W US2025023652 W US 2025023652W WO 2025217163 A2 WO2025217163 A2 WO 2025217163A2
Authority
WO
WIPO (PCT)
Prior art keywords
aav
engineered
capsid
aavhu
aavrh
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/US2025/023652
Other languages
English (en)
Other versions
WO2025217163A3 (fr
Inventor
Jason Wu
Nuria BOTTICELLO-ROMERO
Benjamin DEVERMAN
Ken Chan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Broad Institute Inc
Original Assignee
Broad Institute Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Broad Institute Inc filed Critical Broad Institute Inc
Publication of WO2025217163A2 publication Critical patent/WO2025217163A2/fr
Publication of WO2025217163A3 publication Critical patent/WO2025217163A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14145—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/40—Vectors comprising a peptide as targeting moiety, e.g. a synthetic peptide, from undefined source

Definitions

  • 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.
  • CNS central nervous system
  • at least one of the capsid’s proteins is modified to include an n-mer motif.
  • AAVs adeno-associated viruses
  • capsid engineering has largely focused on targeting specific organs such as the central nervous system (CNS), liver, retina, or muscle.
  • CNS central nervous system
  • Applicants used recently 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.
  • BI306 sequence 1.1
  • BI309 sequence 2.1
  • AAV-PHP.eB a mouse CNS-targeting capsid
  • This enhanced tropism was absent in transgenic mice treated with wild type AAV9 or in mice not expressing the human receptor.
  • 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.
  • 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.
  • AAV engineered adeno- associated virus
  • 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 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. 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).
  • the AAV9 capsid polypeptide includes a K449R mutation, or at an analogous position of a capsid polypeptide of another AAV serotype.
  • 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.
  • the capsid polypeptide includes a VP1, VP2, or VP3 polypeptide, or a combination thereof.
  • the other AAV serotype includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rhlO.
  • 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.
  • 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.
  • the transgene is operably linked to a regulatory sequence that promotes expression in the nervous system.
  • the techniques described herein relate to pharmaceutical composition
  • pharmaceutical composition comprising the recombinant engineered AAV particle of any one of any of those described herein and an acceptable carrier.
  • 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.
  • CNS central nervous system
  • the techniques described herein relate to a method, wherein the pharmaceutical composition is administered systemically or directly to the CNS.
  • 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.
  • 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.
  • 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
  • the other AAV serotype includes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV rh.74, or AAV rh.10.
  • 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.
  • 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.
  • 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.
  • 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.
  • the training includes unsupervised learning, supervised learning, semi-supervised learning, reinforcement learning, transfer learning, incremental learning, curriculum learning, learning to learn, or contrastive learning.
  • 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.
  • 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).
  • 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.
  • CNNs convolutional neural networks
  • RNNs recurrent neural networks
  • LSTMs long short-term memory models
  • GRUs gated recurrent units
  • capsule networks attention mechanisms, or transformer networks.
  • the CD59 protein targeting machine learning model is pre-trained, and further trained to predict CD59 protein targeting by a plurality of sequences.
  • FIG. 1A-1B Receptor-dependent organ targeting of engineered AAVs.
  • 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.
  • FIG. 2 - BI306 transduces CHO cells stably expressing the human or macaque orthologs of the target receptor more efficiently than AAV9.
  • 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).
  • 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.
  • FIG. 5 Library performance in vivo of hCD59-binding capsids:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 with lei 1 vg of hCD59-binding AAVs intravenously, and expression of luciferase and mScarlet was evaluated 2-3 weeks following the injection.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • miR122BS is a binding site for microRNA-122 to down-regulate transgene mRNA in the liver.
  • mice were dosed with lei 1 vg of Capsid l.LCAG- NLS-mScarlet-P2A-Luc.
  • NLS is a nuclear localization sequence
  • P2A-Luc encodes for a self cleaving peptide followed by luciferase. Expression of mScarlet was evaluated three weeks following the second injection.
  • FIG. 19 In vivo validation of cross-species affinity of hCD59 binding capsid 1.1:
  • 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.
  • 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.
  • FIG. 20A-20C Human receptor-targeted AAVs efficiently transduced the brain and muscle but were de-targeted from the liver in transgenic mice.
  • 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).
  • FIG. 22 (SEQ ID NO: 6028-6040) CD59 targeting moiety - example insertions between residues 558 and 559 AAV9 VP1.
  • FIG. 23 (SEQ ID NO: 1, 6041-6051) Example serotype sequence alignment.
  • FIG. 24 - (SEQ ID NO: 1, 6052-6054) CD59 binding moiety insertion site in AAV9
  • 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,
  • subject refers 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.
  • 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.
  • “at least one of A and 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.
  • 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.
  • 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 more symptom(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.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
  • a transgene expression vector 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.
  • a transgene expression vector 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.
  • operably linked refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments.
  • the term refers to the functional relationship of a transcriptional regulatory sequence and a transgene to be transcribed.
  • 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.
  • 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.
  • 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.
  • recombinant refers to nucleic acids or polypeptides that are genetically engineered.
  • a "transgene” is a polynucleotide sequence that may encode an RNA (mRNA) that is translated into protein.
  • a transgene may comprise a cDNA sequence.
  • 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).
  • the transgene nucleotide sequence may comprise one or more introns.
  • the transgene can be polycistronic (e.g., two coding regions separated by internal ribosome entry site (IRES)).
  • a transgene may encode more than one protein.
  • 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).
  • 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.
  • capsid mutants 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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, embodiments disclosed 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions 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.
  • the CD59 targeting moiety binds to a CD59 polypeptide.
  • 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.
  • the n-mer is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids in length.
  • the n-mer motif has a length of at least 7 amino acids.
  • a composition comprising a targeting moiety effective to increase transduction of CNS tissues comprises a n-mer motif, the n-mer motif comprising or consisting of an amino acid sequence of any of those described herein.
  • 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.
  • 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.
  • 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.
  • the transduction and/or transduction efficiency is increased or enhanced in cells of the CNS.
  • the transduction and /or transduction efficiency is increased or enhanced in endothelial cells.
  • the composition comprising a n- mer is selective to a target cell as compared to other cell types and/or other virus particles.
  • ‘selective’ and ‘cell-selective’ refers to preferential targeting for cells as compared to other cell types.
  • 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.
  • 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.
  • a target cell e.g., endothelial cells across the CNS e.g., the brain
  • other cells types e.g. hepatocytes
  • virus particles e.g., AAVs not containing the CD59 targeting moiety
  • 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, the CD59 targeting moiety is part of (e g., inserted between consecutive amino acids of) a viral capsid protein, including an AAV capsid protein.
  • 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
  • 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[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),
  • 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.
  • 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.
  • GPI-anchored protein is CD59, which is a membrane attack complexinhibiting protein. It is broadly expressed in human tissue.
  • CD59 located on chromosome 11 (i.e., 11 pl3), is a protein that in humans is encoded by the CD59 gene (Gene Identifier).
  • the CD59 targeting moiety binds to the CNS. In an embodiment, the CD59 targeting moiety binds to one or more of the CD59.
  • 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
  • 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.
  • the engineered viral capsids can be variants of a wild-type viral capsid.
  • the engineered AAV capsids can be variants of wild-type AAV capsids.
  • the wild-type AAV capsids can be composed of VP1, VP2, VP3 capsid proteins or a combination thereof.
  • 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.
  • 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.
  • AAVhu .t 19 AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.
  • 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.
  • AAV-PAEC 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
  • 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.
  • the CD59 targeting moieties comprises of modifying the AAV capsid polypeptide.
  • 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.
  • 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 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.
  • 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.
  • the Xi, X2, X3, X4, X5, Xe, and X 7 modify 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.
  • the CD59 targeting moiety is inserted or substituted in loop IV and/or loop VIII.
  • 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.
  • 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.
  • AAV-PAEC 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
  • 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.
  • the CD59 targeting moiety is incorporated into a viral protein, such as a capsid protein, including but not limited to adenoviral or AAV proteins.
  • the n-mer is located between two amino acids of the viral protein such that the CD59 targeting moiety is external (i.e., is presented on the surface of) to a viral capsid.
  • 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.
  • the n-mer can be inserted between two consecutive amino acids in a variable amino acid region in a viral capsid protein.
  • the n-mer can be inserted between two consecutive amino acids in a variable amino acid region in an AAV capsid protein.
  • 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.
  • 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.
  • the CD59 targeting moiety is inserted or substituted in loop IV and/or loop VIII.
  • the n-mer or 7-mer is a CD59 targeting moiety.
  • 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).
  • the engineered capsid is a modified AAV3 capsid and can have a n-mer motif inserted after or a neighbor of amino acid 586.
  • the engineered capsid is a modified AAV4 capsid and can have a n-mer motif inserted after or a neighbor of amino acid 586.
  • the engineered capsid is a modified AAV5 capsid and can have a n-mer motif inserted after or a neighbor of amino acid 575.
  • 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.
  • 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.
  • the engineered capsid is a modified AAV9 capsid and can have a n-mer inserted in between amino acid 588 and 589.
  • 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.
  • the engineered capsid can have a 7-mer motif inserted between two consecutive amino 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.
  • 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.
  • AAVhu .t 19 AAVrh.2, AAVrh.2R, AAVrh.8, AAVrh.8R, AAVrh.10, AAVrh.12, AAVrh.13, AAVrh.
  • 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.
  • AAV-PAEC 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
  • 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.
  • 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 the insertion site.
  • 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.
  • 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.
  • the AAV capsid protein is selected from SEQ ID NO: 1.
  • the n-mer comprises EFNNGSD (SEQ ID NO: 89) or GAASLMP (SEQ ID NO: 109).
  • the CD59 targeting moiety comprises of the amino acid sequence of one of SEQ ID Nos: 89-5983.
  • the CD59 targeting moiety can include a polypeptide, a polynucleotide, a lipid, a polymer, a sugar, or a combination thereof.
  • the engineered viral capsid and/or capsid proteins can be encoded by one or more engineered viral capsid polynucleotides.
  • the engineered viral capsid polynucleotide is an engineered AAV capsid polynucleotide or engineered adenovirus capsid polynucleotide.
  • an engineered viral capsid polynucleotide e.g., an engineered AAV capsid polynucleotide or engineered adenovirus capsid polynucleotide
  • the poly adenylation signal can be an SV40 polyadenylation signal.
  • 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.
  • 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.
  • the engineered AAV capsid encoding polynucleotide can be operably coupled to a polyadenylation tail.
  • the poly adenylation tail can be an SV40 polyadenylation tail.
  • the AAV capsid encoding polynucleotide can be operably coupled to a promoter.
  • the regulatory sequence that regulates the expression of the transgene is a promoter and can be a tissue- or cell type-specific promoter.
  • the tissue-specific promoter is specific for muscle (e.g., cardiac, skeletal, and/or smooth muscle), neurons or other nervous system cells (e.g., astrocytes, glial cells, Schwann cells, ependymal cells, pericyte, oligodendrocyte, oligodendrocyte progenitor), 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.
  • muscle e.g., cardiac, skeletal, and/or smooth muscle
  • neurons or other nervous system cells e.g.,
  • 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).
  • 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.
  • 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.
  • CNS central nervous system
  • 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 tension (1987) Cell, 51 :7-19; Llewellyn et al. (2010) Nat.
  • NSE neuron-specific enolase
  • AADC aromatic amino acid decarboxylase
  • a neurofilament promoter see, e.g., GenBank HUMNFL, L04147
  • a synapsin promoter see, e.g., GenBank HUMSYNIB, M553O1
  • 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.
  • GnRH promoter see, e.g., Radovick et al., (1991) Proc. Natl. Acad. Sci. USA, 88:3402- 3406
  • L7 promoter see, e.g., Oberdick et al., (1990) Science, 248:223-226
  • DNMT promoter see, e.g., Badge et al., (1988) Proc. Natl. Acad. Sci.
  • enkephalin promoter see, e.g., Comb et al., (1988) EMBO J., 17:3793- 3805
  • MBP myelin basic protein
  • CMV enhancer/platelet-derived growth factor-p promoter see, e.g., Liu et al., (2004) Gene Ther., 11 :52-60
  • SYN minimal human synapsin 1 promoter
  • 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.
  • MBP myelin basic protein
  • 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).
  • the promoter can be a fugu SST (somatostatin) promoter (Nathanson, et al. Frontiers in Neural Circuits 3: 19).
  • 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.
  • tissue-specific promoters can be found in the TiProD (Tissue specific promoter database webpage tiprod.bioinf.med.uni-goettingen.de).
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the viral capsid protein may comprise one or more mutations relative to wild type.
  • 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 -
  • 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.
  • AAV-PAEC 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,
  • the K449R substituted AAV capsid is selected from SEQ
  • the viral capsid protein may comprise additional targeting motifs that are in addition to the CD59 targeting moiety of the present disclosure.
  • the additional targeting moieties can be antibodies or fragments thereof.
  • 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.
  • the binding partner is unique to a cell type or cell state or a to a group of related cell types or cell states.
  • the binding partner is a receptor, channel, or other complex present on the surface of a target cell.
  • 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.
  • “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 processes can 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.
  • 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.
  • 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.
  • DARPins designed ankyrin repeat proteins
  • the additional targeting moiety is a receptor ligand or binding protein.
  • the additional targeting moiety is attached or otherwise coupled to the capsid surface.
  • the additional targeting moiety is encoded by a vector that produces a capsid of the present invention described herein.
  • 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).
  • a ligand e.g., a 7-mer covalently linked to a primary amino group of a capsid polypeptide 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).
  • 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.
  • a targeting ligand e.g., the CD59 targeting moiety
  • 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).
  • 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.
  • 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).
  • 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.
  • the CD59 targeting moiety encoded in the vector system binds to CD59.
  • 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.
  • the vector can also be referred to and considered an engineered vector or system thereof although not specifically noted as such.
  • 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 that can express one or more components of the engineered viral capsid, particle, or other compositions described herein.
  • vectors containing one or more of the polynucleotide sequences described herein are included in a vector or vector system.
  • 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.
  • the vector can include an engineered viral (e.g., AAV) capsid polynucleotide having a 3’ polyadenylation signal.
  • the 3’ polyadenylation is an SV40 polyadenylation signal.
  • the vector does not have splice regulatory elements.
  • the vector includes one or more minimal splice regulatory elements.
  • the vector can further include a modified splice regulatory element, wherein the modification inactivates the splice regulatory element.
  • 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.
  • the polynucleotide sequence can be sufficient to induce splicing is a splice acceptor or a splice donor.
  • the viral (e.g., AAV) capsid polynucleotide is an engineered viral (e.g., AAV) capsid polynucleotide as described elsewhere herein.
  • the vector does not include one or more minimal splice regulatory elements, modified splice regulatory agent, splice acceptor, and/or splice donor.
  • 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.
  • engineered viral e.g., AAV
  • compositions e.g., polypeptides, particles, etc.
  • Other uses for the vectors and vector systems described herein are also within the scope of this disclosure.
  • the term is a tool that allows or facilitates the transfer of an entity from one environment to another.
  • 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.
  • a vector is capable of replication when associated with the proper control elements.
  • 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.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector 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)).
  • viruses 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).
  • vectors e.g., non-episomal mammalian vectors
  • Other 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.
  • 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.
  • 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.
  • a nucleic acid e.g., a polynucleotide
  • 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.
  • 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).
  • the vector can be a bicistronic vector.
  • a bicistronic vector can be used for one or more elements of the engineered viral (e.g., AAV) capsid system described herein.
  • 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.
  • 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.
  • 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.
  • AAV engineered viral
  • CD59 targeting moiety of the present disclosure described herein e.g., nucleic acid transcripts, proteins, enzymes, and combinations thereof
  • 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.
  • the suitable host cell is a eukaryotic cell.
  • 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.
  • 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.
  • the host cell is a suitable yeast cell.
  • the yeast cell can be from Saccharomyces cerevisiae.
  • the host cell is a suitable mammalian cell. Many types of mammalian cells have been developed to express vectors.
  • Suitable mammalian cells include, but are not limited to, HEK293, Chinese Hamster Ovary Cells (CHOs), mouse myeloma cells, HeLa, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, MCF-7, Y79, SO- Rb50, HepG G2, DIKX-X11, J558L, Baby hamster kidney cells (BHK), and chicken embryo fibroblasts (CEFs).
  • Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • the vector can be a yeast expression vector.
  • yeast expression 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).
  • 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.
  • 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).
  • CEN centromeric
  • ARS autonomous replication sequence
  • a promoter such as an RNA Polymerase III promoter
  • a terminator such as an RNA polymerase III terminator
  • an origin of replication e.g., auxotrophic, antibiotic, or other selectable markers
  • marker gene e.g., auxotrophic, antibiotic, or other selectable markers.
  • expression vectors for use in yeast may include plasmids, yeast artificial chromosomes, 2p plasmids, yeast integrative plasmids, yeast replicative plasmids, shuttle vectors, and
  • 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 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).
  • the vector is a mammalian expression vector.
  • the mammalian expression vector is capable of expressing one or more polynucleotides and/or polypeptides in a mammalian cell.
  • 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.
  • 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.
  • 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.
  • 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.
  • a regulatory element 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.
  • Vectors may be introduced and propagated in a prokaryote or prokaryotic cell.
  • 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).
  • 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.
  • the vector can be a fusion vector or fusion expression vector.
  • 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.
  • 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.
  • 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.
  • a proteolytic cleavage site 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
  • GST glutathione S-transferase
  • 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).
  • 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).
  • an engineered viral e.g., AAV
  • 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.
  • AAV engineered viral
  • 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.
  • 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).
  • the engineered polynucleotides of the present invention can be operably linked to and expressed from the same promoter.
  • 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.
  • the polynucleotides and/or vectors thereof described herein can include one or more regulatory elements that can be operatively linked to the polynucleotide.
  • 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).
  • IRS internal ribosomal entry sites
  • 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.
  • 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).
  • 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.
  • 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.
  • pol III promoters include, but are not limited to, U6 and Hl promoters.
  • pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) (see, e.g., Boshart et al, Cell, 41 :521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • 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).
  • 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.
  • the vector can contain a minimal promoter.
  • the minimal promoter is the Mecp2 promoter, tRNA promoter, or U6.
  • the minimal promoter is tissue specific.
  • the length of the vector polynucleotide the minimal promoters and polynucleotide sequences is less than 4.4Kb.
  • 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.
  • 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.
  • 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.
  • the regulated promoter is a tissue specific promoter as previously discussed elsewhere herein.
  • Regulated promoters include conditional promoters and inducible promoters.
  • 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.
  • 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)), central nervous 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 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
  • 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.
  • 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).
  • 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)
  • a negative/conditional inducible promoter e.g.,
  • the inducer can be a compound, environmental condition, or other stimulus.
  • 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.
  • 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.
  • engineered polynucleotide of the present disclosure e.g., an engineered viral (e.g., AAV) capsid polynucleotide
  • 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.
  • a rAAV vector including an rAAV vector genome as described herein, comprises at least one synthetic AAV ITR, wherein one or more CpG islands (a cytosine base followed immediately by a guanine base (a CpG) in which the cytosines in such arrangement tend to be methylated) that typically occur at, or near the transcription start site in an ITR are deleted and/or substituted.
  • 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.
  • AAV ITR2 is known to contain 16 CpG islands of which one or more, or all 16 can be deleted.
  • 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.
  • 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.
  • 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.
  • oversize vectors Several 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.
  • 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.
  • SD splice donor
  • SA splice acceptor
  • 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.
  • a third dual AAV strategy 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.
  • AP alkaline phosphatase
  • One or more of the engineered polynucleotides of the present disclosure 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.
  • 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).
  • the selectable marker or tag is a polynucleotide barcode or unique molecular identifier (UMI).
  • 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.
  • 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,
  • 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.
  • the vector or vector system can include one or more polynucleotides encoding one or more CD59 targeting moieties.
  • 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.
  • 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.
  • the engineered polynucleotide(s) of the present disclosure e.g., an engineered viral (e.g., AAV) capsid polynucleotide(s)
  • products expressed therefrom include the CD59 targeting moiety and can be targeted to specific cells, tissues, organs, etc.
  • 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.
  • the specific cells are CNS cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • In vitro translation can be stand-alone (e.g., translation of a purified polyribonucleotide) or linked/coupled to transcription.
  • 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.).
  • RNA or DNA starting material 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.
  • 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.
  • polynucleotides of the engineered AAV capsid system described herein can be codon optimized.
  • 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.
  • 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.
  • codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • 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).
  • 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.
  • one or more codons e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • 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.
  • 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.
  • the vector polynucleotide can be codon optimized for expression in a specific celltype, tissue type, organ type, and/or subject type.
  • 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.
  • 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, bone cells, tendon cells, cartilage cells), blood cells, stem cells and other progenitor cells, immune system cells, germ cells, and combinations thereof.
  • 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, bone cells
  • the polynucleotide is codon optimized for a specific tissue type.
  • 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.
  • the polynucleotide is codon optimized for a specific organ.
  • 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.
  • codon optimized sequences are within the ambit of the ordinary skilled artisan in view of the description herein.
  • 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.
  • the vector is a non-viral vector or carrier.
  • non- viral vectors can have the advantage(s) of reduced toxicity and/or immunogenicity and/or increased bio-safety as compared to viral vectors.
  • 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.
  • an engineered capsid polynucleotide e.g., an engineered AAV capsid polynucle
  • Non-viral vectors and carriers include naked polynucleotides, chemicalbased carriers, polynucleotide (non-viral) based vectors, and particle-based carriers.
  • vector 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.
  • one or more engineered AAV capsid polynucleotides or other polynucleotides of the present disclosure described elsewhere herein can be included in a naked polynucleotide.
  • naked polynucleotide 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.
  • 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.
  • 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.
  • the naked polynucleotide contains only the engineered AAV capsid polynucleotide(s) or other polynucleotides of the present disclosure.
  • 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.
  • 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,
  • the non-viral polynucleotide vector can have a conditional origin of replication.
  • the non-viral polynucleotide vector can be an ORT plasmid.
  • the non-viral polynucleotide vector can have a minimalistic immunologically defined gene expression.
  • the non-viral polynucleotide vector can have one or more post-segregationally killing system genes.
  • the non-viral polynucleotide vector is AR-free.
  • the non-viral polynucleotide vector is a minivector.
  • the non-viral polynucleotide vector includes a nuclear localization signal.
  • the non-viral polynucleotide vector can include one or more CpG motifs.
  • 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.
  • S/MARs can facilitate a once-per-cell-cycle replication to maintain the non- viral polynucleotide vector as an episome in daughter cells.
  • 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.
  • 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.
  • the non-viral vector is a transposon vector or system thereof.
  • transposon also referred to as transposable element
  • 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.
  • the non-viral polynucleotide vector can be a retrotransposon vector.
  • the retrotransposon vector includes long terminal repeats.
  • the retrotransposon vector does not include long terminal repeats.
  • the non-viral polynucleotide vector can be a DNA transposon vector.
  • DNA transposon vectors can include a polynucleotide sequence encoding a transposase.
  • the transposon vector is configured as a non-autonomous transposon vector, meaning that the transposition does not occur spontaneously on its own.
  • the transposon vector lacks one or more polynucleotide sequences encoding proteins required for transposition.
  • the non-autonomous transposon vectors lack one or more Ac elements.
  • 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.
  • TIRs transposon terminal inverted repeats
  • 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.
  • the transposon vector or system thereof can be configured as a gene trap.
  • 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.
  • 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
  • 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.
  • 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.
  • Tcl/mariner superfamily see e.g., Ivies et al. 1997. Cell. 91(4): 501-510
  • piggyBac piggyBac superfamily
  • Tol2 superfamily hAT
  • Frog Prince Tcl/mariner
  • 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.
  • any one given chemical carrier can include features from multiple categories.
  • particle 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.
  • the non-viral carrier can be an inorganic particle.
  • the inorganic particle can be a nanoparticle.
  • the inorganic particles can be configured and optimized by varying size, shape, and/or porosity.
  • the inorganic particles are optimized to escape from the reticulo endothelial system.
  • 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 and magnetite), quantum dots, fullerenes (e.g., carbon nanoparticles, nanotubes, nanostrings, and the like), and combinations thereof.
  • suitable inorganic non-viral carriers are discussed elsewhere herein.
  • the non-viral carrier can be lipid-based. Suitable lipid-based carriers are also described in greater detail herein.
  • 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).
  • 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).
  • 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).
  • the lipid-based non-viral carrier can be a solid lipid particle or nanoparticle.
  • the non-viral carrier can be peptide-based.
  • 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.
  • 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).
  • 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.
  • PEI polyethylenimine
  • PLA poly (DL-lactide)
  • PLGA poly (DL-Lactide-co-glycoside)
  • 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.
  • 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 specific molecule or composition (e.g., calcium, NaCl, and the like), pressure and the like.
  • 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.
  • the particle can include a polymer that can be selected from the group of polymethacrylates and polyacrylates.
  • 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.
  • the non-viral carrier can be a polymer-based carrier.
  • 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.
  • the vector is a viral vector.
  • viral vector 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).
  • a polynucleotide such as an engineered AAV capsid polynucleotide, cargo, or other composition or molecule of the present disclosure
  • 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.
  • 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.
  • the viral vectors are configured to produce replication incompetent viral particles for improved safety of these systems.
  • the vector can be an adenoviral vector.
  • 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.
  • the polynucleotide to be delivered via the adenoviral particle can be up to about 8 kb.
  • 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.
  • the vector can encode the engineered AAV capsids, said capsids forming adenoviral particles.
  • 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).
  • 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.
  • 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.
  • 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).
  • the vector is a hybrid-adenoviral vector or system thereof.
  • Hybrid adenoviral vectors are composed of the high transduction efficiency of a gene-deleted adenoviral vector and the long-term genome-integrating potential of adeno-associated, and transposon based- gene transfer.
  • 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.
  • a hybrid-adenoviral vector can include one or more features of a retrovirus and/or an adeno-associated virus.
  • 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.
  • 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.
  • the engineered vector or system thereof can be an adeno-associated vector (AAV).
  • AAV adeno-associated vector
  • 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).
  • AAVs have some deficiency in their replication and/or pathogenicity and thus can be safer than adenoviral vectors.
  • the AAV can integrate into a specific site on chromosome 19 of a human cell with no observable side effects.
  • 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.
  • the AAV vector or system thereof can be operably linked to a regulatory sequence, said regulatory sequence encoding one or more regulatory molecules.
  • the regulatory molecules can be promoters, enhancers, repressors and the like, which are described in greater detail elsewhere herein.
  • the AAV vector or system thereof can include one or more polynucleotides that can encode one or more regulatory proteins.
  • the promoter can be a tissue specific promoter as previously discussed.
  • the tissue specific promoter can drive expression of an engineered capsid AAV capsid polynucleotide described herein.
  • 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.
  • 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.
  • adenovirus helper factors can include, but are not limited, El A, E1B, E2A, E4ORF6, and VA RNAs.
  • a producing host cell line expresses one or more of the adenovirus helper factors.
  • the AAV vector or system thereof can be configured to produce AAV particles having a specific serotype.
  • the serotype can be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV8, AAV9 or any combinations thereof.
  • the AAV can be AAV1, AAV2, AAV5, AAV9 or any combination thereof.
  • 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.
  • 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.
  • 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.
  • the nucleotide sequences of the genomes of the AAV serotypes are known in the art.
  • 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.
  • 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.
  • 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.
  • the tropism of the AAV serotype can be modified by an engineered AAV capsid described 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.
  • 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).
  • wild-type AAV9 is biased towards muscle and brain in humans (see e.g., Srivastava. 2017. Curr. Opin. Virol. 21 :75-80.)
  • 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.
  • an engineered capsid and/or capsid protein variant of a wild-type AAV serotype can have a different tropism than the wild-type reference AAV serotype.
  • an engineered AAV capsid and/or capsid protein variant of AAV9 can have specificity for a tissue other than muscle or brain in humans.
  • 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.
  • pRep2/Cap5 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.
  • 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.
  • 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.
  • 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.
  • the tropism of the resulting AAV particle will be that of the engineered AAV capsid.
  • the AAV vector or system thereof is AAV rh.74 or AAV rh.10.
  • the AAV vector or system thereof is configured as a “gutless” vector, similar to that described in connection with a retroviral vector.
  • 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.
  • 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.
  • 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.
  • additional modifications may be implemented to further increase the efficacy of the AAV.
  • 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.
  • 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.
  • modified ITRs are no longer suitable substrates for the Rep68 and Rep78 proteins.
  • scAAV specific self-complementary AAV
  • 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.
  • the cis-elements are optimized for targeted delivery.
  • the ciselements are optimized because the packaging capacity of AAVs is restricted.
  • small cis-elements replace long promoter sequences for the delivery of large therapeutic transgenes (e.g., 4.4-4.5 kbs).
  • Example approach 1 takes advantage of an AAV genome concatemerized via the homologous recombination of ITR sequences.
  • 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.
  • transgene fragments of different lengths are packaged into different AAV virions at undefined locations on the vector genome. Either homologous recombination of the overlapping regions of the different AAV vector genomes or annealing of different AAV vector genomes at complementary regions via single- stranded templates produces the transgene cassette. In an embodiment, overlapping fragments may be added to the end of the individual AAV vectors to encourage homologous recombination.
  • 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.
  • example approach 4 an AAV genome is cross-packaged into the capsids of other parvoviruses thus creating chimeric vectors.
  • 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.
  • the vectors described herein can be constructed using any suitable process or technique.
  • 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.
  • AAV vectors 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.
  • the vector can have one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a “cloning site”).
  • one or more insertion sites e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors.
  • 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.
  • 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)).
  • 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.
  • 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
  • helper polynucleotides e.g., a vector that carries the AAV Rep-Cap encoding polyn
  • 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.
  • exons e.g., exogenous exons or exons that are part of a CDS of a trans-splicing molecule
  • second RNA molecule e.g., a pre-mRNA molecule, e.g., an endogenous pre-mRNA molecule
  • 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 to its 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).
  • target gene e.g., pre-mRNA
  • a splicing domain e.g., a splicing domain having a 3' or 5' splice site
  • 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
  • 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.
  • the engineered AAV vectors and systems thereof described herein can be produced by any of these methods.
  • 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).
  • nucleic acids e.g., engineered AAV capsid system transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.
  • virus particles such as from viral vectors and systems thereof.
  • 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.
  • 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.
  • the route of administration, formulation and dose can be as in US Patent No. 8,454,972 and as in clinical trials involving AAV.
  • the route of administration, formulation and dose can be as in US Patent No. 8,404,658 and as in clinical trials involving adenovirus.
  • the route of administration, formulation and dose can be as in US Patent No 5,846,946 and as in clinical studies involving plasmids.
  • doses can be based on or extrapolated to an average 70 kg individual (e.g., a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed.
  • the viral vectors can be injected into or otherwise delivered to the tissue or cell of interest.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • engineered AAV capsid system components e.g., polynucleotides encoding engineered AAV capsid and/or capsid proteins
  • particle 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.
  • any of the of the engineered AAV capsid system components e.g., polypeptides, polynucleotides, vectors, and combinations thereof described herein
  • particle delivery can be selected and be advantageous for delivery of the polynucleotide or vector components. It will be appreciated that in embodiments, particle delivery can also be advantageous for other engineered capsid system molecules and formulations described elsewhere herein.
  • 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.
  • 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.
  • the engineered AAV particles can include 1-60 engineered AAV capsid proteins described herein.
  • 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.
  • the engineered AAV particles can contain 0-59 wild-type AAV capsid proteins.
  • 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.
  • 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.
  • 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 the like), chemical compounds, and combinations thereof.
  • 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.
  • 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.
  • the cargo is capable of treating or preventing a CNS disease or disorder, details of which are described herein.
  • 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.
  • 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.
  • 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.
  • the cargo is a cargo polynucleotide that can be packaged into an engineered viral particle and subsequently delivered to a cell.
  • delivery is cell selective, e.g., neurons and glial cells of the CNS.
  • 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.
  • the cargo polynucleotide can be capable of modifying a polynucleotide (e.g., gene or transcript) of a cell to which it is delivered.
  • a polynucleotide e.g., gene or transcript
  • 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.
  • 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.
  • 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).
  • 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. Length of protuberant region is independent from total length of siRNA molecule.
  • siRNAs may act by post-transcriptional degradation or silencing of target messenger.
  • the exogenous polynucleotides encode shRNAs. In shRNAs, the antiparallel strands that form siRNA are connected by a loop or hairpin region.
  • 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.
  • 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.
  • 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,
  • 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).
  • RNAi RNA interference
  • siRNA small interfering RNA
  • shRNA short-hairpin RNA
  • 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.
  • 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.
  • 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.
  • the flanking retroelement polypeptide encoding polynucleotide UTR(s) are from PNMA, Arc, PEG10 or other Sushi Class polypeptide.
  • the inclusion of the 3’ UTR, the 5 ’UTR, or both can increase packaging and/or delivery of the cargo that they flank.
  • 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.
  • 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.
  • engineered viral e.g., AAV
  • the cargo molecule is or encodes a gene editing system or component thereof.
  • the cargo molecule is or encodes a CRISPR-Cas system molecule or a component thereof.
  • the cargo molecule is a polynucleotide that encodes one or more components of a gene modification system (such as a CRISPR-Cas system).
  • 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.
  • 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 (such as single stranded RNA viruses).
  • viruses such as single stranded RNA viruses
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions 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.
  • 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.
  • 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 promote tissue 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.
  • the one or more polypeptides may comprise one or more antibodies.
  • 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).
  • 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.
  • 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.
  • 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.
  • the antibody is a fragment or portion thereof.
  • the antibody is an epitope binding protein or portion thereof.
  • the term “binding portion” of an antibody 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 DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins. Binding fragments include Fab, Fab', F(ab')2, Fabc, Fd, dAb, Fv, single chains, single-chain antibodies, e.g., scFv, and single domain antibodies.
  • the cargo or antibody is an antibody fragment or portion.
  • the cargo is an epitope binding protein.
  • 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
  • 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).
  • antigen binding i.e., specific binding
  • the antibody is a single-chain antibody (scFvs).
  • singlechain variable fragment 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 more serine or threonine residues to increase or modify solubility.
  • the VH and light (VL) can be linked via the linker in any order.
  • 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.
  • VHH heavy chain antibody
  • sdAbs single-domain antibodies
  • 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.
  • VHH is used interchangeably with “nanobody.”
  • the -12-15 kDa variable domains of these antibodies can be produced recombinantly and can recognize antigen in the absence of the remainder of the antibody heavy chain.
  • 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.
  • the heavy chain antibody is an antibody derived from cartilaginous fishes (immunoglobulin new antigen receptor (IgNAR)) or camelid ungulates.
  • camelids include dromedaries, camels, llamas and alpacas.
  • 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 ).
  • immunoglobulin class refers to the five classes of immunoglobulin that have been identified in humans and higher mammals, IgG, IgM, IgA, IgD, and IgE.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the term “conformation” refers to the tertiary structure of a protein or polypeptide (e.g., an antibody, antibody chain, domain or region thereof).
  • light (or heavy) chain conformation refers to the tertiary structure of a light (or heavy) chain variable region
  • antibody conformation or “antibody fragment conformation” refers to the tertiary structure of an antibody or fragment thereof.
  • 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).
  • Curr Opin Biotechnol 2007, 18:295-304 include 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.
  • 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.
  • 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.
  • “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 10 7 M 1 (or a dissociation coefficient of I pM or less or a dissociation coefficient of Inm or less) typically bind with correspondingly greater specificity.
  • 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 or less, 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).
  • 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.
  • 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 BIACORETM method. The dissociation constant, Kd, and the association constant, Ka, are quantitative measures of affinity.
  • 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.
  • 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.
  • donor antibody such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and capacity.
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • 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.
  • a “blocking” antibody or an antibody “antagonist” is one which inhibits or reduces biological activity of the antigen(s) it binds.
  • the blocking antibodies or antagonist antibodies or portions thereof described herein completely inhibit the biological activity of the antigen(s).
  • Antibodies may act as agonists or antagonists of the recognized polypeptides.
  • 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
  • receptor activation can 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.
  • 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.
  • 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.
  • receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex.
  • 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.
  • antibodies which activate the receptor are also included in the disclosure. 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 agonists and antagonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Pat. No. 5,811,097; Deng et al., Blood 92 (6): 1981-1988 (1998); Chen et al., Cancer Res. 58(16): 3668-3678 (1998); Harrop et al., J. Immunol. 161(4) : 1786- 1794 (1998); Zhu et al., Cancer Res.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • a conserved N- terminal domain also referred to herein as a PLMP domain
  • IscB proteins may also further comprise a conserved C-terminal domain.
  • 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.
  • IscB nucleic acid-guided nucleases may comprise CRISPR- associated IscB nucleases.
  • the IscB nucleases are CRISPR-associated proteins, e.g., the loci of the nucleases are associated with an CRISPR array.
  • 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.
  • IsrBs are homologs of IscB nucleases.
  • IsrB nucleases comprise the PLMP and RuvC domains but do not comprise a HNH domain.
  • 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.
  • the IsrB is an OMEGA RNA guided nickase.
  • the OMEGA RNA guided IsrB nicks a DNA target.
  • the DNA target is a dsDNA and the nicks occurs on the non-target strand of the dsDNA target.
  • 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.
  • 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.
  • the IshB, or IscB homolog comprises a PLMP domain and an HNH domain, but does not comprise a RuvC domain.
  • IshB nucleases may be part of the IS605 OrfB family of transposases.
  • the IshB nuclease is from Actinoplanes lobatus and has the Genbank accession number MBB4752409.
  • the RefSeq database accession number for the nuclease with accession number MBB4752409 is WP_188124268 and the INSDC number is GGN95087.
  • 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.
  • 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.
  • 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.
  • the RuvC-III sub-domain forms the C-terminus of the TnpB nuclease.
  • 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.
  • the TnpB nuclease is from Ktedonobacter racemifer, or comprises a conserved RNA region with similarity to the 5’ ITR of K. racemifer TnpB loci.
  • 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.
  • 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.
  • 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.
  • 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.
  • the RuvC-III sub-domain forms the C-terminus of the Fanzor nuclease.
  • engineered cells that can include one or more of the engineered AAV capsid polynucleotides, polypeptides, vectors, and/or vector systems.
  • one or more of the engineered AAV capsid polynucleotides can be expressed in the engineered cells.
  • the engineered cells can be capable of producing engineered AAV capsid proteins and/or engineered AAV capsid particles that are described elsewhere herein.
  • 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.
  • a cargo molecule e.g., a cargo polynucleotide
  • 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.
  • 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.
  • 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.
  • 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 can include one or more of the engineered AAV capsid system polynucleotides, polypeptides, vectors, and/or vector systems described elsewhere herein.
  • 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.
  • producer cells Such cells are also referred to herein as “producer cells”.
  • modified 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.
  • isolated cells can be modified prior to receiving an engineered AAV capsid molecule.
  • 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.
  • 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.
  • the organism is a host of AAV.
  • 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.
  • 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).
  • 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.
  • the engineered cell can be a cell line.
  • 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
  • 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.
  • a muscle cell e.g. cardiac muscle, skeletal muscle, and/or smooth muscle
  • bone cell e.g. cardiac muscle, skeletal muscle, and/or smooth muscle
  • immune cell including but not limited to B cells, macrophages, T-cells, CAR-T cells, and the like
  • kidney cells including but not limited to B cells, macrophages, T-cells, CAR-T cells, and the like
  • kidney cells including but not limited to B cells, macrophag
  • the engineered cell can be a fungus cell.
  • 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.
  • 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.
  • the yeast cell is an S. cerevisiae, Kluyveromyces marxianus, or Issatchenkia orientalis cell.
  • Other yeast cells may include without limitation Candida spp. (e.g., Candida albicans), Yarrowia spp. (e.g., Yarrowia lipolytica), Pichia spp.
  • the fungal cell is a fdamentous fungal cell.
  • filamentous fungal cell refers to any type of fungal cell that grows in filaments, i.e., hyphae or mycelia.
  • 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).
  • the fungal cell is an industrial strain.
  • “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.
  • the fungal cell is a polyploid cell.
  • 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.
  • the fungal cell is a diploid cell.
  • 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).
  • 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.
  • the fungal cell is a haploid cell.
  • a “haploid” cell may refer to any cell
  • 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).
  • 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.
  • the engineered cell is a cell obtained from a subject.
  • the subject is a healthy or non-diseased subject.
  • 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.
  • the cargo polynucleotides of the produced engineered AAV capsid particle can be capable of transferring the desired characteristic to a recipient cell.
  • 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.
  • 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.
  • the engineered cells can be used to produce engineered viral (e.g., AAV) capsid polynucleotides, vectors, and/or particles.
  • the engineered viral (e.g., AAV) capsid polynucleotides, vectors, and/or particles are produced, harvested, and/or delivered to a subject in need thereof.
  • the engineered cells are delivered to a subject.
  • Other uses for the engineered cells are described elsewhere herein.
  • the engineered cells can be included in formulations and/or kits described elsewhere herein.
  • 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.
  • 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 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.
  • compositions, polynucleotides, polypeptides, particles, cells, vector systems and combinations thereof described herein can be contained in a formulation, such as a pharmaceutical formulation.
  • the formulations can be used to generate polypeptides and other particles that include one or more CNS-specific CD59 targeting moieties described herein.
  • the formulations can be delivered to a subject in need thereof.
  • 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.
  • 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.
  • pharmaceutical formulations containing an amount of one or more of the polypeptides, polynucleotides, vectors, cells, or combinations thereof described herein.
  • 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.
  • 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.
  • the amount can range from about 1 cell to 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x IO 10 or more cells. In embodiments where the pharmaceutical formulation contains one or more cells, the amount can range from about 1 cell to 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x IO 10 or more cells per nL, pL, mL, or L.
  • the formulation can contain 1 to 1 x 10 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x IO 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 , 1 x 10 16 , 1 x IO 17 , 1 x 10 18 , 1 x 10 19 , or 1 x IO 20 transducing units (TU)/mL of the engineered AAV capsid particles.
  • TU transducing units
  • the formulation can be 0.1 to 100 mL in volume and can contain 1 to 1 x 10 1 , 1 x 10 2 , 1 x 10 3 , 1 x 10 4 , 1 x 10 5 , 1 x 10 6 , 1 x 10 7 , 1 x 10 8 , 1 x 10 9 , 1 x IO 10 , 1 x 10 11 , 1 x 10 12 , 1 x 10 13 , 1 x 10 14 , 1 x 10 15 , 1 x 10 16 , 1 x 10 17 , 1 x 10 18 , 1 x 10 19 , or 1 x IO 20 transducing units (TU)/mL of the engineered AAV capsid particles.
  • TU transducing units
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • interleukins e.g., IL-2, IL-7, and IL- 12
  • cytokines e.g., interferons (e.g., IFN-a, IFN
  • 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.
  • 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
  • metamizole nabumetone
  • phenazone phenazone
  • quinine quinine
  • 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.
  • benzodiazepines e.g., alprazolam, bromazepam, chlordiazepoxide, clonazepam,
  • 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, prothipend
  • 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).
  • 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)
  • non-steroidal anti-inflammatories e.g., ibuprofen, naproxen, ketoprofen, and nimesulide
  • COX-2 inhibitors e.g., rofecoxib, celecoxib, and etoricoxib
  • immune selective anti-inflammatory derivatives e.g., submandibular gland peptide-T and its derivatives
  • 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
  • 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), echinoc ameb
  • 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, daca
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the AAV formulation is administered by intrathecally in equal portions to the cistema magna and the lumbar spine.
  • 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.
  • 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).
  • more than one administration 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.
  • treatment of a disease or disorder may comprise a one-time administration of an effective dose of a pharmaceutical composition virus vector disclosed herein.
  • 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.
  • rAAV particles may be administered to multiple locations, for example, 1, 2, 3, 4, or 5 locations simultaneously or staggered over time.
  • the more than one administration may include immuno-suppression or immunomodulatory agents (e.g., steroids, anti-B cell antibodies, rapamycin or other mTOR inhibitors).
  • the immuno-suppression or immunomodulatory agent is an inhibitor, see e.g., WO2021067598A1, hereby incorporated by reference.
  • the immuno-suppression or immunomodulatory agent is an antibody, see e.g., EP3909602A1, hereby incorporated by reference.
  • the immuno-suppression or immunomodulatory agent is a protease or glycosidase, see e g., W02020016318A1, hereby incorporated by reference.
  • the immuno-suppression or immunomodulatory agent is a steroid, see e.g., WO2021163322, hereby incorporated by reference.
  • the timing of administration can vary from individual to individual, depending upon such factors as the severity of an individual's symptoms.
  • 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.
  • 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.
  • PF68 pluronic acid
  • the active ingredient will be in the form of an aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • 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.
  • Dosage forms adapted for parenteral administration and/or adapted for any type of injection 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.
  • the dosage form can also be prepared to prolong or sustain the release of any ingredient.
  • the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be the ingredient whose release is delayed.
  • the release of an optionally included auxiliary ingredient is delayed.
  • Suitable methods for delaying the release of an ingredient include, but are not limited to, coating or embedding the ingredients in material in polymers, wax, gels, and the like. Delayed release dosage formulations can be prepared as described in standard references such as “Pharmaceutical dosage form tablets,” eds. Liberman et. al.
  • Dosage forms adapted for nasal or inhalation administration include aerosols, solutions, suspension drops, gels, or dry powders.
  • 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.
  • 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.
  • 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
  • the dosage forms can be aerosol formulations suitable for administration by inhalation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the pharmaceutical formulation is a dry powder inhalable formulation.
  • 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.
  • the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein is in a particle-size reduced form.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • effective amount 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.
  • “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.
  • 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 .
  • the one or more therapeutic effects comprise transducing the CNS.
  • 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).
  • the effective amount can be about IxlO 1 particles per pL, nL, pL, mL, or L to 1X1O 20 / particles per pL, nL, pL, mL, or L or more, such as about IxlO 1 , IxlO 2 , IxlO 3 , IxlO 4 , IxlO 5 , IxlO 6 , IxlO 7 , IxlO 8 , IxlO 9 , IxlO 10 , IxlO 11 , IxlO 12 , IxlO 13 , IxlO 14 , IxlO 15 , IxlO 16 , IxlO 17 , IxlO 18 , IxlO 19 , to/or about IxlO 20 particles per pL, nL, pL, mL, or L.
  • the effective titer can be about 1X10 1 transforming units per pL, nL, pL, mL, or L to 1X1O 20 / transforming units per pL, nL, pL, mL, or L or more, such as about IxlO 1 , IxlO 2 , IxlO 3 , IxlO 4 , IxlO 5 , IxlO 6 , IxlO 7 , IxlO 8 , IxlO 9 , IxlO 10 , IxlO 11 , IxlO 12 , IxO 13 , IxlO 14 , IxlO 15 , IxlO 16 , IxlO 17 , IxlO 18 , IxlO 19 , to/or about IxlO 20 transforming units per pL, nL, pL, mL, or L or any numerical value or subrange within these ranges.
  • 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,
  • the amount or effective amount, particularly where an infective particle is being delivered can be expressed as vector genomes per kilogram bodyweight (vg/kg).
  • the effective amount can be about IxlO 1 vg/kg or more, such as about IxlO 2 , IxlO 3 , IxlO 4 , IxlO 5 , IxlO 6 , IxlO 7 , IxlO 8 , IxlO 9 , IxlO 10 , IxlO 11 , IxlO 12 , IxlO 13 , IxlO 14 , IxlO 1 ’, IxlO 16 , IxlO 17 , IxlO 18 , IxlO 19 , to/or about IxlO 20 such that the effective amount can be anything in between, for example, 0.1 to 10 (O.lxlO 12 to 10xl0 12 ).
  • the pharmaceutical composition is delivered at a dosage between 0.1 x 10 12 vg/kg to 1 x 10 14 vg/kg. In an embodiment, the dosage is between 0.1 x 10 12 vg/kg to 100 x 10 12 vg/kg. In an embodiment, the dosage is between I x lO 12 vg/kg to 10 x 10 12 vg/kg. In an embodiment, the dosage is 5 x 10 12 vg/kg.
  • 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,
  • 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
  • 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.
  • 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.
  • 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.
  • 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,
  • the effective amount of the secondary active agent is any non-zero amount ranging from about 0 to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
  • Kits 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 % w/w, v/v, or w/v of the total pharmaceutical formulation or any numerical value or subrange within these ranges.
  • kits that contain one or more of the one or more of the compositions, polypeptides, polynucleotides, vectors, cells, or other components described herein and combinations thereof and pharmaceutical formulations described herein.
  • one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof described herein can be presented as a combination kit.
  • the terms "combination kit” or “kit of parts” refers to the compounds, or formulations and additional components that are used to package, screen, test, sell, market, deliver, and/or administer the combination of elements or a single element, such as the active ingredient, contained therein.
  • the combination kit can contain one or more of the components (e.g., one or more of the one or more of the polypeptides, polynucleotides, vectors, cells, and combinations thereof) or formulation thereof can be provided in a single formulation (e.g., a liquid, lyophilized powder, etc.), or in separate formulations.
  • the separate components or formulations can be contained in a single package or in separate packages within the kit.
  • the kit can also include instructions in a tangible medium of expression that can contain information and/or directions regarding the content of the components and/or formulations contained therein, safety information regarding the content of the components(s) and/or formulation(s) contained therein, information regarding the amounts, dosages, indications for use, screening methods, component design recommendations and/or information, recommended treatment regimen(s) for the components(s) and/or formulations contained therein.
  • tangible medium of expression refers to a medium that is physically tangible or accessible and is not a mere abstract thought or an unrecorded spoken word.
  • “Tangible medium of expression” includes, but is not limited to, words on a cellulosic or plastic material, or data stored in a suitable computer readable memory form. The data can be stored on a unit device, such as a flash memory drive or CD-ROM or on a server that can be accessed by a user via, e.g., a web interface.
  • the disclosure provides a kit comprising one or more of the components described herein.
  • the kit comprises a vector system and instructions for using the kit.
  • the vector system includes a regulatory element operably linked to one or more engineered polynucleotides, such as those containing a selective CD59 targeting moiety, as described elsewhere herein and, optionally, a cargo molecule, which can optionally be operably linked to a regulatory element.
  • the one or more engineered polynucleotides such as those containing a selective CD59 targeting moiety, as described elsewhere herein and, can be included on the same or different vectors as the cargo molecule in embodiments containing a cargo molecule within the kit.
  • the kit comprises a vector system and instructions for using the kit.
  • the vector system comprises (a) a first regulatory element operably linked to a direct repeat sequence and one or more insertion sites for inserting one or more guide sequences up- or downstream (whichever applicable) of the direct repeat sequence, wherein when expressed, the guide sequence directs sequence-specific binding of a Cas9 CRISPR complex to a target sequence in a eukaryotic cell, wherein the Cas9 CRISPR complex comprises a Cas9 enzyme complexed with the guide sequence that is hybridized to the target sequence; and/or (b) a second regulatory element operably linked to an enzyme-coding sequence encoding said Cas9 enzyme comprising a nuclear localization sequence.
  • a tracr sequence may also be provided.
  • the kit comprises components (a) and (b) located on the same or different vectors of the system.
  • component (a) further comprises two or more guide sequences operably linked to the first regulatory element, wherein when expressed, each of the two or more guide sequences direct sequence specific binding of a CRISPR complex to a different target sequence in a eukaryotic cell.
  • the Cas9 enzyme comprises one or more nuclear localization sequences of sufficient strength to drive accumulation of said CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.
  • the CRISPR enzyme is a type V or VI CRISPR system enzyme.
  • the CRISPR enzyme is a Cas9 enzyme.
  • the Cas9 enzyme is derived from Francisella tularensis 1, Francisella tularensis subsp. novi ci da, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium GW201 l_GWA2_33_10, Parcubacteria bacterium GW2011_GWC2_44_17, Smithella sp. SCADC, Acidaminococcus sp.
  • BV3L6 Lachnospiraceae bacterium MA2020, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237, Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens, or Porphyromonas macacae Cas9 (e.g., modified to have or be associated with at least one DD), and may include further alteration or mutation of the Cas9, and can be a chimeric Cas9.
  • the DD-CRISPR enzyme is codon-optimized for expression in a eukaryotic cell.
  • the DD-CRISPR enzyme directs cleavage of one or two strands at the location of the target sequence.
  • the DD-CRISPR enzyme lacks or substantially DNA strand cleavage activity (e.g., no more than 5% nuclease activity as compared with a wild-type enzyme or enzyme not having the mutation or alteration that decreases nuclease activity).
  • the first regulatory element is a polymerase III promoter.
  • the second regulatory element is a polymerase II promoter.
  • the guide sequence is at least 16, 17, 18, 19, 20, 25 nucleotides, or between 16-30, or between 16-25, or between 16-20 nucleotides in length.
  • compositions including one or more of the cell-selective CD59 targeting moieties, engineered AAV capsid system polynucleotides, polypeptides, vector(s), engineered cells, engineered AAV capsid particles can be used generally to package and/or deliver one or more cargos to neurons, glial cells, and endothelial cells of the CNS.
  • delivery is done in cell-selective manner based upon the selectivity of the CD59 targeting moiety.
  • this is conferred by the tropism of the engineered AAV capsid, which can be influenced at least in part by the inclusion of one or CD59 targeting moieties described elsewhere herein.
  • compositions including one or more of the CNS CD59 targeting moieties can be administered to a subject or a cell, tissue, and/or organ and facilitate the transfer and/or integration of the cargo to the recipient cell.
  • engineered cells capable of producing compositions such as polypeptides and other particles (e.g., engineered AAV capsids and viral particles), containing one or more of the CD59 targeting moieties can be generated from the polynucleotides, vectors, and vector systems etc., described herein.
  • the engineered AAV capsid system molecules e.g., polynucleotides, vectors, and vector systems, etc .
  • the polynucleotides, vectors, and vector systems etc., described herein capable of generating the compositions, such as polypeptides and other particles (e.g., engineered AAV capsids and viral particles), containing one or more of the CD59 targeting moieties can be delivered to a cell or tissue, in vivo, ex vivo, or in vitro.
  • the composition when delivered to a subject, can transform a subject’s cell in vivo or ex vivo to produce an engineered cell that can be capable of making a composition described herein that contains one or more of the cell-selective CD59 targeting moieties described herein, including but not limited to the engineered AAV capsid particles, which can be released from the engineered cell and deliver cargo molecule(s) to a recipient cell in vivo or produce personalized engineered compositions (e.g., AAV capsid particles) for reintroduction into the subject from which the recipient cell was obtained.
  • the engineered AAV capsid particles e.g., AAV capsid particles
  • an engineered cell can be delivered to a subject, where it can release produced compositions of the present disclosure (including but not limited to engineered AAV capsid particles) such that they can then deliver a cargo (e.g., a cargo polynucleotide(s)) to a recipient cell.
  • compositions of the present disclosure including but not limited to engineered AAV capsid particles
  • a cargo e.g., a cargo polynucleotide(s)
  • These general processes can be used in a variety of ways to treat and/or prevent disease or a symptom thereof in a subject, generate model cells, generate modified organisms, provide cell selection and screening assays, in bioproduction, and in other various applications.
  • compositions such as polypeptides and other particles (e.g., engineered AAV capsids and viral particles), containing one or more of the CD59 targeting moieties can be delivered to neural cells of the CNS.
  • compositions containing one or more CD59 targeting moieties can be delivered to glial cells of the CNS.
  • the engineered AAV capsid polynucleotides, vectors, and systems thereof can be used to generate engineered AAV capsid variant libraries that can be mined for variants with a desired cell -selectivity.
  • the description provided herein as supported by the various Examples can demonstrate that one having a desired cell-selectivity in mind could utilize the present disclosure as described herein to obtain a capsid with the desired cell-selectivity.
  • AAV adeno-associated virus
  • AAV capsid variants have unique antigenic profiles and demonstrate distinct cellular tropisms driven by differences in receptor binding.
  • AAV capsids can be chemically modified to alter tropism, produced as hybrid vectors combining the properties of multiple serotypes, and carry peptide insertions that introduce novel receptor-binding activity.
  • directed evolution of shuffled genome libraries can identify engineered variants with unique properties, and rational modification of the viral capsid can alter tropism, reduce blockage by neutralizing antibodies, or enhance transduction efficiency, (reviewed by Castle, M.
  • CD59 targeting moieties of the capsid polypeptides disclosed herein enhance AAV transduction in the CNS.
  • the modified AAV capsid facilitates AAV transduction to at least one region of the brain, muscle, and lungs.
  • the modified AAV capsid facilitates AAV transduction to the forebrain, the midbrain or the hindbrain.
  • the modified AAV capsid facilitates AAV transduction to at least one of the following brain regions chosen from the occipital lobes, the temporal lobe, the parietal lobe, the frontal lobe, the cerebral cortex, the cerebellum, the hypothalamus, the thalamus, the pituitary gland, the pineal gland, the amygdala, the hippocampas and the mid-brain.
  • the modified AAV capsid facilitates AAV transduction to at least two of the following brain regions chosen from the occipital lobes, the temporal lobe, the parietal lobe, the frontal lobe, the cerebral cortex, the cerebellum, the hypothalamus, the thalamus, the pituitary gland, the pineal gland, the amygdala, the hippocampas and the mid-brain.
  • the modified AAV capsid facilitates AAV transduction to at least three of the following brain regions chosen from the occipital lobes, the temporal lobe, the parietal lobe, the frontal lobe, the cerebral cortex, the cerebellum, the hypothalamus, the thalamus, the pituitary gland, the pineal gland, the amygdala, the hippocampas and the mid-brain.
  • the modified AAV capsid facilitates AAV transduction of the endothelial cells of the CNS.
  • MicroRNAs are indeed small, non-coding RNA molecules that play a crucial role in regulating gene expression at the post-transcriptional level. They bind to complementary sequences on messenger RNAs (mRNAs), typically resulting in gene silencing either by degrading the mRNA or by inhibiting its translation into proteins.
  • mRNAs messenger RNAs
  • AAV transgene expression in the various regions of the brain can be further fine-tuned by the incorporation of specific miRNA binding sites into the transgene's 5' or 3'-UTR.
  • AAV transgene expression can be suppressed in those cells where the microRNA is expressed and the corresponding miRNA binding site is placed in the 5' or 3'- UTR.
  • a summary of miRNA gene expression in specific cell types of the CNS can be found in the Table below, reproduced from Zolboot, N., Du, J. X., Zampa, F. & Lippi, G. MicroRNAs instruct and maintain cell type diversity in the nervous system. Front. Mol. Neurosci. 14, 646072 (2021).
  • the transgene's 5' or 3'-UTR may contain one or more miR-122 binding sites to suppress spurious transgene expression and potential toxicity in the liver (see, for example, Qiao, C. et al. Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver. Gene Ther. 18, 403-410 (2011)).
  • the transgene's 5' or 3'-UTR may contain one or more miR-183 binding sites to suppress spurious transgene expression and potential toxicity in the liver (see, for example, Hordeaux, J.; Buza, E.
  • compositions used in methods disclosed herein are capable of increasing transduction of neurons and/or glial cells of the CNS, allowing for delivery of cargo and therapeutics directly to such cell types.
  • a method is disclosed wherein the cargo is one or more polypeptides.
  • a method is disclosed wherein the disease or disorder is a cancer, neurological disorder, or infection.
  • methods of treatment comprise administering a composition as detailed herein to a subject in need thereof.
  • the cancer is a neuroepithelial cancer.
  • the cancer is a neuroepithelial tumor, for example, Astrocytic tumors, e.g., Diffuse Astrocytoma (fibrillary, protoplasmic, gemistocytic, mixed), Anaplastic (malignant) astrocytoma, Glioblastoma (giant cell, gliosarcoma variants), Pilocytic astrocytoma, Pleomorphic xanthoastrocytoma, or Subependymal giant cell astrocytoma; Oligodendroglial tumors, e.g., Oligodendroglioma, Anaplastic (malignant) Oligodendroglioma, Ependymal tumors, Ependymoma (cellular, papillary, clear cell, tanycytic), Anaplastic (
  • the cancer is a primary cancer metastasized to brain or other region of the central nervous system.
  • the neurological disorder is caused by a neurodegenerative or a neurodevelopmental disease.
  • Example neurodegenerative diseases include, but are not limited to, Alzheimer’s disease and other memory disorders, amyotrophic lateral sclerosis (ALS), ataxia, Huntington’s disease, Parkinson’s disease, Friedreich’s ataxia, motor neuron disease, multiple system atrophy, progressive supranuclear palsy.
  • neurodevelopmental diseases include, but are not limited to, attention-deficit/hyperactivity disorder (ADHD), autism, learning disabilities, intellectual disability (also known as mental retardation), conduct disorders, cerebral palsy, speech and language disorders, Tourette syndrome, Schizophrenia, Rett syndrome, Angelman’s syndrome, Fragile X syndrome, and impairments in vision and hearing.
  • ADHD attention-deficit/hyperactivity disorder
  • autism learning disabilities
  • intellectual disability also known as mental retardation
  • conduct disorders cerebral palsy
  • speech and language disorders Tourette syndrome, Schizophrenia, Rett syndrome, Angelman’s syndrome
  • Fragile X syndrome and impairments in vision and hearing.
  • the disease is a lysosomal storage disease.
  • the lysosomal storage disease may include Gangliosidosis generalized GM1 type 1; type 2; type3 , Krabbe’s disease, Metachromatic leukodystrophy, Fabry’s disease, Gaucher disease, Niemann-Pick disease A, Niemann-Pick disease B, Niemann-Pick disease Cl, Niemann-Pick disease C2, Tay-Sachs disease, Sandhoff disease, GM2-activator deficiency, Multiple sulfatase deficiency, Oligosaccharidosis, Alpha mannosidosis, Schindler’s disease, Aspartylglucosaminuria, Fucosidosis, Mucopolysaccharidosis, Hurler syndrome, Scheie syndrome, Hurler-Scheie syndrome, Hunter syndrome, SanFilippo syndrome A, SanFilippo syndrome B, SanFilippo syndrome C, SanFilippo syndrome D, Morquio syndrome A, Morquio syndrome B,
  • the disease is a lysosomal storage disease.
  • the lysosomal storage disease may include Gaucher’s or Parkinson’s disease.
  • Gaucher’s disease may be Type II or Type III Gaucher’s disease.
  • a method of creating humanized transgenic nonhuman animals comprising: delivering to one or more cells of a non-human animal a vector system or a recombinant viral particle comprising a recombinant viral genome, wherein: the vector system or recombinant viral genome encodes a human CD59 polypeptide, wherein the encoded human CD59 polypeptide is under the control of a tissue-specific promoter or miRNA binding element that has selective activity in the desired cell, tissue, or organ.
  • the one or more cells are endothelial cells.
  • the one or more cells are CNS cells.
  • the one or more cells are of the CNS, lungs, kidneys, liver, or any combination thereof.
  • the endothelial cells are of the CNS.
  • the recombinant viral particle optionally an AAV viral particle, comprises a capsid polypeptide, optionally an AAV capsid polypeptide, wherein the capsid polypeptide comprises a CNS specific CD59 targeting moiety.
  • a humanized transgenic non-human animal comprising: one or more cells expressing a human CD59 polypeptide, optionally wherein the one or more cells are CNS cells.
  • the transgenic non-human animal is a rodent, optionally a mouse.
  • the humanized non-human animal has a suppressed immune system.
  • the modification may occur ex vivo or in vitro, for instance in a cell culture and in some instances not in vivo. In other embodiments, it may occur in vivo.
  • the disclosure provides a method of modifying an organism or a non-human organism by manipulation of a target sequence in a genomic locus of interest comprising: Delivering, e.g., via particle(s) or nanoparticle(s) or vector(s) (e.g., viral vector, e.g., AAV, adenovirus) a non-naturally occurring or engineered composition.
  • a single cell or a population of cells may preferably be modified ex vivo and then reintroduced, e.g., transplanted to make transgenic organisms that express CD59 in certain cells.
  • the disclosure in an embodiment, comprehends a method of modifying a eukaryote, such as a transgenic eukaryote comprising delivering, e.g., via vector(s) and/or particle(s) and/or nanoparticles a non-naturally occurring or engineered composition.
  • the system may comprise one, two, three or four different vectors; and the system may comprise one, two, three or four different nanoparticle complex(es) delivering the component(s) of the system.
  • Components I, II, III and IV may thus be located on one, two, three or four different vectors, and may be delivered by one, two, three or four different particle or nanoparticle complex(es) or AAVs or components I, II, III and IV can be located on same or different vector(s) / parti cle(s) / nanoparti cle(s), with all combinations of locations envisaged. And complexes that target the CNS or CNS tissue or CNS cells are advantageous.
  • vectors are delivered to the eukaryotic cell in a transgenic eukaryote.
  • said modifying takes place in said eukaryotic cell in a cell culture.
  • the disclosure provides a method of generating a model eukaryotic cell or a model transgenic eukaryote comprising one or more human proteins.
  • the present disclosure provides a transgenic eukaryote, e.g., mouse.
  • the present disclosure provides a constitutive transgenic eukaryote, e.g., mouse line obtained by crossing of the transgenic mouse with another mouse line.
  • progeny (or progenies) derived from said transgenic eukaryote, e.g., mouse line may be successfully bred over at least five generations without exhibiting increased levels of genome instability or cellular toxicity.
  • the present disclosure provides a method for simultaneously introducing multiple mutations ex vivo in a tissue, organ or a cell line (of the CNS), or in vivo in the same.
  • transgenic non-human eukaryote e.g., animal model with multiple mutations in any number of loci can be envisioned and are within the scope of the present disclosure.
  • transgenic non-human eukaryote e.g., animal model provides a valuable tool for research purposes, e.g., viral transduction, and opens the door for developing and testing new therapeutic interventions for targeting specific tissue involving mutations at multiple loci.
  • Such uses are within the scope of the present disclosure.
  • the eukaryotic cell can comprise a constitutive promoter, or a tissue specific promoter, or an inducible promoter; and, the eukaryotic cell can be part of a non-human transgenic eukaryote, e.g., a non-human mammal, primate, rodent, mouse, rat, rabbit, canine, dog, cow, bovine, sheep, ovine, goat, pig, fowl, poultry, chicken, fish, insect or arthropod; advantageously a mouse.
  • a non-human transgenic eukaryote e.g., a non-human mammal, primate, rodent, mouse, rat, rabbit, canine, dog, cow, bovine, sheep, ovine, goat, pig, fowl, poultry, chicken, fish, insect or arthropod; advantageously a mouse.
  • the isolated eukaryotic cell or the non-human transgenic eukaryote can express an additional protein or enzyme, such as CD59 ; and, the expression of CD59 can be driven by coding therefor functionally or operatively linked to a constitutive promoter, or a tissue specific promoter, or an inducible promoter.
  • the eukaryotic cell can be a mammalian cell, e.g., a mouse cell, such as a mouse cell that is part of a transgenic mouse having cells that express CD59 receptor .
  • Transgenic non-human eukaryotic organisms e.g., animals are also provided in an aspect of practice of the instant disclosure.
  • Preferred examples include animals comprising CD59, in terms of polynucleotides encoding CD59or the protein itself.
  • the disclosure involves a constitutive or conditional or inducible CD59 non-human eukaryotic organism, such as an animal, e.g., a primate, rodent, e.g., mouse, rat and rabbit, are preferred; and can include a canine or dog, livestock (cow / bovine, sheep / ovine, goat or pig), fish, fowl or poultry, e.g., chicken, and an insect or arthropod, with it mentioned that it is advantageous if the animal is a model as to a human or animal protein, cell, or tissue, as use of the non-human eukaryotic organisms in condition modeling, e.g., via inducing a plurality, are preferred.
  • a constitutive or conditional or inducible CD59 non-human eukaryotic organism such as an animal, e.g., a primate, rodent, e.g., mouse, rat and rabbit, are preferred; and can include a canine or dog, livestock (cow
  • transgenic mice with the constructs, as exemplified herein one may inject pure, linear DNA into the pronucleus of a zygote from a pseudo pregnant female, e.g. a CB56 female. Founders may then be identified, genotyped, and backcrossed to CB57 mice. The constructs may then be cloned and optionally verified, for instance by Sanger sequencing. Knock ins are envisaged (alone or in combination).
  • the disclosure involves a non-human eukaryote, animal, mammal, primate, rodent, etc. or cell thereof or tissue thereof that may be used as a model.
  • a method of the disclosure may be used to create a non-human eukaryote, e.g., an animal, mammal, primate, rodent or cell that comprises a modification in one or more nucleic acid sequences associated or correlated with a cell or tissue of such (such as the CNS).
  • the cell may be in vivo or ex vivo in the cases of multicellular organisms.
  • a cell line may be established if appropriate culturing conditions are met and preferably if the cell is suitably adapted for this purpose (for instance a stem cell).
  • cell lines are also envisaged.
  • the disclosure can involve cells, e.g., non-human eukaryotic, e.g., animal, such as mammal, e.g., primate, rodent, mouse, rat, rabbit, etc., or even human cells, transformed to contain CD59, e g., such cells as to which a vector that contains nucleic acid molecule(s) encoding a CD59, e.g., with nucleic acid(s) encoding a promoter and at least one NLS, advantageously two or more NLSs, or such cells that have had their genome altered, e.g., through the vector being an integrating virus or through such cells being stem cells or cells that give rise to a cell line or a living organism (but where
  • Such cells are then transplanted into or onto an animal suitable for expressing a cell or tissue.
  • the cells proliferate on or in the non-human eukaryote, e.g., animal model.
  • the non-human eukaryote, e.g., animal model, having the proliferated heterologous transplanted CD59-containing cells is then administered RNA(s) or vector(s), e g., AAV, adenovirus containing or providing RNA(s), e.g., under the control of a promoter such as a U6 promoter and/or particle/ s) and/or nanoparticle(s).
  • the non- human eukaryote e.g., animal model can then be used for testing, e.g., as to potential therapy and/or putative treatment via a possibly pharmaceutically active compound.
  • the administering can be at or to or for body delivery to the proliferated heterologous transplanted CD59-containing cells, e.g., direct injection at or near such proliferated heterologous transplanted CD59-containing cells, or inj ection or other administration in such a way that the RNA(s) are delivered into the proliferated heterologous transplanted CD59-containing cells, e.g., injection into the bloodstream whereby bodily functions transport to the proliferated heterologous transplanted CD59-containing cells.
  • barcoding techniques of WO/2013/138585 Al can be adapted or integrated into the practice of the disclosure.
  • WO/2013/138585 Al provides methods for simultaneously determining the effect of a test condition on viability or proliferation of each of a plurality of genetically heterogeneous cell types. The number of living cells in the sample after exposure to the test condition as compared to the reference number of cells indicates the effect of the test condition on viability or proliferation of each cell type.
  • WO/2013/138585 Al also provides methods for simultaneously determining the effect of a test condition on viability or proliferation of each of a plurality of genetically heterogeneous cell types. Referenced patent applications are hereby incorporated by reference.
  • a method of screening an AAV library for a recombinant AAV particle which binds to CD59 and/or has increased tropism for the CNS comprising assaying the AAV library of any of those described herein for increased binding of a CD59 and/or tropism for the CNS relative to an AAV vector with a reference capsid and selecting those recombinant AAV vectors which have increased binding of CD59 and/or tropism for the CNS.
  • the composition is a viral particle comprising one or more capsid proteins each comprising the candidate CD59 targeting moiety.
  • at least one of the one or more compositions furthers comprises a cargo.
  • the cargo is or encodes a therapeutic nucleic acid or polypeptide, a selectable marker, or a control polypeptide or nucleic acid.
  • a method of screening CD59 targeting moieties including incorporated within an AAV, capable of transduction of CNS tissues via binding to a CD59 in humanized transgenic non-human animals comprising: (a) introducing a plurality of vector systems to one or more humanized transgenic non-human animal that express the human CD59 and (b) detecting the CD59 targeting moiety that binds to a CD59.
  • the engineered AAV capsid system vectors, engineered cells, and/or engineered AAV capsid particles described herein can be used in a screening assay and/or cell selection assay.
  • the engineered delivery system vectors, engineered cells, and/or engineered AAV capsid particles can be delivered to a subject and/or cell.
  • the cell is a eukaryotic cell.
  • the cell can be in vitro, ex vivo, in situ, or in vivo.
  • the engineered AAV capsid system molecules, vectors, engineered cells, and/or engineered AAV capsid particles described herein can introduce an exogenous molecule or compound to subject or cell to which they are delivered.
  • the presence of an exogenous molecule or compound can be detected which can allow for identification of a cell and/or attribute thereof.
  • the delivered molecules or particles can impart a gene or other nucleotide modification (e.g., mutations, gene or polynucleotide insertion and/or deletion, etc.).
  • the nucleotide modification can be detected in a cell by sequencing.
  • the nucleotide modification can result in a physiological and/or biological modification to the cell that results in a detectable phenotypic change in the cell, which can allow for detection, identification, and/or selection of the cell.
  • the phenotypic change can be cell death, such as embodiments where binding of a CRISPR complex to a target polynucleotide results in cell death.
  • Embodiments of the disclosure allow for selection of specific cells without requiring a selection marker or a two-step process that may include a counterselection system.
  • the cell(s) may be prokaryotic or eukaryotic cells.
  • the disclosure provides for a method of selecting one or more cell(s) by introducing one or more mutations in a gene in the one or more cell(s), the method comprising: introducing one or more vectors, which can include one or more engineered delivery system molecules or vectors described elsewhere herein, into the cell(s), wherein the one or more vectors can include a CRISPR enzyme and/or drive expression of one or more of: a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template; or other polynucleotide to be inserted into the cell and/or genome thereof; wherein, for example that which is being expressed is within and expressed in vivo by the CRISPR enzyme and/or the editing template, when included, comprises the one or more mutations that abolish CRISPR enzyme cleavage; allowing homologous recombination of the editing template with the target polynucleotide in the cell(s) to be selected; allowing a CRISPR complex to
  • the screening methods involving the engineered AAV capsid system molecules, vectors, engineered cells, and/or engineered AAV capsid particles can be used in detection methods such as fluorescence in situ hybridization (FISH).
  • FISH fluorescence in situ hybridization
  • one or more components of an engineered CRISPR-Cas system that includes a catalytically inactive Cas protein can be delivered by an engineered AAV capsid system molecule, engineered cell, and/or engineered AAV capsid particle described elsewhere herein to a cell and used in a FISH method.
  • the CRISPR-Cas system can include an inactivated Cas protein (dCas) (e.g., a dCas9), which lacks the ability to produce DNA double-strand breaks may be fused with a marker, such as fluorescent protein, such as the enhanced green fluorescent protein (eEGFP) and co-expressed with small guide RNAs to target pericentric, centric and teleomeric repeats in vivo.
  • dCas inactivated Cas protein
  • eEGFP enhanced green fluorescent protein
  • the dCas system can be used to visualize both repetitive sequences and individual genes in the human genome.
  • Such new applications of labelled dCas, dCas CRISPR-Cas systems, engineered AAV capsid system molecules, engineered cells, and/or engineered AAV capsid particles can be used in imaging cells and studying the functional nuclear architecture, especially in cases with a small nucleus volume or complex 3-D structures.
  • a similar approach involving a polynucleotide fused to a marker can be delivered to a cell via an engineered AAV capsid system molecule, vector, engineered cell, and/or engineered AAV capsid particle described herein and integrated into the genome of the cell and/or otherwise interact with a region of the genome of a cell for FISH analysis.
  • a marker e.g., a fluorescent marker
  • Similar approaches for studying other cell organelles and other cell structures can be accomplished by delivering to the cell (e.g., via an engineered delivery AAV capsid molecule, engineered cell, and/or engineered AAV capsid particle described herein) one or more molecules fused to a marker (such as a fluorescent marker), wherein the molecules fused to the marker are capable of targeting one or more cell structures.
  • a marker such as a fluorescent marker
  • the engineered AAV capsid system molecules and/or engineered AAV capsid particles can be used in a screening assay inside or outside of a cell.
  • the screening assay can include delivering a CRISPR-Cas cargo molecule(s) via an engineered AAV capsid particle.
  • the disclosure provides a cell from or of an in vitro method of delivery, wherein the method comprises contacting the delivery system with a cell, optionally a eukaryotic cell, whereby there is delivery into the cell of constituents of the delivery system, and optionally obtaining data or results from the contacting, and transmitting the data or results.
  • the disclosure provides a cell from or of an in vitro method of delivery, wherein the method comprises contacting the delivery system with a cell, optionally a eukaryotic cell, whereby there is delivery into the cell of constituents of the delivery system, and optionally obtaining data or results from the contacting, and transmitting the data or results; and wherein the cell product is altered compared to the cell not contacted with the delivery system, for example altered from that which would have been wild type of the cell but for the contacting.
  • the cell product is non-human or animal.
  • the cell product is human.
  • a host cell is transiently or non-transiently transfected with one or more vectors described herein.
  • a cell is transfected as it naturally occurs in a subject optionally to be reintroduced therein.
  • a cell that is transfected is taken from a subject.
  • the engineered AAV capsid system molecule(s) and/or engineered AAV capsid particle(s) directly to the host cell.
  • the engineered AAV capsid system molecule(s) can be delivered together with one or more cargo molecules to be packaged into an engineered AAV capsid particle.
  • Machine learning is a field of study within artificial intelligence that allows computers to learn functional relationships between inputs and outputs without being explicitly programmed.
  • Machine learning involves a module comprising algorithms that may learn from existing data by analyzing, categorizing, or identifying the data.
  • Such machine-learning algorithms operate by first constructing a model from training data to make predictions or decisions expressed as outputs.
  • the training data includes data for one or more identified features and one or more outcomes, for example a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein and one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • Data supplied to a machine learning algorithm can be considered a feature, which can be described as an individual measurable property of a phenomenon being observed.
  • the concept of feature is related to that of an independent variable used in statistical techniques such as those used in linear regression.
  • the performance of a machine learning algorithm in pattern recognition, classification and regression is highly dependent on choosing informative, discriminating, and independent features.
  • Features may comprise numerical data, categorical data, time-series data, strings, graphs, or images.
  • Features of the invention may further include a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein.
  • AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein may include any of the sequences described herein.
  • the input may include EFNNGSD (SEQ ID NO: 89) or GAASLMP (SEQ ID NO: 109).
  • Classification problems also referred to as categorization problems
  • Training data teaches the classifying algorithm how to classify.
  • features to be categorized may include a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein, which can be provided to the classifying machine learning algorithm and then placed into categories of, for example, one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • Regression algorithms aim at quantifying and correlating one or more features.
  • Training data teaches the regression algorithm how to correlate the one or more features into a quantifiable value.
  • features such as a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein can be provided to the regression machine learning algorithm resulting in one or more continuous values, for example, of one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • the machine learning module can be trained using techniques such as unsupervised, supervised, semi-supervised, reinforcement learning, transfer learning, incremental learning, curriculum learning techniques, and/or learning to learn. Training typically occurs after selection and development of a machine learning module and before the machine learning module is operably in use.
  • the training data used to teach the machine learning module can comprise input data such as a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein and the respective target output data such as one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • unsupervised learning is implemented.
  • Unsupervised learning can involve providing all or a portion of unlabeled training data to a machine learning module.
  • the machine learning module can then determine one or more outputs implicitly based on the provided unlabeled training data.
  • supervised learning is implemented.
  • Supervised learning can involve providing all or a portion of labeled training data to a machine learning module, with ilie machine learning module determining one or more outputs based on the provided labeled training data, and the outputs are either accepted or corrected depending on the agreement to the actual outcome of the training data.
  • supervised learning of machine learning system(s) can be governed by a set of rules and/or a set of labels for the training input, and the set of rules and/or set of labels may be used to correct inferences of a machine learning module.
  • semi-supervised learning is implemented.
  • Semi-supervised learning can involve providing all or a portion of training data that is partially labeled to a machine learning module.
  • semi-supervised learning is used for a portion of labeled training data
  • unsupervised learning is used for a portion of unlabeled training data.
  • reinforcement learning is implemented. Reinforcement learning can involve first providing all or a portion of the training data to a machine learning module and as the machine learning module produces an output, the machine learning module receives a “reward” signal in response to a correct output.
  • the reward signal is a numerical value and the machine learning module is developed to maximize the numerical value of the reward signal.
  • reinforcement learning can adopt a value function that provides a numerical value representing an expected total of the numerical values provided by the reward signal over time.
  • Transfer learning is implemented. Transfer learning techniques can involve providing all or a portion of a first training data to a machine learning module, then, after training on the first training data, providing all or a portion of a second training data.
  • a first machine learning module can be pre-trained on data from one or more computing devices. The first trained machine learning module is then provided to a computing device, where the computing device is intended to execute the first trained machine learning model to produce an output. Then, during the second training phase, the first trained machine learning model can be additionally trained using additional training data, where the training data can be derived from kernel and non-kernel data of one or more computing devices.
  • This second training of the machine learning module and/or the first trained machine learning model using the training data can be performed using either supervised, unsupervised, or semi-supervised learning.
  • transfer learning techniques can involve one, two, three, or more training attempts.
  • Incremental learning techniques can involve providing a trained machine learning module with input data that is used to continuously extend the knowledge of the trained machine learning module.
  • Another machine learning training technique is curriculum learning, which can involve training the machine learning module with training data arranged in a particular order, such as providing relatively easy training examples first, then proceeding with progressively more difficult training examples.
  • difficulty of training data is analogous to a curriculum or course of study at a school.
  • learning to learn is implemented.
  • Learning to learn, or metalearning comprises, in general, two levels of learning: quick learning of a single task and slower learning across many tasks.
  • a machine learning module is first trained and comprises of a first set of parameters or weights. During or after operation of the first trained machine learning module, the parameters or weights are adjusted by the machine learning module. This process occurs iteratively on the success of the machine learning module.
  • an optimizer or another machine learning module, is used wherein the output of a first trained machine learning module is fed to the optimizer that constantly learns and returns the final results.
  • Other techniques for training the machine learning module and/or trained machine learning module are possible as well. Contrastive Learning
  • contrastive learning is implemented.
  • Contrastive learning is a self-supervised model of learning in which training data is unlabeled and is considered as a form of learning in-between supervised and unsupervised learning.
  • This method learns by contrastive loss, which separates unrelated (i.e., negative) data pairs and connects related (i.e., positive) data pairs.
  • contrastive loss which separates unrelated (i.e., negative) data pairs and connects related (i.e., positive) data pairs.
  • to create positive and negative data pairs more than one view of a datapoint, such as rotating an image or using a different time-point of a video, is used as input.
  • Positive and negative pairs are learned by solving dictionary look-up problem.
  • the two views are separated into query and key of a dictionary.
  • a query has a positive match to a key and negative match to all other keys.
  • the machine learning module learns by connecting queries to their keys and separating queries from their non-keys.
  • a loss function such as those described herein, is used to minimize the distance between positive data pairs (e.g., a query to its key) while maximizing the distance between negative data points. See e.g., Tian, Yonglong, etal. "What makes for good views for contrastive learning?.” Advances in Neural Information Processing Systems 33 (2020): 6827- 6839.
  • the machine learning module is pre-trained.
  • a pre-trained machine learning model is a model that has been previously trained to solve a similar problem.
  • the pretrained machine learning model is generally pre-trained with similar input data to that of the new problem.
  • a pre-trained machine learning model further trained to solve a new problem is generally referred to as transfer learning, which is described herein.
  • transfer learning which is described herein.
  • a pre-trained machine learning model is trained on a large dataset of related information. The pre-trained model is then further trained and tuned for the new problem.
  • Using a pre-trained machine learning module provides the advantage of building a new machine learning module with input neurons/nodes that are already familiar with the input data and are more readily refined to a particular problem.
  • a machine learning module previously trained using a plurality of AAV capsid polypeptide sequences comprising a targeting moieties may be further trained to estimate one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off- target tissues.
  • a machine learning module previously trained using a plurality of AAV capsid polypeptide sequences comprising a targeting moieties may be further trained to estimate one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off- target tissues.
  • a trained machine learning module can be provided to a computing device where a trained machine learning module is not already resident, in other words, after training phase has been completed, the trained machine learning module can be downloaded to a computing device.
  • a first computing device storing a trained machine learning module can provide the trained machine learning module to a second computing device.
  • Providing a trained machine learning module to the second computing device may comprise one or more of communicating a copy of trained machine learning module to the second computing device, making a copy of trained machine learning module for the second computing device, providing access to trained machine learning module to the second computing device, and/or otherwise providing the trained machine learning system to the second computing device.
  • a trained machine learning module can be used by the second computing device immediately after being provided by the first computing device. In some examples, after a trained machine learning module is provided to the second computing device, the trained machine learning module can be installed and/or otherwise prepared for use before the trained machine learning module can be used by the second computing device.
  • a trained machine learning module can receive input data and operably generate results. As such, the input data can be used as an input to the trained machine learning module for providing corresponding results to kernel components and non-kemel components. For example, a trained machine learning module can generate results in response to requests.
  • a trained machine learning module can be executed by a portion of other software. For example, a trained machine learning module can be executed by a result daemon to be readily available to provide results upon request.
  • a machine learning module and/or trained machine learning module can be executed and/or accelerated using one or more computer processors and/or on-device coprocessors.
  • Such on-device co-processors can speed up training of a machine learning module and/or generation of results.
  • trained machine learning module can be trained, reside, and execute to provide results on a particular computing device, and/or otherwise can make results for the particular computing device.
  • Input data can include data from a computing device executing a trained machine learning module and/or input data from one or more computing devices.
  • a trained machine learning module can use results as input feedback.
  • a trained machine learning module can also rely on past results as inputs for generating new results.
  • input data can comprise a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein and, when provided to a trained machine learning module, results in output data such as one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • linear regression machine learning is implemented.
  • LiR is typically used in machine learning to predict a result through the mathematical relationship between an independent and dependent variable, such as a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein and one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues, respectively.
  • a simple linear regression model would have one independent variable (x) and one dependent variable (y).
  • the machine learning algorithm tries variations of the tuning variables m and b to optimize a line that includes all the given training data.
  • the tuning variables can be optimized, for example, with a cost function.
  • a cost function takes advantage of the minimization problem to identify the optimal tuning variables.
  • the minimization problem preposes the optimal tuning variable will minimize the error between the predicted outcome and the actual outcome.
  • An example cost function may comprise summing all the square differences between the predicted and actual output values and dividing them by the total number of input values and results in the average square error.
  • the machine learning module may use, for example, gradient descent methods.
  • An example gradient descent method comprises evaluating the partial derivative of the cost function with respect to the tuning variables. The sign and magnitude of the partial derivatives indicate whether the choice of a new tuning variable value will reduce the cost function, thereby optimizing the linear regression algorithm. A new tuning variable value is selected depending on a set threshold. Depending on the machine learning module, a steep or gradual negative slope is selected.
  • Both the cost function and gradient descent can be used with other algorithms and modules mentioned throughout. For the sake of brevity, both the cost function and gradient descent are well known in the art and are applicable to other machine learning algorithms and may not be mentioned with the same detail.
  • LiR models may have many levels of complexity comprising one or more independent variables. Furthermore, in an LiR function with more than one independent variable, each independent variable may have the same one or more tuning variables or each, separately, may have their own one or more tuning variables. The number of independent variables and tuning variables will be understood to one skilled in the art for the problem being solved.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein are used as the independent variables to train a LiR machine learning module, which, after training, is used to estimate, for example, one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • logistic regression machine learning is implemented.
  • Logistic Regression often considered a LiR type model, is typically used in machine learning to classify information, such as a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein into categories such as one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • LoR takes advantage of probability to predict an outcome from input data. However, what makes LoR different from a LiR is that LoR uses a more complex logistic function, for example a sigmoid function.
  • the cost function can be a sigmoid function limited to a result between 0 and 1.
  • the tuning variable(s) of the cost function are optimized (typically by taking the log of some variation of the cost function) such that the result of the cost function, given variable representations of the input features, is a number between 0 and 1, preferably falling on either side of 0.5.
  • gradient descent may also be used in LoR cost function optimization and is an example of the process.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein are used as the independent variables to train a LoR machine learning module, which, after training, is used to estimate, for example, one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • a Bayesian Network is implemented.
  • BNs are used in machine learning to make predictions through Bayesian inference from probabilistic graphical models.
  • input features are mapped onto a directed acyclic graph forming the nodes of the graph.
  • the edges connecting the nodes contain the conditional dependencies between nodes to form a predicative model.
  • For each connected node the probability of the input features resulting in the connected node is learned and forms the predictive mechanism.
  • the nodes may comprise the same, similar or different probability functions to determine movement from one node to another.
  • the nodes of a Bayesian network are conditionally independent of its non-descendants given its parents thus satisfying a local Markov property.
  • the first method involves computing the joint probability of a particular assignment of values for each variable.
  • the joint probability can be considered the product of each conditional probability and, in some instances, comprises the logarithm of that product.
  • the second method is Markov chain Monte Carlo (MCMC), which can be implemented when the sample size is large.
  • MCMC is a well-known class of sample distribution algorithms and will not be discussed in detail herein.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein are mapped to the BN graph to train the BN machine learning module, which, after training, is used to estimate one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off- target tissues.
  • RF random forest
  • RF consists of an ensemble of decision trees producing individual class predictions. The prevailing prediction from the ensemble of decision trees becomes the RF prediction.
  • Decision trees are branching flowchart-like graphs comprising of the root, nodes, edges/branches, and leaves.
  • the root is the first decision node from which feature information is assessed and from it extends the first set of edges/branches.
  • the edges/branches contain the information of the outcome of a node and pass the information to the next node.
  • the leaf nodes are the terminal nodes that output the prediction.
  • Decision trees can be used for both classification as well as regression and is typically trained using supervised learning methods. Training of a decision tree is sensitive to the training data set.
  • An individual decision tree may become over or under-fit to the training data and result in a poor predictive model.
  • Random forest compensates by using multiple decision trees trained on different data sets.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein are used to train the nodes of the decision trees of a RF machine learning module, which, after training, is used to estimate one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • the RF has any two or more decision trees.
  • the RF has 2 trees, 3 trees, 4 trees, 5 trees, 6 trees, 7 trees, 8 trees, 9 trees, 10 trees, 11 trees, 12 trees, 13 trees, 14 trees, 15 trees, 16 trees, 17 trees, 18 trees, 19 trees, 20 trees, 21 trees, 22 trees, 23 trees,
  • gradient boosting is implemented.
  • Gradient boosting is a method of strengthening the evaluation capability of a decision tree node.
  • a tree is fit on a modified version of an original data set. For example, a decision tree is first trained with equal weights across its nodes. The decision tree is allowed to evaluate data to identify nodes that are less accurate. Another tree is added to the model and the weights of the corresponding underperforming nodes are then modified in the new tree to improve their accuracy. This process is performed iteratively until the accuracy of the model has reached a defined threshold or a defined limit of trees has been reached. Less accurate nodes are identified by the gradient of a loss function. Loss functions must be differentiable such as a linear or logarithmic functions.
  • the modified node weights in the new tree are selected to minimize the gradient of the loss function.
  • a decision tree is implemented to determine one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues and gradient boosting is applied to the tree to improve its ability to accurately determine the one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • NNs are implemented.
  • NNs are a family of statistical learning models influenced by biological neural networks of the brain.
  • NNs can be trained on a relatively large dataset (e g., 50,000 or more) and used to estimate, approximate, or predict an output that depends on a large number of inputs/features.
  • NNs can be envisioned as so- called “neuromorphic” systems of interconnected processor elements, or “neurons”, and exchange electronic signals, or “messages”.
  • connections in NNs that carry electronic “messages” between “neurons” are provided with numeric weights that correspond to the strength or weakness of a given connection.
  • the weights can be tuned based on experience, making NNs adaptive to inputs and capable of learning.
  • an NN for one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues is defined by a set of input neurons that can be given input data such as a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein.
  • the input neuron weighs and transforms the input data and passes the result to other neurons, often referred to as “hidden” neurons. This is repeated until an output neuron is activated.
  • the activated output neuron produces a result.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein are used to train the neurons in a NN machine learning module, which, after training, is used to estimate one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • a NN layer (e.g., input, hidden, and output) has 1 or more neurons.
  • the NN layer has 1-500 neurons.
  • the NN layer has about 1 neuron, 5 neurons, 10 neurons, 15 neurons, 20 neurons, 25 neurons, 30 neurons, 35 neurons, 40 neurons, 45 neurons, 50 neurons, 55 neurons, 60 neurons, 65 neurons, 70 neurons, 75 neurons, 80 neurons, 85 neurons, 90 neurons, 95 neurons, 100 neurons, 105 neurons, 110 neurons, 115 neurons, 120 neurons, 125 neurons, 130 neurons, 135 neurons, 140 neurons, 145 neurons, 150 neurons, 155 neurons, 160 neurons, 165 neurons, 170 neurons, 175 neurons, 180 neurons, 185 neurons, 190 neurons, 195 neurons, 200 neurons, 205 neurons, 210 neurons, 215 neurons, 220 neurons, 225 neurons, 230 neurons, 235 neurons, 240 neurons, 245 neurons, 250 neurons, 255 neurons, 260 neurons, 265 neurons, 270 neurons, 275 neurons, 280 neurons, 2
  • convolutional autoencoder is implemented.
  • a CAE is a type of neural network and includes, in general, two main components. First, the convolutional operator that filters an input signal to extract features of the signal. Second, an autoencoder that learns a set of signals from an input and reconstructs the signal into an output. By combining these two components, the CAE learns the optimal filters that minimize reconstruction error resulting an improved output. CAEs are trained to only learn filters capable of feature extraction that can be used to reconstruct the input.
  • convolutional autoencoders implement unsupervised learning.
  • the convolutional autoencoder is a variational convolutional autoencoder.
  • features from a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein are used as an input signal into a CAE which reconstructs that signal into an output such as one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • Deep learning expands the neural network by including more layers of neurons.
  • a deep learning module is characterized as having three “macro” layers: (1) an input layer which takes in the input features, and fetches embeddings for the input, (2) one or more intermediate (or hidden) layers which introduces nonlinear neural net transformations to the inputs, and (3) a response layer which transforms the final results of the intermediate layers to the prediction.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein are used to train the neurons of a deep learning module, which, after training, is used to estimate one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • CNN Convolutional Neural Network
  • CNNs is a class of NNs further attempting to replicate the biological neural networks, but of the animal visual cortex.
  • CNNs process data with a grid pattern to learn spatial hierarchies of features.
  • NNs are highly connected, sometimes fully connected, CNNs are connected such that neurons corresponding to neighboring data (e.g., pixels) are connected. This significantly reduces the number of weights and calculations each neuron must perform.
  • input data such as a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein
  • a CNN typically, comprises of three layers: convolution, pooling, and fully connected.
  • the convolution and pooling layers extract features and the fully connected layer combines the extracted features into an output, such as one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • the convolutional layer comprises of multiple mathematical operations such as of linear operations, a specialized type being a convolution.
  • the convolutional layer calculates the scalar product between the weights and the region connected to the input volume of the neurons. These computations are performed on kernels, which are reduced dimensions of the input vector.
  • the kernels span the entirety of the input.
  • the rectified linear unit i.e., ReLu
  • applies an elementwise activation function e.g., sigmoid function
  • CNNs can optimized with hyperparameters.
  • there three hyperparameters are used: depth, stride, and zero-padding.
  • Depth controls the number of neurons within a layer. Reducing the depth may increase the speed of the CNN but may also reduce the accuracy of the CNN.
  • Stride determines the overlap of the neurons.
  • Zero-padding controls the border padding in the input.
  • the pooling layer down-samples along the spatial dimensionality of the given input (i.e., convolutional layer output), reducing the number of parameters within that activation.
  • kernels are reduced to dimensionalities of 2x2 with a stride of 2, which scales the activation map down to 25%.
  • the fully connected layer uses inter-layer-connected neurons (i.e., neurons are only connected to neurons in other layers) to score the activations for classification and/or regression. Extracted features may become hierarchically more complex as one layer feeds its output into the next layer. See O’Shea, K.; Nash, R. An Introduction to Convolutional Neural Networks. arXiv 2015 and Yamashita, R., el al Convolutional neural networks: an overview and application in radiology. Insights Imaging 9, 611-629 (2016).
  • convolutional autoencoder is implemented.
  • a CAE is a type of neural network and comprises, in general, two main components. First, the convolutional operator that filters an input signal to extract features of the signal. Second, an autoencoder that learns a set of signals from an input and reconstructs the signal into an output. By combining these two components, the CAE leams the optimal filters that minimize reconstruction error resulting an improved output. CAEs are trained to only learn filters capable of feature extraction that can be used to reconstruct the input.
  • convolutional autoencoders implement unsupervised learning.
  • the convolutional autoencoder is a variational convolutional autoencoder.
  • features from a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein are used as an input signal into a CAE which reconstructs that signal into an output such as one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off- target tissues.
  • features from a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein are used as an input signal into a CAE which reconstructs that signal into an output such as one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • a recurrent neural network is implemented.
  • RNNs are class of NNs further attempting to replicate the biological neural networks of the brain.
  • RNNs comprise of delay differential equations on sequential data or time series data to replicate the processes and interactions of the human brain.
  • RNNs have “memory” wherein the RNN can take information from prior inputs to influence the current output.
  • RNNs can process variable length sequences of inputs by using their “memory” or internal state information. Where NNs may assume inputs are independent from the outputs, the outputs of RNNs may be dependent on prior elements with the input sequence.
  • a RNN determines one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • RNN recurrent neural network
  • LSTM long short-term memory
  • LSTM are a class of RNNs designed to overcome vanishing and exploding gradients.
  • long term dependencies become more difficult to capture because the parameters or weights either do not change with training or fluctuate rapidly. This occurs when the RNN gradient exponentially decreases to zero, resulting in no change to the weights or parameters, or exponentially increases to infinity, resulting in large changes in the weights or parameters. This exponential effect is dependent on the number of layers and multiplicative gradient.
  • LSTM overcomes the vanishing/exploding gradients by implementing “cells” within the hidden layers of the NN.
  • the “cells” comprise three gates: an input gate, an output gate, and a forget gate.
  • the input gate reduces error by controlling relevant inputs to update the current cell state.
  • the output gate reduces error by controlling relevant memory content in the present hidden state.
  • the forget gate reduces error by controlling whether prior cell states are put in “memory” or forgotten.
  • the gates use activation functions to determine whether the data can pass through the gates. While one skilled in the art would recognize the use of any relevant activation function, example activation functions are sigmoid, tanh, and RELU. See Zhu, Xiaodan, et al. "Long short-term memory over recursive structures.” International Conference on Machine Learning. PMLR, 2015.
  • the machine learning network comprises a generative adversarial network (GAN).
  • GAN generative adversarial network
  • a generative model in general, can summarize the distribution of input variables and generates new input variables in the input distribution.
  • a (GAN) may have learned a distribution, such as a Gaussian distribution, for a variable.
  • the GAN can then summarize the data distribution, and then generate new input variables that fit into the distribution.
  • GANs may include two components: a generator and a discriminator.
  • a generator creates new input variables from the learned space.
  • the generator takes a, for example, fixed-length random vector, which is drawn from a Gaussian distribution, as input and generates a sample in the domain.
  • the multidimensional vector space forms a compressed representation of the data distribution.
  • a discriminator determines whether an input variable is real (i.e., a user input) or fake (i.e., generated) using a binary class label.
  • a discriminator can be used in transfer learning.
  • the discriminator “trains” the generator to produce input variables that are indiscernible from the user input data. See e.g., Goodfellow, I. J.; Pouget-Abadie, J.; Mirza, M.; Xu, B.; Warde- Farley, D.; Ozair, S.; Courville, A.; Bengio, Y. Generative Adversarial Networks. arXiv 2014.
  • the input data comprises of a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein and the GAN generates one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • the generative adversarial network comprises a Wasserstein Generative Adversarial Network (WGAN).
  • WGAN modifies a GAN by using a critic mechanism that scores the realness or fakeness of an input instead of a discriminator that may classify the realness or fakeness of an input.
  • the advantage to a critic over a discriminator may be, for example, minimizing the distance between the distribution of the data observed in the user input dataset and the distribution observed in generated input. In some instances, a continuous distribution is desired when a finer sensitivity to the input is needed.
  • the critic model comprises of a linear activation function.
  • the input data comprises of one or more target nucleic acid sequences and the WGAN generates one or more target binding oligonucleotides.
  • Diffusion Models are implemented.
  • a DM which can also be referred to as a diffusion probabilistic model or score-based generative model, is a type of generative model/latent variable model.
  • Generative models are a type of classification models used to create (i.e., generate) a target variable from an observable variable given a joint probability distribution.
  • a DM includes three (3) main components: the forward module (i.e., diffusion module), the reverse module (i.e., reverse diffusion module), and the sampling module.
  • the forward module takes the input and perturbs (e.g., adds noise to) the data in a stepwise method.
  • the input data is an unequal distribution data
  • the forward module perturbs this distribution until the input data resembles a balanced distribution of data (e.g., Gaussian distribution, can be isotropic or non-isotropic).
  • Data perturbation can include the addition of data, removal of data, shifting/rearrangement of data, or combination thereof.
  • the forward module can either perturb the input data in discrete or continuous steps. In an embodiment, the perturbation to the input data is performed randomly.
  • the forward module can be configured to apply a particular type of perturbation based on the input data and the amount of perturbation per step. It is understood by one of ordinary skill in the art that the configuration is dependent on the input and output. The type of perturbation and the amount of perturbation per step can vary with the application of the described methods and systems. In an embodiment, the configuration is learned (e.g., by a neural network) or empirically designed.
  • the reverse module then takes the perturbed data as input and trains a denoising or deperturbation network (e.g., a neural network, an encoder/decoder network,) to learn how to reorganize the balanced distribution of data back into its original form.
  • Data reorganization can include the addition of data, removal of data, shifting/rearrangement of data, or combination thereof.
  • the reverse module reorganizes the distribution of data in stepwise method and the steps can be either discrete or continuous.
  • the reverse module can take a perturbed input and generate an output by reorganizing the data according to the learned distribution.
  • the sampling module uses the denoising/de-perturbation network trained by the reverse module to generate unique outputs from input data.
  • one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues is provided as input data to the forward process, which sufficiently perturbs the distribution of the one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • the reverse module learns how to return the perturbed data back to the original one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • the sampling module can then be given a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein and generate one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • ARs are implemented.
  • An AR is a type of time series model, which can learn and use relationships between a first set of data (i.e., the input) and a successive set of data (i.e., the output).
  • An AR can include an autocorrelation module (ACP), which describes the successive set of data to the first set of data.
  • ACP autocorrelation module
  • the relationship between the first set of data and successive set of data is generally linear, such as the relationship between a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein and one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues, and the AR can be used to model that linear relationship.
  • the AR can be an n-order autoregression models (i.e., wherein n is first-, second-, third-, . . ., n-), such that the output depends on n-related inputs.
  • An AR is generally trained by maximizing the likelihood score between an input and an output using an autoregressive factorization module.
  • Another example of an AR can be described as a Linear Regression Model (LiRM) wherein the dependent variable(s) are the first set of data.
  • the tuning variables are trained to generate the independent variable(s), which is the successive data.
  • the tuning variables determine the strength and direction of the relationship between the first set of data and the successive set of data as well as the reduce background noise/error.
  • ARs described as a LiRM are a unique class of LiRM wherein the dependent variables are constrained to data defined by time series.
  • the AR is vector autoregression (VAR) model or conditional autoregressive model (CAR) model.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein is processed by an AR to generate one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • an AR to generate one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • Matrix Factorization is implemented. Matrix Factorization machine learning exploits inherent relationships between two entities drawn out when multiplied together. Generally, the input features are mapped to a matrix F which is multiplied with a matrix R containing the relationship between the features and a predicted outcome. The resulting dot product provides the prediction. The matrix R is constructed by assigning random values throughout the matrix. In this example, two training matrices are assembled. The first matrix X contains training input features and the second matrix Z contains the known output of the training input features. First the dot product of R and X are computed and the square mean error, as one example method, of the result is estimated.
  • the values in R are modulated and the process is repeated in a gradient descent style approach until the error is appropriately minimized.
  • the trained matrix R is then used in the machine learning model.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein are used to train the relationship matrix R in a matrix factorization machine learning module.
  • the relationship matrix R and input matrix F which comprises vector representations of a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein, results in the prediction matrix P comprising one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • a hidden Markov model is implemented.
  • a HMM takes advantage of the statistical Markov model to predict an outcome.
  • a Markov model assumes a Markov process, wherein the probability of an outcome is solely dependent on the previous event. In the case of HMM, it is assumed an unknown or “hidden” state is dependent on some observable event.
  • a HMM comprises a network of connected nodes. Traversing the network is dependent on three model parameters: start probability; state transition probabilities; and observation probability. The start probability is a variable that governs, from the input node, the most plausible consecutive state. From there each node i has a state transition probability to node j.
  • the state transition probabilities are stored in a matrix Mij wherein the sum of the rows, representing the probability of state i transitioning to state j, equals 1.
  • the observation probability is a variable containing the probability of output o occurring.
  • N o j wherein the probability of output o is dependent on state j.
  • the state and output probabilities are computed. This can be accomplished with, for example, an inductive algorithm.
  • the state sequences are ranked on probability, which can be accomplished, for example, with the Viterbi algorithm.
  • the model parameters are modulated to maximize the probability of a certain sequence of observations.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein are used to train the nodes/states of the HMM machine learning module, which, after training, is used to estimate one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off- target tissues.
  • the HMM can have any number of states.
  • the HMM has 1 states, 2 states, 3 states, 4 states, 5 states, 6 states, 7 states, 8 states, 9 states, 10 states, 11 states, 12 states, 13 states, 14 states, 15 states, 16 states, 17 states, 18 states, 19 states, 20 states, 21 states, 22 states, 23 states, 24 states, 25 states, 26 states, 27 states, 28 states, 29 states, 30 states, 31 states, 32 states, 33 states, 34 states, 35 states, 36 states, 37 states, 38 states, 39 states, 40 states, 41 states, 42 states, 43 states, 44 states, 45 states, 46 states, 47 states, 48 states, 49 states, 50 states, or any range between any two number of states listed.
  • the HMM has 1-5 states, 1-10 states, 1-15 states, 1-20 states, 1-25 states, 1-30 states, 1-35 states, 1-40 states, 1-45 states, or 1-50 states.
  • the HMM has 0-5 states, 5-10 states, 10-15 states, 15-20 states, 20- 25 states, 25-30 states, 30-35 states, 35-40 states, 40-45 states, 45-50 states, or 50-55 states.
  • support vector machines are implemented.
  • SVMs separate data into classes defined by n-dimensional hyperplanes (n-hy perplane) and are used in both regression and classification problems.
  • Hyperplanes are decision boundaries developed during the training process of a SVM. The dimensionality of a hyperplane depends on the number of input features. For example, a SVM with two input features will have a linear (1-dimensional) hyperplane while a SVM with three input features will have a planer (2-dimensional) hyperplane.
  • a hyperplane is optimized to have the largest margin or spatial distance from the nearest data point for each data type. In the case of simple linear regression and classification a linear equation is used to develop the hyperplane.
  • a kernel is a function that transforms the input features into higher dimensional space. Kernel functions can be linear, polynomial, a radial distribution function (or gaussian radial distribution function), or sigmoidal.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein are used to train the linear equation or kernel function of the SVM machine learning module, which, after training, is used to estimate one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • K-means clustering is implemented.
  • KMC assumes data points have implicit shared characteristics and “clusters” data within a centroid or “mean” of the clustered data points.
  • KMC adds a number of k centroids and optimizes its position around clusters. This process is iterative, where each centroid, initially positioned at random, is repositioned towards the average point of a cluster. This process concludes when the centroids have reached an optimal position within a cluster. Training of a KMC module is typically unsupervised.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein are used to train the centroids of a KMC machine learning module, which, after training, is used to estimate one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • K-nearest neighbor is implemented.
  • KNN shares similar characteristics to KMC.
  • KNN assumes data points near each other share similar characteristics and computes the distance between data points to identify those similar characteristics but instead of k centroids, KNN uses k number of neighbors.
  • the k in KNN represents how many neighbors will assign a data point to a class, for classification, or object property value, for regression. Selection of an appropriate number of k is integral to the accuracy of KNN. For example, a large k may reduce random error associated with variance in the data but increase error by ignoring small but significant differences in the data. Therefore, a careful choice of k is selected to balance overfitting and underfitting.
  • the distance between neighbors is computed. Common methods to compute this distance are Euclidean, Manhattan or Hamming to name a few.
  • neighbors are given weights depending on the neighbor distance to scale the similarity between neighbors to reduce the error of edge neighbors of one class “out-voting” near neighbors of another class.
  • A is 1 and a Markov model approach is utilized.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein are used to train a KNN machine learning module, which, after training, is used to estimate one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • the machine learning module may communicate with one or more other systems.
  • an integration system may integrate the machine learning module with one or more email servers, web servers, one or more databases, or other servers, systems, or repositories.
  • one or more functionalities may require communication between a user and the machine learning module.
  • Any one or more of the module described herein may be implemented using hardware (e.g., one or more processors of a computer/machine) or a combination of hardware and software.
  • any module described herein may configure a hardware processor (e.g., among one or more hardware processors of a machine) to perform the operations described herein for that module.
  • any one or more of the modules described herein may comprise one or more hardware processors and may be configured to perform the operations described herein.
  • one or more hardware processors are configured to include any one or more of the modules described herein.
  • modules described herein as being implemented within a single machine, database, or device may be distributed across multiple machines, databases, or devices.
  • the multiple machines, databases, or devices are communicatively coupled to enable communications between the multiple machines, databases, or devices.
  • the modules themselves are communicatively coupled (e.g., via appropriate interfaces) to each other and to various data sources, to allow information to be passed between the applications so as to allow the applications to share and access common data.
  • the machine learning module comprises multimodal translation (MT), also known as multimodal machine translation or multimodal neural machine translation.
  • MT comprises of a machine learning module capable of receiving multiple (e.g., two or more) modalities.
  • the multiple modalities comprise of information connected to each other.
  • the machine learning a plurality of AAV capsid polypeptide sequences comprising a targeting moiety comprising information on a CD59 protein. The machine learning module then determines one or more sequences from the plurality of sequences having increased binding and/or decreases transduction of off-target tissues corresponding to the CD59 protein.
  • the MT may comprise of a machine learning method further described herein.
  • the MT comprises a neural network, deep neural network, convolutional neural network, convolutional autoencoder, recurrent neural network, or an LSTM.
  • a machine learning method comprising multiple modalities is embedded as further described herein.
  • the embedded data is then received by the machine learning module.
  • the machine learning module processes the embedded data (e.g. encoding and decoding) through the multiple layers of architecture then determines the one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues corresponding the modalities comprising the input.
  • the machine learning methods further described herein may be engineered for MT wherein the inputs described herein comprise of multiple modalities of a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein.
  • the inputs described herein comprise of multiple modalities of a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein.
  • the machine learning module may use embedding to provide a lower dimensional representation, such as a vector, of features to organize them based off respective similarities.
  • these vectors can become massive.
  • particular values may become very sparse among a large number of values (e.g., a single instance of a value among 50,000 values). Because such vectors are difficult to work with, reducing the size of the vectors, in some instances, is necessary.
  • a machine learning module can learn the embeddings along with the model parameters.
  • features such as a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein can be mapped to vectors implemented in embedding methods.
  • embedded semantic meanings are utilized. Embedded semantic meanings are values of respective similarity. For example, the distance between two vectors, in vector space, may imply two values located elsewhere with the same distance are categorically similar. Embedded semantic meanings can be used with similarity analysis to rapidly return similar values.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein is embedded.
  • the methods herein are developed to identify meaningful portions of the vector and extract semantic meanings between that space.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein is processed with a natural language processing (NPL) model.
  • NPL is a computational approach to evaluating text.
  • General applications of NPL may comprise information retrieval, information extraction, question-answering, summarization, machine translation, dialogue systems.
  • Information extraction comprises of recognition, tagging, and extraction into a structured representation.
  • key elements of information such as persons, companies, locations, and/or organizations, are extracted from collections of text.
  • the key elements of information may include physical properties (e.g., quantifiable/measurable) recited in the text and/or subjective properties (e.g., feelings).
  • Question-answering comprises of generating a list of documents corresponding to a user’s query.
  • QA may comprise generating either just the text of the answer or answer-providing passages.
  • Summarization comprises of reducing a large amount of text into a smaller amount of text.
  • summarization may comprise of an abbreviated narrative representation of the original document.
  • Machine translation comprises of either rule-based or probabilistic methods (e.g., machine learning) for translating text to speech or vice versa, or from one language to another.
  • MT attempts to capture contextual, idiomatic and pragmatic nuances of language.
  • Dialogue systems comprise of conversational communications through communication modes such as text, speech, and images.
  • NPL may comprise of different levels of language.
  • ascending levels of language may comprise of phonology, morphology, lexical, syntactic, semantic, discourse, and pragmatic.
  • the phonology level comprises of the interpretation of speech sounds.
  • phonological analysis may comprise of phonetic rules, phonemic rules, and prosodic rules.
  • Morphology comprises of the nature of words, which comprise of morphemes (i.e., smallest units of meaning).
  • the suffix -ed to a verb indicates the action of the verb took place in the past.
  • the lexical level comprises of interpreting the meaning of individual words. For example, words may be assigned part-of-speech tags based on context.
  • the syntactic level comprises of analyzing the words in a sentence to extract the grammatical structure of the sentence. For example, syntactic processes attempt to compute the meaning of a sentence from the order and dependency of the words in a sentence.
  • the semantic level comprises of determining the meaning of a sentence by analyzing the interactions between word-level meanings in a sentence.
  • a semantic process may comprise disambiguation wherein words with multiple meanings are reduced into a singular meaning based on the context of the sentence.
  • the discourse level comprises of determining the meaning of more than one sentence.
  • discourse processing may comprise of anaphora resolution and discourse/text structure recognition.
  • the pragmatic level comprises of determining the use of language based on the context of the text, not necessarily the content of the text. For example, pragmatic processes deduce extra meanings read into text that are not necessarily encoded into the text.
  • NPL may also comprise different approaches to language processing.
  • the approaches may comprise of symbolic, statistical, connectionist, and hybrid.
  • Symbolic approaches comprise of using explicit representations of facts through well-understood knowledge representation schemes to analyze linguistic phenomena.
  • symbolic approaches may comprise of logic or rule-based systems.
  • Statical approaches comprise of using text corpa to determine generalized models of linguistic phenomena.
  • a statical approach may use a HMM to determine speech recognition, lexical acquisition, parsing, part-of-speech tagging, collocations, statistical machine translation, statistical grammar learning, etc.
  • Connectionist approach comprises of combining statistical learning with various theories of representation.
  • connectionist approach may comprise of a network of interconnected local processing units with knowledge stored as weights in the connections between units wherein local interactions may result in an observed global behavior. See e.g., Liddy, E D. 2001. Natural Language Processing. In Encyclopedia of Library and Information Science, 2nd Ed. NY. Marcel Decker, Inc.
  • NLP tasks may comprise: sentence boundary detection (e.g., abbreviations and titles - ‘m.g.,’ ‘Dr.’); tokenization (e.g., hyphens, forward slashes - ‘ 10 mg/day,’ ‘N-acetylcysteine’); part-of-speech assignment to individual words (e.g., homographs and gerunds); morphological decomposition (e.g., lemmatization); shallow parsing (chunking) (e.g., identifying phrases); problem-specific segmentation (e.g., segmenting text into meaningful groups); spelling/grammatical error identification and recovery (e.g., recovering false positives) ; named entity recognition (e g., identifying persons, locations, diseases, genes, or medication); word sense disambiguation (e.g., determining a homograph's correct meaning); negation and uncertainty identification (e.g., inferring whether a named entity is present or absent); relationship extraction (e g.
  • a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein is processed with Latent Dirichlet Allocation (LDA).
  • LDA is a topic model for classifying text, wherein a document or more generally a set of text represents a random mixture over latent topics and each topic is characterized by a distribution of words. LDA is capable of identifying similar groups of text and associating them with certain topics. Generally, topics are identified by searching for groups of text in a document and taking a probability distribution that a group of text belongs to a topic and is likely to be found in the document.
  • LDA is used on a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein to identify one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues.
  • a number of topics are selected to be determined from a plurality of AAV capsid polypeptide sequences comprising a targeting moiety for binding to a CD59 protein data.
  • the topics may comprise any topic related to one or more sequences from the plurality of sequences having increased binding to the CD59 protein and/or one or more sequences from the plurality of sequences that decreases transduction of off-target tissues described herein.
  • the LDA model then needs to be trained to learn the selected topics. First, a set of training text is used as input for the LDA model. The text is randomly distributed among the selected topics. In an iterative process, the LDA model determines the proportion of text in a set that are currently assigned to a selected topic, then determines the proportion of assignments to the selected topic over all the sets and reassigns the word to a different topic based off a computed probability. This process is complete once a steady state of acceptable assignments is determined.
  • the LDA model can then be used to determine topics from user data, which can then be passed to the machine learning network. See e.g., Blei, David M., Andrew Y. Ng, and Michael I. Jordan. “Latent dirichlet allocation.” Journal of machine Learning research 3. Jan (2003): 993-1022 incorporated herein by reference.
  • Example 1 An AAV Capsid Programmed to Bind Human and Macaque Receptors Mediates Enhanced Gene Delivery to the CNS and Skeletal Muscle in vivo
  • AAVs Natural adeno-associated viruses
  • capsid engineering has largely focused on targeting specific organs, such as the CNS, liver, retina, or muscle.
  • Applicants used the recently described receptortargeting platform (Huang et al., 2023) to target peptide-modified AAV9 capsids to a human receptor that is broadly expressed across diverse cell types, including the central nervous system (CNS) and muscle.
  • CNS central nervous system
  • BI306 and BI309 Two of the capsids tested, BI306 and BI309, efficiently transduced the CNS to an extent comparable to AAV-PHP.eB, a mouse CNS-targeting capsid (Chan et al., 2017) when intravenously administered to mice overexpressing the human receptor in the brain microvasculature (Fig. 1 A). This enhanced tropism was absent in mice treated with wild type AAV9 or in mice not expressing the human receptor. When BI306 was intravenously administered to mice expressing the human receptor in cardiac and skeletal muscle in addition to the CNS, it achieved substantially enhanced muscle transduction compared to wild type mice or transgenic mice injected with AAV9 (Fig. IB).
  • BI306 was also de-targeted from the liver compared to AAV9 (Fig. IB). Finally, Applicants demonstrated that BI306 can interact with both human and macaque, but not mouse, orthologs of the same receptor (Fig. 2). The findings show that BI306 is a cross-species receptor-targeting AAV that can mediate enhanced gene delivery in a receptor-dependent manner. Based on the ubiquitous expression of the receptor across the human body, it is likely that BI306 and similar capsids can mediate enhanced gene delivery to the cells of multiple organs after systemic delivery.
  • capsids have several novel features and advantages: 1. hCD59 is broadly expressed protein in humans, so the capsids are expected to have broadly enhanced tropisms. 2. GPI-anchored proteins are a known protein target for mouse-specific BBB crossing engineered AAVs, but no AAV so far has been reported to interact with human CD59. 3. Applicants have demonstrated that systemic injection of the AAVs can efficiently target both the brain and muscle in mice that exogenously express hCD59 on target tissue. 4. One of the hCD59-binding capsids, named BI306, also interacts with the macaque protein ortholog of CD59 and can transduce the CNS in mice expressing CD59 from either human or macaque. 5.
  • BI306 is partially liver detargeted in mice, which is beneficial for reducing toxicity and for improved targeting other organs. 6. By specifically targeting a human receptor, the likelihood of the AAVs functioning in humans is more favorable than other engineered CNS-targeting capsids that do not translate beyond animal models. 7. These capsids and their potential for broadly enhanced tropisms are well suited to the treatment of many genetic disorders including, but not limited to, Fabry disease, Gaucher disease, Friedreich's ataxia, spinal muscular atrophy, Huntington's disease, prion disease, and Hunter syndrome.
  • Adeno-associated virus (AAV) vectors are one of the most commonly used methods for therapeutic gene delivery. Gene therapies for multisystemic disorders require efficient gene delivery to many organs, though natural adeno-associated viruses (AAVs) have limited tissue tropisms especially beyond the liver. AAV engineering by modifying the capsid sequences of native AAV serotypes has had promising outcomes in targeting AAVs to specific organs such as the central nervous system (CNS) or muscle in mice or non-human primates, but not for system- wide transduction. Engineered AAVs often do not translate across species, so it is difficult to determine whether or not these AAVs would be as effective in humans.
  • CNS central nervous system
  • Engineered AAVs often do not translate across species, so it is difficult to determine whether or not these AAVs would be as effective in humans.
  • MAC complement membrane attack complex
  • the capsid BI306 is able to interact with both human and macaque CD59 protein orthologs, in addition it is de-targeted from the liver in mice compared the parental AAV9 serotype. No other AAV to Applicants knowledge has been engineered to interact with hCD59.
  • An AAV targeting hCD59 has the potential to target multiple organs in the human body, expanding its utility in gene therapy.
  • Several genetic disorders such as certain lysosomal storage disorders (i.e., Fabry, Gaucher, Hunter, etc.) affect many different organs, and genes implicated in disease (SMN, FXN, HTT, CFTR, UBE3A, etc.) are expressed in many tissues in addition to the most affected organs. Therefore, an hCD59 targeting AAV has broad potential for therapeutic gene delivery to treat a number of diseases.
  • Macromolecules including AAVs may target organs through both direct receptor- mediated uptake or transport across endothelial cells between vessels and tissue. Therefore, the variety of cells in which CD59 is expressed expands the potential for hCD59-targeting AAVs for delivery of therapeutic genes to a wide range of cell types.
  • a random NNK library of 7-mer peptide substitution in place of amino acids 451-460 was first screened for binding to a Fc- hCD59 fusion protein with a method previously described (Huang et al. 2023).
  • AAV sequences that bound to Fc-hCD59 were built into a second-round synthetic library.
  • the second-round library was screened for binding and transduction in HEK293T cells transiently transfected with hCD59.
  • the library was screened for enrichment of brain transduction in mice bred from the cross (LSL)-hCD59 x Tie2-Cre (strain#’ s 035504 and 008863, respectively).
  • LSL-hCD59 mice have Cre-dependent expression of human CD59 and Tie2-Cre expressed Cre in endothelial cells.
  • the progeny mice thus express hCD59 in endothelial cell, including in the brain vasculature.
  • Top enriched sequences were selected and validated in vitro and in vivo. In vitro validation was performed by measuring transduction in both transfected HEK cells as well as CHO cells stably transfected with hCD59.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Veterinary Medicine (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Capsides d'AAV9 modifiées par un peptide modifié par les demandeurs pour une transduction d'organe améliorée à l'échelle du système par ingénierie directe d'une interaction de liaison avec la glycoprotéine CD59 liée au GPI humain. Les virus adéno-associés (AAV) sont utilisés dans de nombreuses thérapies géniques expérimentales et approuvées. Cependant, les AAV natifs présentent des tropismes de tissu limités ainsi que des rendements de distribution extra-hépatiques comparativement faibles. Tandis que l'ingénierie des capsides s'est largement centrée sur le ciblage d'organes spécifiques, le traitement de troubles multisystème nécessite une distribution de gène efficace à de nombreux organes. Les demandeurs décrivent de nouveaux capsides modifiés qui se lient à CD59.
PCT/US2025/023652 2024-04-08 2025-04-08 Nouveaux capsides aav se liant à cd59 humain Pending WO2025217163A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463631415P 2024-04-08 2024-04-08
US63/631,415 2024-04-08

Publications (2)

Publication Number Publication Date
WO2025217163A2 true WO2025217163A2 (fr) 2025-10-16
WO2025217163A3 WO2025217163A3 (fr) 2025-11-20

Family

ID=95560599

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2025/023652 Pending WO2025217163A2 (fr) 2024-04-08 2025-04-08 Nouveaux capsides aav se liant à cd59 humain

Country Status (1)

Country Link
WO (1) WO2025217163A2 (fr)

Citations (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4797368A (en) 1985-03-15 1989-01-10 The United States Of America As Represented By The Department Of Health And Human Services Adeno-associated virus as eukaryotic expression vector
US4873316A (en) 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
EP0404097A2 (fr) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application
US5173414A (en) 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
WO1993011161A1 (fr) 1991-11-25 1993-06-10 Enzon, Inc. Proteines multivalentes de fixation aux antigenes
WO1993024641A2 (fr) 1992-06-02 1993-12-09 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Virus adeno-associe a sequences terminales inversees utilisees comme promoteur
WO1996040281A2 (fr) 1995-06-07 1996-12-19 Alliance Pharmaceutical Corp. Emulsions gazeuses stabilisees avec des ethers fluores ayant des coefficients d'ostwald faibles
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
US5811097A (en) 1995-07-25 1998-09-22 The Regents Of The University Of California Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling
US5846946A (en) 1996-06-14 1998-12-08 Pasteur Merieux Serums Et Vaccins Compositions and methods for administering Borrelia DNA
US6750059B1 (en) 1998-07-16 2004-06-15 Whatman, Inc. Archiving of vectors
US20040171156A1 (en) 1995-06-07 2004-09-02 Invitrogen Corporation Recombinational cloning using nucleic acids having recombination sites
US20050123596A1 (en) 2003-09-23 2005-06-09 Kohane Daniel S. pH-triggered microparticles
US7541446B2 (en) 1999-06-07 2009-06-02 Tet Systems Holding Gmbh & Co. Kg Tet repressor-based transcriptional regulatory proteins
US20100003218A1 (en) 2008-05-28 2010-01-07 Dongsheng Duan Hybrid-aav vectors to deliver large gene expression cassette
US7745592B2 (en) 2001-05-01 2010-06-29 National Research Council Of Canada Cumate-inducible expression system for eukaryotic cells
US7776321B2 (en) 2001-09-26 2010-08-17 Mayo Foundation For Medical Education And Research Mutable vaccines
US20110027239A1 (en) 2009-07-29 2011-02-03 Tissue Genesis, Inc. Adipose-derived stromal cells (asc) as delivery tool for treatment of cancer
WO2011028929A2 (fr) 2009-09-03 2011-03-10 The Regents Of The University Of California Promoteur sensible aux nitrates
US7947823B2 (en) 2003-07-09 2011-05-24 Universita Degli Studi Di Roma “La Sapienza” siRNA expression system
US8404658B2 (en) 2007-12-31 2013-03-26 Nanocor Therapeutics, Inc. RNA interference for the treatment of heart failure
US8454972B2 (en) 2004-07-16 2013-06-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Method for inducing a multiclade immune response against HIV utilizing a multigene and multiclade immunogen
WO2013138585A1 (fr) 2012-03-16 2013-09-19 The Broad Institute, Inc. Procédés multiplex pour analyser simultanément des populations de cellules mélangées
WO2014093622A2 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Délivrance, fabrication et optimisation de systèmes, de procédés et de compositions pour la manipulation de séquences et applications thérapeutiques
US20150232883A1 (en) 2013-12-12 2015-08-20 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using particle delivery components
US20150267202A1 (en) 2004-09-16 2015-09-24 Sarepta Therapeutics, Inc. ANTISENSE ANTIVIRAL COMPOUND AND METHOD FOR TREATING ss/RNA VIRAL INFECTION
US20160251398A1 (en) 2005-07-13 2016-09-01 Sarepta Therapeutics, Inc. Antibacterial antisense oligonucleotide and method
US20170079916A1 (en) 2015-09-23 2017-03-23 Massachusetts Institute Of Technology Compositions and methods for modified dendrimer nanoparticle delivery
WO2017106304A1 (fr) 2015-12-15 2017-06-22 Sarepta Therapeutics, Inc. Conjugués peptide-oligonucléotide
US20180216111A1 (en) 2015-02-27 2018-08-02 Sarepta Therapeutics, Inc. Antisense-induced exon2 inclusion in acid alpha-glucosidase
US20190247516A1 (en) 2017-11-10 2019-08-15 Norwegian University Of Science And Technology (Ntnu) Neuronal enhancers
US20190284555A1 (en) 2016-04-18 2019-09-19 Sarepta Therapeutics, Inc. Antisense oligomers and methods of using the same for treating diseases associated with the acid alpha-glucosidase gene
US10494645B2 (en) 2013-04-18 2019-12-03 Fondazione Telethon Effective delivery of large genes by dual AAV vectors
WO2020016318A1 (fr) 2018-07-17 2020-01-23 INSERM (Institut National de la Santé et de la Recherche Médicale) Compositions et procédés pour augmenter ou améliorer la transduction de vecteurs de thérapie génique et pour éliminer ou réduire des immunoglobulines
US20200140492A1 (en) 2017-06-27 2020-05-07 Regeneron Pharmaceuticals, Inc. Tropism-Modified Recombinant Viral Particles and Uses Thereof for the Targeted Introduction of Genetic Material into Human Cells
WO2020180928A1 (fr) 2019-03-04 2020-09-10 University Of Florida Research Foundation, Incorporated Promoteur d'opsine humain amélioré pour expression spécifique dans les bâtonnets
US10898585B2 (en) 2017-04-14 2021-01-26 Ptc Therapeutics .Inc. Gene therapy for AADC deficiency
WO2021067598A1 (fr) 2019-10-04 2021-04-08 Ultragenyx Pharmaceutical Inc. Procédés pour une utilisation thérapeutique améliorée d'aav recombinant
WO2021163322A1 (fr) 2020-02-14 2021-08-19 Ultragenyx Pharmaceutical Inc. Thérapie génique pour le traitement d'un trouble du déficit en cdkl5
EP3909602A1 (fr) 2014-04-25 2021-11-17 University of Florida Research Foundation, Inc. Méthodes permettant qu'un sujet reçoive des doses multiples de virus adéno-associé recombinant
US20210380969A1 (en) 2018-10-02 2021-12-09 Voyager Therapeutics, Inc. Redirection of tropism of aav capsids
WO2022087494A1 (fr) 2020-10-23 2022-04-28 The Broad Institute, Inc. Nucléases iscb reprogrammables et leurs utilisations
US11382988B2 (en) 2019-11-08 2022-07-12 Coave Therapeutics Modified adeno-associated virus vectors and delivery thereof into the central nervous system
WO2022159892A1 (fr) 2021-01-25 2022-07-28 The Broad Institute, Inc. Polypeptides tnpb reprogrammables et leur utilisation
US11648319B2 (en) 2016-06-09 2023-05-16 Centre National De La Recherche Scientifique (Cnrs) rAAV with chemically modified capsid
WO2023114872A2 (fr) 2021-12-14 2023-06-22 The Broad Institute, Inc. Polynucléotides fanzor reprogrammables et leurs utilisations
US20240091381A1 (en) 2022-05-13 2024-03-21 Ascidian Therapeutics, Inc. Abca4 trans-splicing molecules

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20220143214A1 (en) * 2019-01-30 2022-05-12 The Broad Institute, Inc. Systems for evolved adeno-associated viruses (aavs) for targeted delivery
JP2025528068A (ja) * 2022-08-03 2025-08-26 ボイジャー セラピューティクス インコーポレイテッド 血液脳関門を通過させるための組成物及び方法

Patent Citations (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4797368A (en) 1985-03-15 1989-01-10 The United States Of America As Represented By The Department Of Health And Human Services Adeno-associated virus as eukaryotic expression vector
US4873316A (en) 1987-06-23 1989-10-10 Biogen, Inc. Isolation of exogenous recombinant proteins from the milk of transgenic mammals
EP0404097A2 (fr) 1989-06-22 1990-12-27 BEHRINGWERKE Aktiengesellschaft Récepteurs mono- et oligovalents, bispécifiques et oligospécifiques, ainsi que leur production et application
US5173414A (en) 1990-10-30 1992-12-22 Applied Immune Sciences, Inc. Production of recombinant adeno-associated virus vectors
WO1993011161A1 (fr) 1991-11-25 1993-06-10 Enzon, Inc. Proteines multivalentes de fixation aux antigenes
WO1993024641A2 (fr) 1992-06-02 1993-12-09 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Virus adeno-associe a sequences terminales inversees utilisees comme promoteur
US5641870A (en) 1995-04-20 1997-06-24 Genentech, Inc. Low pH hydrophobic interaction chromatography for antibody purification
WO1996040281A2 (fr) 1995-06-07 1996-12-19 Alliance Pharmaceutical Corp. Emulsions gazeuses stabilisees avec des ethers fluores ayant des coefficients d'ostwald faibles
US20040171156A1 (en) 1995-06-07 2004-09-02 Invitrogen Corporation Recombinational cloning using nucleic acids having recombination sites
US5811097A (en) 1995-07-25 1998-09-22 The Regents Of The University Of California Blockade of T lymphocyte down-regulation associated with CTLA-4 signaling
US5846946A (en) 1996-06-14 1998-12-08 Pasteur Merieux Serums Et Vaccins Compositions and methods for administering Borrelia DNA
US6750059B1 (en) 1998-07-16 2004-06-15 Whatman, Inc. Archiving of vectors
US7541446B2 (en) 1999-06-07 2009-06-02 Tet Systems Holding Gmbh & Co. Kg Tet repressor-based transcriptional regulatory proteins
US7745592B2 (en) 2001-05-01 2010-06-29 National Research Council Of Canada Cumate-inducible expression system for eukaryotic cells
US7776321B2 (en) 2001-09-26 2010-08-17 Mayo Foundation For Medical Education And Research Mutable vaccines
US7947823B2 (en) 2003-07-09 2011-05-24 Universita Degli Studi Di Roma “La Sapienza” siRNA expression system
US20050123596A1 (en) 2003-09-23 2005-06-09 Kohane Daniel S. pH-triggered microparticles
US8454972B2 (en) 2004-07-16 2013-06-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Method for inducing a multiclade immune response against HIV utilizing a multigene and multiclade immunogen
US20150267202A1 (en) 2004-09-16 2015-09-24 Sarepta Therapeutics, Inc. ANTISENSE ANTIVIRAL COMPOUND AND METHOD FOR TREATING ss/RNA VIRAL INFECTION
US20160251398A1 (en) 2005-07-13 2016-09-01 Sarepta Therapeutics, Inc. Antibacterial antisense oligonucleotide and method
US8404658B2 (en) 2007-12-31 2013-03-26 Nanocor Therapeutics, Inc. RNA interference for the treatment of heart failure
US20100003218A1 (en) 2008-05-28 2010-01-07 Dongsheng Duan Hybrid-aav vectors to deliver large gene expression cassette
US20110027239A1 (en) 2009-07-29 2011-02-03 Tissue Genesis, Inc. Adipose-derived stromal cells (asc) as delivery tool for treatment of cancer
WO2011028929A2 (fr) 2009-09-03 2011-03-10 The Regents Of The University Of California Promoteur sensible aux nitrates
WO2013138585A1 (fr) 2012-03-16 2013-09-19 The Broad Institute, Inc. Procédés multiplex pour analyser simultanément des populations de cellules mélangées
WO2014093622A2 (fr) 2012-12-12 2014-06-19 The Broad Institute, Inc. Délivrance, fabrication et optimisation de systèmes, de procédés et de compositions pour la manipulation de séquences et applications thérapeutiques
US10494645B2 (en) 2013-04-18 2019-12-03 Fondazione Telethon Effective delivery of large genes by dual AAV vectors
US20150232883A1 (en) 2013-12-12 2015-08-20 The Broad Institute Inc. Delivery, use and therapeutic applications of the crispr-cas systems and compositions for targeting disorders and diseases using particle delivery components
EP3909602A1 (fr) 2014-04-25 2021-11-17 University of Florida Research Foundation, Inc. Méthodes permettant qu'un sujet reçoive des doses multiples de virus adéno-associé recombinant
US20180216111A1 (en) 2015-02-27 2018-08-02 Sarepta Therapeutics, Inc. Antisense-induced exon2 inclusion in acid alpha-glucosidase
US20170079916A1 (en) 2015-09-23 2017-03-23 Massachusetts Institute Of Technology Compositions and methods for modified dendrimer nanoparticle delivery
WO2017106304A1 (fr) 2015-12-15 2017-06-22 Sarepta Therapeutics, Inc. Conjugués peptide-oligonucléotide
US20190284555A1 (en) 2016-04-18 2019-09-19 Sarepta Therapeutics, Inc. Antisense oligomers and methods of using the same for treating diseases associated with the acid alpha-glucosidase gene
US11648319B2 (en) 2016-06-09 2023-05-16 Centre National De La Recherche Scientifique (Cnrs) rAAV with chemically modified capsid
US10898585B2 (en) 2017-04-14 2021-01-26 Ptc Therapeutics .Inc. Gene therapy for AADC deficiency
US20200140492A1 (en) 2017-06-27 2020-05-07 Regeneron Pharmaceuticals, Inc. Tropism-Modified Recombinant Viral Particles and Uses Thereof for the Targeted Introduction of Genetic Material into Human Cells
US20190247516A1 (en) 2017-11-10 2019-08-15 Norwegian University Of Science And Technology (Ntnu) Neuronal enhancers
WO2020016318A1 (fr) 2018-07-17 2020-01-23 INSERM (Institut National de la Santé et de la Recherche Médicale) Compositions et procédés pour augmenter ou améliorer la transduction de vecteurs de thérapie génique et pour éliminer ou réduire des immunoglobulines
US20210380969A1 (en) 2018-10-02 2021-12-09 Voyager Therapeutics, Inc. Redirection of tropism of aav capsids
WO2020180928A1 (fr) 2019-03-04 2020-09-10 University Of Florida Research Foundation, Incorporated Promoteur d'opsine humain amélioré pour expression spécifique dans les bâtonnets
WO2021067598A1 (fr) 2019-10-04 2021-04-08 Ultragenyx Pharmaceutical Inc. Procédés pour une utilisation thérapeutique améliorée d'aav recombinant
US11382988B2 (en) 2019-11-08 2022-07-12 Coave Therapeutics Modified adeno-associated virus vectors and delivery thereof into the central nervous system
WO2021163322A1 (fr) 2020-02-14 2021-08-19 Ultragenyx Pharmaceutical Inc. Thérapie génique pour le traitement d'un trouble du déficit en cdkl5
WO2022087494A1 (fr) 2020-10-23 2022-04-28 The Broad Institute, Inc. Nucléases iscb reprogrammables et leurs utilisations
WO2022159892A1 (fr) 2021-01-25 2022-07-28 The Broad Institute, Inc. Polypeptides tnpb reprogrammables et leur utilisation
WO2023114872A2 (fr) 2021-12-14 2023-06-22 The Broad Institute, Inc. Polynucléotides fanzor reprogrammables et leurs utilisations
US20240091381A1 (en) 2022-05-13 2024-03-21 Ascidian Therapeutics, Inc. Abca4 trans-splicing molecules

Non-Patent Citations (173)

* Cited by examiner, † Cited by third party
Title
"Pharmaceutical dosage form tablets", 1989, MARCEL DEKKER, INC.
"Pharmaceutical dosage forms and drug delivery systems", 1995, WILLIAMS AND WILKINS
"Remington - The science and practice of pharmacy", 2000, LIPPINCOTT WILLIAMS & WILKINS
ALTAE-TRAN, H.; ET AL.: "The Widespread IS200/IS605 Transposon Family Encodes Diverse Programmable RNA-Guided Endonucleases.", SCIENCE, vol. 374, 2021, pages 57 - 65, XP055901842, DOI: 10.1126/science.abj6856
AMALFITANO ET AL., J. VIROL., vol. 72, 1998, pages 926 - 933
AMRANN ET AL., GENE, vol. 69, 1988, pages 301 - 315
BADGE ET AL., PROC. NATL. ACAD. SCI., vol. 85, 1988, pages 3648 - 3652
BALAGUE ET AL., BLOOD, vol. 95, 2000, pages 820 - 828
BARTUNEK ET AL., CYTOKINE, vol. 8, no. 1, 1996, pages 14 - 20
BENNETZENHALL, J BIOL CHEM., vol. 257, no. 6, 25 March 1982 (1982-03-25), pages 3026 - 31
BINZ ET AL.: "Engineering novel binding proteins from non-immunoglobulin domains.", NAT BIOTECHNOL, vol. 23, 2005, pages 1257 - 1268
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423
BIRDWALKER, TRENDS BIOTECHNOL., vol. 9, no. 4, April 1991 (1991-04-01), pages 132 - 7
BLEI, DAVID M.ANDREW Y. NGMICHAEL 1. JORDAN: "Latent dirichlet allocation", JOURNAL OF MACHINE LEARNING RESEARCH, 2003, pages 993 - 1022
BOSHART ET AL., CELL, vol. 41, 1985, pages 521 - 530
BOSMAN, FBUYSE, MADEGRIECK, RCELIS, LDE BOER, MVAN DOORSSELAERE, VSABLON, E: "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., vol. 22, no. 1, 2001, pages 11 - 24
BOUNDY ET AL., J. NEUROSCI., vol. 18, 1998, pages 9989
BOYNE, A. R.DANOS, O. F.VOLLES, M. JGUO, X.: "Regulation of gene expression by aptamer-mediated modulation of alternative splicing", US PATENT, 2022
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
BUCK ET AL., INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 12, 2020, pages 21
BUCKHOLZ, R.GGLEESON, M.A., BIOTECHNOLOGY (NY, vol. 9, no. 11, 1991, pages 1067 - 72
BÜNING, H.SRIVASTAVA, A.: "Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors.", MOLECULAR THERAPY - METHODS & CLINICAL DEVELOPMENT, vol. 12, 2019, pages 248 - 265, XP055593479, DOI: 10.1016/j.omtm.2019.01.008
BYRNERUDDLE, PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 5473 - 5477
CALAMEEATON, ADV. IMMUNOL., vol. 43, 1988, pages 235 - 275
CAMPBELLGOWRI, PLANT PHYSIOL., vol. 92, no. 1, pages 1 - 11
CAMPESTILGHMAN, GENES DEV., vol. 3, 1989, pages 537 - 546
CARLSON ET AL., J. BIOL. CHEM., vol. 272, no. 17, 1997, pages 11295 - 11301
CASANOVA ET AL., GENESIS, vol. 31, 2001, pages 37
CASTLE, M. J.TURUNEN, H. T.VANDENBERGHE, L. H.WOLFE, J. H: "Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids.", METHODS MOL. BIOL., vol. 1382, 2016, pages 133 - 149, XP055944095, DOI: 10.1007/978-1-4939-3271-9_10
CHEN ET AL., CANCER RES., vol. 58, no. 15, 1998, pages 3209 - 3214
CHOI ET AL., CURR GENE THER, vol. 5, no. 3, 2005, pages 299 - 310
CIDECIYAN ET AL., N ENGL J MED., vol. 361, 2009, pages 725 - 727
COMB ET AL., EMBO J., vol. 17, 1988, pages 3793 - 3805
COONEY ET AL., MOL. THER., vol. 23, no. 4, 2015, pages 667 - 674
CRANE ET AL., GENE THER., vol. 19, no. 4, 2012, pages 443 - 452
CROYLE ET AL., GENE THER., vol. 12, 2005, pages 579 - 587
DE GRAAF ET AL., METHODS MOL BIOL., vol. 178, 2002, pages 379 - 87
DENG ET AL., BLOOD, vol. 92, no. 6, 1998, pages 1981 - 1988
DEVERMAN ET AL., NATURE BIOTECH., vol. 34, no. 2, pages 204 - 209
DIAMANT N ET AL.: "Patient contrastive learning: A performant, expressive, and practical approach to electrocardiogram modeling.", PLOS COMPUT BIOL., vol. 18, no. 2, 14 February 2022 (2022-02-14), pages e1009862
DIMATTIA ET AL., J. VIROL., vol. 86, no. 12, 2012, pages 6947 - 6958
DOSHI, A.SADEGHI, F.VARADARAJAN, N.CIRINO, P. C.: "Small-molecule inducible transcriptional control in mammalian cells.", CRIT. REV. BIOTECHNOL., vol. 40, 2020, pages 1131 - 1150, XP009538876, DOI: 10.1080/07388551.2020.1808583
EDLUND ET AL., SCIENCE, vol. 230, 1985, pages 912 - 916
EHRHARDT ET AL., MOL. THER., vol. 156, 2007, pages 1834 - 1841
FLOTTE ET AL., HUM. GENE. THER., vol. 7, 1996, pages 1145 - 1159
FOLEY CP ET AL.: "Intra-arterial delivery of AAV vectors to the mouse brain after mannitol mediated blood brain barrier disruption.", J CONTROL RELEASE., vol. 196, 28 December 2014 (2014-12-28), pages 71 - 78, XP029112386, DOI: 10.1016/j.jconrel.2014.09.018
GAO ET AL., J. VIROL., vol. 78, 2004, pages 6381 - 6388
GEBAUERSKERRA: "Engineered protein scaffolds as next-generation antibody therapeutics.", CURR OPIN CHEM BIOL., vol. 13, 2009, pages 245 - 55
GILLDAMLE: "Biopharmaceutical drug discovery using novel protein scaffolds.", CURR OPIN BIOTECHNOL, vol. 17, 2006, pages 653 - 658
GLASCOCK, J. J. ET AL.: "Delivery of Therapeutic Agents Through Intracerebroventricular (ICV) and Intravenous (IV) Injection in Mice.", J. VIS. EXP, vol. 56, pages 29968
GOODFELLOW, I. J.POUGET-ABADIE, J.MIRZA, M.XU, BWARDE-FARLEY, DOZAIR, S.COURVILLE, ABENGIO, Y, GENERATIVE ADVERSARIAL NETWORKS. ARXIV, 2014
GOSSEN ET AL., TIBS, vol. 18, 1993, pages 471475
GOSSEN, M. ET AL., SCIENCE, vol. 268, 1995, pages 1766 - 1769
GOSSEN, MH. BUJARD, PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 5547 - 5551
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., vol. 24, 2016, pages 287 - 297, XP055436946, DOI: 10.1038/mt.2015.187
GRIMM, D. ET AL., J. VIROL., vol. 82, 2008, pages 5887 - 5911
HARDEE ET AL., GENES, vol. 8, no. 2, 2017, pages 65
HARROP ET AL., J. IMMUNOL., vol. 160, no. 7, 1998, pages 3170 - 3179
HENRYMACKENZIE: "Antigen recognition by single-domain antibodies: structural latitudes and constraints.", MABS., vol. 10, no. 6, 2018, pages 815 - 826
HERMONATMUZYCZKA, PNAS, vol. 81, 1984, pages 6466 - 6470
HIRANO ET AL., NATURE, vol. 610, 2022, pages 575 - 581
HOLLINGER ET AL., PNAS, vol. 90, 1993, pages 6444
HOLLINGER, PHILIPPPROSPERO, TWINTER, G: "Diabodies'': small bivalent and bispecific antibody fragments''.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA., vol. 90, no. 14, July 1993 (1993-07-01), pages 6444 - 8
HORDEAUX, JBUZA, E. LJEFFREY, BSONG, CJAHAN, T.YUAN, YZHU, YBELL, PLI, MCHICHESTER, J. A.: "MicroRNA-Mediated Inhibition of Transgene Expression Reduces Dorsal Root Ganglion Toxicity by AAV Vectors in Primates.", SCIENCE TRANSLATIONAL MEDICINE, 2020, pages 12
HU, M. C. -T.N. DAVIDSON, CELL, vol. 49, 1987, pages 603 - 612
HUSTON ET AL., PNAS, vol. 85, 1988, pages 5879
HUSTON, J. S.LEVINSON, DMUDGETT-HUNTER, M.TAI, M. S.NOVOTNY, J.MARGOLIES, M. N.CREA, R.: "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, vol. 85, no. 16, 1988, pages 5879 - 5883, XP000872837, DOI: 10.1073/pnas.85.16.5879
IVICS ET AL., CELL, vol. 91, no. 4, 1997, pages 501 - 510
JIANG ET AL., SCIENCE ADVANCES, vol. 39, 2023, pages 9
KANEDA ET AL., NEURON, vol. 6, 1991, pages 583 - 594
KARVELIS ET AL., NATURE, vol. 599, 2021, pages 692 - 696
KAUFMAN ET AL., EMBO J., vol. 6, 1987, pages 187 - 195
KAY ET AL., NAT. GENET., vol. 24, 2000, pages 257 - 261
KINOSHITA, T.: "Glycosylphosphatidylinositol (GPI) Anchors: Biochemistry and Cell Biology: Introduction to a Thematic Review Series.", JOURNAL OF LIPID RESEARCH, vol. 57, 2016, pages 4 - 5
KOIDEKOIDE: "Monobodies: antibody mimics based on the scaffold of the fibronectin type III domain.", METHODS MOL BIOL, vol. 352, 2007, pages 95 - 109, XP009102789
KOIRALA ET AL., ADV. EXP. MED. BIOL., vol. 801, 2014, pages 703 - 709
KOLMAR: "Alternative binding proteins: biological activity and therapeutic potential of cystine-knot miniproteins.", FEBS J, vol. 275, 2008, pages 2684 - 2690, XP055417456, DOI: 10.1111/j.1742-4658.2008.06440.x
KOTIN, HUMAN GENE THERAPY, vol. 5, 1994, pages 793 - 801
KUBOMITANI, J. VIROL., vol. 77, no. 5, 2003, pages 2964 - 2971
KUGLER ET AL., GENE THER., vol. 10, no. 4, 2003, pages 337 - 47
KUGLER ET AL., VIROLOGY, vol. 311, no. 1, 2003, pages 89 - 95
KUIJANHERSKOWITZ, CELL, vol. 30, 1982, pages 933 - 943
LAI ET AL., DNA CELL. BIOL., vol. 21, 2002, pages 895 - 913
LE GALL, FKIPRIYANOV, SMMOLDENHAUER, G;LITTLE, M: "Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding", FEBS LETTERS., vol. 453, no. 1, 1999, pages 164 - 168, XP071239699, DOI: 10.1016/S0014-5793(99)00713-9
LI, CSAMULSKI, R.J.: "Engineering adeno-associated virus vectors for gene therapy.", NAT REV GENET, vol. 21, 2020, pages 255 - 272
LIAUTARD ET AL., CYTOKINE, vol. 9, no. 4, 1997, pages 233 - 241
LIDDY, E.D.: "Natural Language Processing. In Encyclopedia of Library and Information Science", 2001, MARCEL DECKER, INC
LIU ET AL., GENE THER., vol. 11, 2004, pages 52 - 60
LLEWELLYN ET AL., NAT. MED., vol. 16, no. 10, 2010, pages 1161 - 1166
LUCKLOWSUMMERS, VIROLOGY, vol. 170, 1989, pages 31 - 39
MAKAROVA ET AL., NAT. REV, vol. 18, 2020, pages 67 - 83
MAKAROVA ET AL., THE CRISPR JOURNAL, 2018
MAKAROVA ET AL.: "Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants", NATURE REVIEWS MICROBIOLOGY, vol. 18, February 2020 (2020-02-01), pages 67 - 81
MAYFORD ET AL., PROC. NATL. ACAD. SCI. USA, vol. 93, 1996, pages 13250
MCLEAN ET AL., NEUROSCI. LETT., vol. 576, 2014, pages 73 - 78
MIRKOVITCH ET AL., CELL, vol. 39, 1984, pages 223 - 232
MISKEY ET AL., NUCLEIC ACID RES., vol. 31, no. 23, 2003, pages 6873 - 6881
MOL. CELL. BIOL, vol. 8, no. 1, 1988, pages 466 - 472
MOL. THEN, vol. 13, no. 1, 2006, pages 67 - 76
MONTEYS, A. M. ET AL., REGULATED CONTROL OF GENE THERAPIES WITH A DRUG INDUCED SWITCH, 2020
MONTEYS, A. M. ET AL.: "Regulated control of gene therapies by drug-induced splicing.", NATURE, 2021, pages 1 - 5
MORRAL ET AL., HUM. GENE THER., vol. 9, 1998, pages 2709 - 2716
MORRAL ET AL., PNAS, vol. 96, 1999, pages 12816 - 12821
MORTON BR, J MOL EVOL., vol. 46, no. 4, April 1998 (1998-04-01), pages 449 - 59
MULLER ET AL., STRUCTURE, vol. 6, no. 9, 1998, pages 1153 - 1167
MURRAY ET AL., NUCLEIC ACIDS RES., vol. 17, no. 2, 25 January 1989 (1989-01-25), pages 477 - 98
MUZYCZKA, J. CLIN. INVEST, vol. 94, 1994, pages 1351
NAKAMURA, Y. ET AL.: "Codon usage tabulated from the international DNA sequence databases: status for the year 2000", NUCL. ACIDS RES, vol. 28, 2000, pages 292, XP002941557, DOI: 10.1093/nar/28.1.292
NATHANSON ET AL., FRONTIERS IN NEURAL CIRCUITS, vol. 3, pages 19
NEHLSEN ET AL., GENE THER. MOL. BIOL., vol. 10, 2006, pages 233 - 244
NIXONWOOD: "Engineered protein inhibitors of proteases.", CURR OPIN DRUG DISCOV DEV, vol. 9, 2006, pages 261 - 268
NO ET AL., PROC. NATL. ACED. SCI. USA, vol. 93, 1996, pages 3346 - 3351
NORD ET AL., NAT. BIOTECHNOL., vol. 15, no. 8, 1997, pages 772 - 777
NYGREN: "Alternative binding proteins: Affibody binding proteins developed from a small three-helix bundle scaffold.", FEBS J, vol. 275, 2008, pages 2668 - 2676
OBERDICK ET AL., SCIENCE, vol. 249, 1990, pages 374 - 379
OH ET AL., GENE THER., vol. 16, 2009, pages 437
O'SHEA, K.NASH, R., AN INTRODUCTION TO CONVOLUTIONAL NEURAL NETWORKS. ARXIV, 2015
OUYANG ET AL., INT J ONCOL, 2016
PARK JBLACKBURN EHWEISSMAN JSQI LSHUANG B.: "Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system.", CELL, vol. 155, no. 7, 2013, pages 1479 - 91, XP028806611, DOI: 10.1016/j.cell.2013.12.001
PETERS ET AL., , PNAS, vol. 35, 2017, pages 114
PINKERT ET AL., GENES DEV., vol. 1, 1987, pages 268 - 277
PITARD ET AL., J. IMMUNOL. METHODS, vol. 205, no. 2, 1997, pages 177 - 190
PLATT FM ET AL.: "Lysosomal storage diseases.", NATURE REVIEWS DISEASE PRIMERS., vol. 4, no. 1, 1 October 2018 (2018-10-01), pages 27
PLUCKTHUN ET AL., ANNU. REV. PHARMACOL. TOXICOL., vol. 55, no. 1, 2015, pages 489 - 511
PRAT ET AL., J. CELL. SCI. III, 1998, pages 237 - 247
PROC. NATL. ACAD. SCI. USA., vol. 78, no. 3, 1981, pages 1527 - 31
QIAO, C. ET AL.: "Liver-specific microRNA-122 target sequences incorporated in AAV vectors efficiently inhibits transgene expression in the liver", GENE THER., vol. 18, 2011, pages 403 - 410, XP055078580, DOI: 10.1038/gt.2010.157
QUEENBALTIMORE, CELL, vol. 33, 1983, pages 741 - 748
RADOVICK ET AL., PROC. NATL. ACAD. SCI. USA, vol. 88, no. 8, 1991, pages 3431 - 3406
RANUM, P. TMONTEYS, A. M.HUNDLEY, A. ADAVIDSON, B. L.: "Compositions and methods for inducible alternative splicing regulation of gene expression.", WORLD PATENT, 2021
ROSEWELL ET AL., J. GENET. SYNDR. GENE THER. SUPPL., vol. 5, 2011, pages 001
SAITO ET AL., NATURE, vol. 620, 2023, pages 660 - 668
SAMULSKI ET AL., J. VIROL., vol. 63, 1989, pages 03822 - 3828
SASAOKA ET AL., MOL. BRAIN RES., vol. 16, 1992, pages 274
SCHULTZ ET AL., GENE, vol. 54, 1987, pages 113 - 123
SEED, NATURE, vol. 329, 1987, pages 840
SHUJI ET AL., MOL. THER., vol. 19, 2011, pages 76 - 82
SILVERMAN ET AL., NAT. BIOTECHNOL., vol. 23, no. 12, 2005, pages 1493 - 1494
SILVERMAN ET AL.: "Multivalent avimer proteins evolved by exon shuffling of a family of human receptor domains.", NAT BIOTECHNOL, vol. 23, 2005, pages 1556 - 1561, XP009088629, DOI: 10.1038/nbt1166
SIMONELLI ET AL., J AM SOC GENE THER., vol. 18, 2010, pages 643 - 650
SKERRA: "Alternative non-antibody scaffolds for molecular recognition.", CURR OPIN BIOTECHNOL, vol. 18, 2007, pages 295 - 304
SKERRA: "Engineered protein scaffolds for molecular recognition.", J MOL RECOGNIT, vol. 13, 2000, pages 167 - 187, XP009019725, DOI: 10.1002/1099-1352(200007/08)13:4<167::AID-JMR502>3.0.CO;2-9
SMITH ET AL., MOL. CELL. BIOL., vol. 3, 1983, pages 2156 - 2165
SRIVASTAVA ET AL., J. VIROL, vol. 45, 1983, pages 555 - 564
SRIVASTAVA., CURR. OPIN. VIROL., vol. 21, 2017, pages 75 - 80
STUDIER ET AL.: "GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY", 1990, ACADEMIC PRESS, pages: 60 - 89
STUMPP ET AL.: "DARPins: a new generation of protein therapeutics.", DRUG DISCOV TODAY, vol. 13, 2008, pages 695 - 701, XP023440383, DOI: 10.1016/j.drudis.2008.04.013
SULUBACAK, U.CAGLAYAN, OGRÖNROOS, SA. ET AL.: "Multimodal machine translation through visuals and speech.", MACHINE TRANSLATION, vol. 34, 2020, pages 97 - 147, XP037249194, DOI: 10.1007/s10590-020-09250-0
TARYMAN ET AL., NEURON, vol. 14, no. 4, 1995, pages 755 - 762
TERAMATO ET AL., LANCET, vol. 355, 2000, pages 1911 - 1912
THRASHER ET AL., NATURE, vol. 443, 2006, pages 5 - 7
TIAN, YONGLONG ET AL.: "What makes for good views for contrastive learning?.", ADVANCES IN NEURAL INFORMATION PROCESSING SYSTEMS, vol. 33, 2020, pages 6827 - 6839
TRATSCHIN ET AL., MOL. CELL. BIOL., vol. 4, 1984, pages 2072 - 2081
TRATSCHIN ET AL., MOL. CELL. BIOL., vol. 5, 1985, pages 3251 - 3260
VERGHESE ET AL., NUCLEIC ACID RES., vol. 42, 2014, pages e53
VIROLOGY, vol. 330, no. 2, 2004, pages 375 - 383
WAGNER ET AL., NAT BIOTECHNOL., vol. 34, no. 11, 2016, pages 1145 - 1160
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
WARD ET AL., NATURE, vol. 341, 1989, pages 544
WEST ET AL., VIROLOGY, vol. 160, 1987, pages 38 - 47
WINOTOBALTIMORE, EMBO J., vol. 8, 1989, pages 729 - 733
WONG ET AL., ADV. GENET., vol. 89, 2015, pages 113 - 152
WORN ET AL., J MOL BIOL., vol. 305, no. 5, 2 February 2001 (2001-02-02), pages 989 - 1010
WU ET AL., MOLECULAR THERAPY, vol. 14, no. 3, 2006, pages 316 - 327
XU ET AL., SCI. CHINA LIFE SCI., vol. 59, 2016, pages 1024 - 1033
YAMASHITA, R. ET AL.: "Convolutional neural networks: an overview and application in radiology.", INSIGHTS IMAGING, vol. 9, 2018, pages 611 - 629, XP055580998, DOI: 10.1007/s13244-018-0639-9
YUSA ET AL., PNAS, vol. 108, no. 4, 2011, pages 1531 - 1536
Z. CHANG ET AL., ON THE DESIGN FUNDAMENTALS OF DIFFUSION MODELS: A SURVEY, 2023
ZAPATA ET AL., PROTEIN ENG., vol. 8, no. 10, 1995, pages 1057 - 62
ZHANG ET AL., PLOS ONE., vol. 8, no. 10, 2013, pages e76771
ZHOU, H.X., J MOL BIOL., vol. 329, no. 1, 23 May 2003 (2003-05-23), pages 1 - 8
ZHU, XIAODAN ET AL.: "Long short-term memory over recursive structures.'' International Conference on Machine Learning.", PMLR, 2015
ZOLBOOT, N.DU, J. X.ZAMPA, F.LIPPI, G: "MicroRNAs instruct and maintain cell type diversity in the nervous system.", FRONT. MOL. NEUROSCI., vol. 14, 2021, pages 646072

Also Published As

Publication number Publication date
WO2025217163A3 (fr) 2025-11-20

Similar Documents

Publication Publication Date Title
US20240084330A1 (en) Compositions and methods for delivering cargo to a target cell
KR20230053591A (ko) 조작된 근육 표적화 조성물
US20250228976A1 (en) Aav capsids that enable cns-wide gene delivery through interactions with the transferrin receptor
JP2022551986A (ja) 修飾筋肉標的化組成物
US20240325568A1 (en) Engineered targeting compositions for endothelial cells of the central nervous system vasculature and methods of use thereof
AU2020347276A1 (en) Engineered adeno-associated virus capsids
US20250120915A1 (en) Engineered delivery vesicles and uses thereof
US20250064976A1 (en) Engineered pnma proteins and delivery systems thereof
WO2023015297A1 (fr) Compositions de ciblage musculaire modifiées
US20250073350A1 (en) Compositions and methods for delivering cargo to a target cell
WO2025217163A2 (fr) Nouveaux capsides aav se liant à cd59 humain
WO2025217174A1 (fr) Modifications de capside d&#39;aav qui permettent une délivrance améliorée de gènes à l&#39;échelle du snc par l&#39;intermédiaire d&#39;interactions avec l&#39;anhydrase carbonique iv
Roy et al. Cell and Gene Therapy: Current Landscape, Emerging Trends and Future Prospects
US20250352670A1 (en) Delivering genes to the brain endothelium to treat lysosomal storage disorder-derived neuropathology
AU2023321621A1 (en) Methods and compositions for transducing hematopoietic cells
Moreno Engineering efficient and safe in situ genome regulation via CRISPR-Cas9 for enabling gene therapies

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 25722411

Country of ref document: EP

Kind code of ref document: A2